REGULATION OF NK1.1 (NKR-PllCD161) EXPRESSION DURING LYMPHOCYTE LINEAGE COMMITMENT IN THE FETAL MOUSE. James Robert Carlyle A thesis submitted in conforrnity with the requirements for the degree of Doctor of Philosophy Graduate Depanment of Immunology University of Toronto O Copyright by James Robert Carlyle 1999
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REGULATION OF NK1.1 (NKR-PllCD161) EXPRESSION
DURING LYMPHOCYTE LINEAGE COMMITMENT IN THE FETAL MOUSE.
James Robert Carlyle
A thesis submitted in conforrnity with the requirements for the degree of Doctor of Philosophy Graduate Depanment of Immunology
University of Toronto
O Copyright by James Robert Carlyle 1999
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ABSTRACT
Regulation of NK 1.1 (NKR-PlKD 16 1 ) Expression During Lymphocyte Lineage Commitment in
the Fetal Mouse. Ph.D. Thesis Abstract. 1999. James Robert Carlyle. Graduate Department of
Immunology. University of Toronto.
T and natural killer (NK) lymphocytes are presumed to share a common intrathymic
precursor. The development of conventional ap T lymphocytes begins within the early fetal
thymus, after the colonization of multipotent CD 1 1 7' (c-kit) hematopoietic precursors. Irrevocable
commitment to the ap T lineage is marked by thymus-induced expression of CD25 (IL-2Ra) and
initiation of genomic rearrangements at the T cell receptor P (TCRP) loci. However. the contribution
of the fetal thymic microenvironment in mediating NK lineage commitment and differentiation has
remained large1 y unappreciated. In particu lar, the development of functional mouse NK ce1 ls
occurs first within the early fetal thymus. Mature fetal thymic NK cells are characterized by
expression of a type iI transmembrane C-type lectin receptor. NK 1.1 (NKR-P 1 /CD 16 1 ). and by
lack of expression of CD 1 1 7 (NK 1.1 '/CD 1 17'). Moreover, NK cell differentiation is preceded by
a thymus-induced developrnental stage (NK 1. I+/CD 1 17") that marks lineage cornmitment of
multipotent hematopoietic precursors to the T and NK cell fates, with the subsequent loss of B
lymphoid potential. Commitment to this T/NK bipotent stage is induced by fetal thymic stroma. but
is not thymus-dependent. Indeed, previousl y-identi fied C D ~ O + (Th y- 1 ) fetal blood
"prothyrnocytes", once thought to represent exclusively T lineage-committed precursors. also
exhibit NK lineage potential and are phenotypically and functionally identical to fetal thymic
NK 1 . I +/CD 1 1 7'' progenitors. Equivalent populations of NK 1.1 +/CD~O+/CD 1 1 7'0 progenitors are
found in the fetal circulation of both normal and athymic nude (nuhu) mice. These findings
indicate that full commitrnent of circulating hematopoietic precursors to the T lineage occurs after
thymus colonization. Interestingly, the NK 1. I antigen expressed by fetal blood cells does not
a.
II
represent the previously-defined NKR-P 1 C (CD 16 I C) gene product: rather, it represents a novel
NKl . 1 antigen expressed in certain mouse strains that is encoded by the closely-related NKR-PlB
(CD161B) gene. In contrast to NKR-PI C, which transduces activating signals by recruiting
protein tyrosine kinases, NKR-PIB transduces inhibitory signais by associating with the protein
tyrosine phosphatase, SHP- 1. Thus, "the NKI . 1 antigen" actually represents two distinct gene
products possessing opposite regulatory functions. These findings suggest a potential role for
NKR-Pl molecules in mediating self/nonself recognition in the immune system.
DEDICATION
To rny parents:
Robert Wayne Carlyle, M.D. (6T3)
of Moosejaw, Saskatchewan
&
Marie Elizabeth (Gogan) Carlyle
of Springhill, Nova Scotia
The work presented in this thesis would not be possible without the expertise and continued
support of my supervisor, Dr. Juan Carlos Zuiiiga-Pfiücker. 1 consider JC a friend in addition to a
mentor. Common phrases heard in the laboratory that re-shaped my way of doing research: "if
you need it, order it"; "1 can't say no to doing an experiment"; and "data. data. show me data!".
In science, freedom of inquiry is the first step to discovery .
1 would also like to thank the people who made it worthwhile beanng the endless pain of
Grad School ... Eric "Earache" Sebzda -- here's to writing Recent Advances exams while drinking
beer in the hot tub at the cottage, to competing for the first and only Ethanol Precipitation Award in
our first month of Grad School, and to al1 the rest (to the reader, for the definition of scientific
irony, read the Special Edition of Eric's thesis) ... Arun "Eight-bail" Mehra -- 1 blame my career on
you; after all, you convinced me to do this insiead of Med School ... Alp "The Bank of' Oran -- i t
wouldn't have been the same without another hurnan presence at the MSB ... Alison "The Brit"
Michie -- when the boss away, at Margarita's we will play ... James "Hollywood" Holloway -- "the
louder you scream, the faster we go" ... Elena "Laney" Ottaway -- you are my Limelight.
This thesis was supported bigtime by a Studentship from the Medical Reseürch Council of
Canada (MRC). In particular, 1 would like to thank the MRC for their extended financial support.
As for the Department and the University. the emancipation of the Graduate Student from the last
sanctioned bastion of slave labour is almost at hand.
This thesis cost approximately CDN $15,000 in "tuition" to produce; that's roughly 50e a
word -- oops, there goes another $2.50.
TABLE OF CONTENTS
............................................................................................................................. ABSTRACT i i
.......................................................................................................................... DEDICATION iv
........................................................................................................... ACKNO WLEDGMENTS 1.
TABLE OF CONTENTS ............ .. ........................................................................................... vi
LIST OF FIGURES ................................................................................................................ x
... .................................................................................... LIST OF TABLES ............................ .. xi11
LIST OF PUBLICATIONS ............... ... ................................................................................ xiv
.................................................................................................................. ABBREVIATIONS xvi
SELECTED CD XOIi4ENCLATURE ........................................................................................ xviii
ERUDITION ......................................................................................................................... x ix
..................................................................................................................... INTRODUCTION I
Origin and formation of the thymiç microcnvironment ........................................................ I
Origin or thymus-colonizing hcmatopoietic prccursors ........................... .. ..................... 2
. . Early thyrnocytc diltcrentiation cvents ............................................................................. 4
Thymic lymphoid progcnitors (TLPs) ............................. .. ................................. 4
Pro-T cells ....................................................................................................... 7
Eririy pre-T cclls ................................................................................................ 8
................................................................................................. Late pre-T cclls 9
................................................................................. Latc thymocyte diffcrentiation evcnts 10
The immaturc DP LO maturc SP transition during T cell development .......................... 1 1
Positive and negative thymocyte selection .......................................................... I I
CD4 versus CD8 thymocytc l ineage commitment .............................................. 13
Models of lymphocyte lineagc cornmitment and differentiation ............................................ 14
CHAPITR 1 . A novel developmental stage in mouse fetal thymopoiesis .......................................... 19
Introduction ................................................................................................................ 20
Identification of N K I . I + / C D ~ 17* ( F ï N K ) progenitors in the mouse fetal thymus .................... 22
F ï N K progenitors serve as precursors for both T and NK cells .............................................. 23
FTNK progenitors give risc to NK cells but not B lymphoid or myeloid lineage cells ................ 27
FTNK cells reprcsent an early developmental stage in thymocyte differentiation ........................ 30
Discussion ............ ... .................................................................................................
CHAPTER II . Intrathymic NK ceIl differentiation in the fetal mouse .................................................
Introduction ....... .. ....... .. .......................................................................................... Mature NK cells (NKI . I+/CDI 1 7 ~ ) develop carly in mousc fetal thymic ontogeny ....................
................................... Fctal thymic NK cells rcsemblc early prccursor thymocytcs .......... ..
Fctal thymic NK cclls cxpress genes associaied with NK cc11 effector function ..........................
Freshly-isalated fetal thymic NK cclls display MHC-unrestricted cytotoxicity ..........................
In vivo adoptive transfcr of precursor-phenotypc thymocytcs ......... .. ..............................,...
Fetal thyrnic NK cclls arc capable of sustained growth in vitro ..........................................
................................................................................................ ............ Discussion ...
CHAPTER III . Rolc of thc fetal thymus in T and NK lineagc commi[mcnt and differcntiation ................
Introduction ................................................................................................................. + .................................................. Identification of NKI.1 cclls in the rnousc feial circulation
....................... . Circulating fetal NKl If cells rescmblc fetal blood "prothymocytes" .....
NK 1 . 1 + fctal blood/splren cells express genes associatcd wiih lymphoid lineage commitment .....
NK 1 . l + fetal blood/splecn cells maintain their TCRP locus in the germline configuration ..........
. ................. Circulating fetal NKI I + cclls are capable of giving rise to T and NK cells in FTOC
. Circulating fetal NK 1 I + cells %ive rise to NK cells but not B cells upon OP9 coculture ............
Discussion ...................................................................................................................
............................. Mode1 of T and NK lineage commitment events in the fetal mouse
vii
CHAPTER IV . An extrathymic (defauk) pathway for thymocyte lineage commitment ..........................
............................................................................................................... Introduction
.......................... . A subset of fetal TLPs spontaneously upregulatcs NKI 1 expression ex vivo
........... Spontaneous upregulation of NKl . 1 is predetermined by exposure to fetal ihymic stroma
. Initiation of NKI I expression in a subset of fetal TLPs occurs in vivo ...................................
Spontaneous upregulation of NKI . 1 represcnts a differentiation event .....................................
Spontaneous uprcgulaiion of NKl . 1 marks lineagc commitment to the T/NK fates ...................
................................................................................................................... Discussion
. ................. .......... CHAPTER V . Mouse NKR-PI B, a novel NKl I antigen with inhibitory function ..
Introduction .................... .. ......................................
. Exprcssion of NKI 1 on ~ ~ 9 0 ' fetal blood cclls is strain-dependent ....................................
............ Expression of NKR-PI family membcrs is strain-spccific .. ................................... Mousc NKR-PIB is a novel NK1.I antigcn ........................... .. ......................................
...................... ............*.......*.. NKR-PI B functions as a killcr-çell inhibitory rcccptor (KIR) ..
................. ....................... NKR-P 1 B binds SHP- I in a phosphorylation-dcpcndent manner ..
................................................................................................................... Discussion
A modcl for NKR-Pl -mediatecf signalling cvcnts .....................................................
A potcntial rolc for NKR-Pl molcculcs in sclflnon-self recognition ...........................
SUMMARY ............. .. ..........................................................................................................
Lincagc commitment and di ffèrentiation events i n ~ h c fetal thymus .........................................
............................................................................. Th y rnic lymphoid progcni tors (TLPs)
T / N K progenitors (FTNKs) .............................................................................................
Pro-T cells ............ ..... ...............................................................................................
Prc-NK / fetal thymic NK cclls ........................................................................................
Intrathymic T and NK lineage differentiaiion events .............................................................
Extrathymic T/NK lineage differentiation: Redefining "prothymocytes" .................................. ...
Vlll
T versus NK cell fate determination: T by design. NK by default? .........................................
"The NK 1 . 1 antigen". 20 years later .................................................................................
............................................................................................. EXPERIMENTAL PROCEDURES
.......................................................................................... ............................. Mice .. Isolation of fetal cclls ....................................................................................................
Flow cytomctric analysis and ceIl sorting ..........................................................................
Fctal thymic organ culture (FTOC) reconstitution ...............................................................
I n vivo adoptive transfer .................................................................................................
..................... ................................ Cell lincs .. OP9 stroma1 ceIl linc coculturc ........................................................................................
i n vitro cell culiurc ........................................................................................................
CFSE vital dyc labclling .............................................................................................. 5 1
Cr-rclcasc ccll-mediated cytotoxicity assay ................................................................... 5 1 ................................................... Cr-rclease anti body-i nduced redirected l ysis (AIRL) assay
......................................................................................................... RT-PCR analysis
RT-PCR for cDNA çloning ............................. ... ......................................... Gcnomic PCR .........................................................................................................
P l a m i d DNA transtcctions ............................................................................................
Immunoprecipitation and Western blotting ........................................................................
REFERENCES ........................................................................................................................
LIST OF FlGURES
Figure 1
Figure 2
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.
Figure 1 1.
Figure 12.
Figure 1 3.
Classical breakdown of TN thymocytes.
Simplified scheme of mouse thymocyte differentiation.
Identification of NK 1.1' cells dunng mouse fetal thymic ontogeny .
FTNK progenitors give rise to both T and NK lymphocytes in FïOC.
F l U K progenitors give rise to NK cells but fail to generate B and myeloid cells
upon OP9 coculture.
Temporal generation of FTNK cells from multipotent FTLP and FL precursors
in FTOC.
FTLP progenitors predominantly differentiate into T-lineage cells in FTOC.
F ï N K progenitors rapidly differentiate into NK-lineage cells upon OP9
coculture.
Model of early lineage commitment and differentiation events in the mouse fetal
thymus.
Fetal thymic NK cells phenotypically resemble early precursor thymocytes.
NK-enriched day 1 5 fetal thymocytes express characteristic gene products of
functional NK cells.
Fetal thymic NK cells mediate MHC-unrestricted cytotoxicity ex vivo.
Precursor-phenotype fetal thymocytes sorted according to NKl . I expression
show distinct reconstitution potential in vivo.
Figure 14.
Figure 1 5.
Figure 16.
Figure 17.
Figure 18.
Figure 19.
Figure 20.
Figure 2 1 .
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Fetal thymic NK cells display sustained growth in vitro.
Identification of NK 1.1 + cells in the rnouse fetal circulation.
NKI . I ' fetal blood and spleen cells resemble fetal blood "prothymocytes".
NKl.I+ fetal blood and spleen cells express genes associated with lymphocyte
lineage commitment.
NK 1. I + fetal blood and spleen cells maintain their TCRp loci in the gemline
configuration.
NKI . I + fetal blood cells generate T and NK cells in FïOC.
NKI . I + fetal blood cells give rise to NK but not B-lineage cells upon OP9
coculture.
Model for T and NK lineage commitment events in the fetal mouse.
F ïLP progenitor thymocytes spontaneously upregulate NK 1.1 expression ex
vivo.
Temporal regulation of NK 1 . I expression on sorted FïLP and F'L progenitors
during short-term in vitro culture.
Spontaneous upregulation of NKl . 1 on FTLPs is not affected by exposure to
exogenous cytokines or stroma1 cells.
Spontaneous upregulation of NK 1.1 on FTLPs is minimally affected by
transcriptional blockade or mitogen-induced activation.
NK1.l upregulation represents a differentiation event and is not due to
outgrowth of NK1. I + cells.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 3 1 .
Figure 32.
Figure 33.
Figure 34.
Figure 35.
Figure 36.
Upregulation of NK1.1 on F n P s marks the loss of B lineage potential.
Expression of NK 1.1 on CD90+ fetal blood celis is strain-dependent.
Expression of NKR-Pl family members is strain-specific. .
Mouse NKR-P I B represents a novel NK 1.1 antigen.
NKR-PIB inhibits NK ce11 function in antibody-induced redirected lysis
(AIRL) assays.
Mouse NKR-P 1 B recmits the SHZ-containing tyrosine phosphatase, SHP- 1 . in a phosphorylation-dependent rnanner.
Proposed molecular mechanism for NKR-P 1 -mediatecl signalling.
Simplified scheme outlining control points in intrathymic T and NK lineage
commi tmen t.
Characterization of fetal thymocyte subsets and ennchment of early precursors
by depletion of lineage-committed progeny.
General developmentai scheme outlining lymphocyte lineage cornmitment events
in the fetal mouse.
LIST OF TABLES
Table 1. Phenotypic charactenzation of fetal thymocyte subsets according to expression
of NK1.1 and CD1 17.
xiii
LIST OF PUBLICATIONS
1 . S.E. Albert, C. McKerlie, A. Pester, B.-J. Edgell, J.R. Carlyle, M. Petric, and J.W.
Chamberlain. 1997. Time-dependent induction of protective anti-influenza immune
responses in human peripheral blood lymphocyte/SCID mice. J. Imrnwiol. 159: 1393-
1403.
2. M.M. Kushida, A. Dey, X.-L. Zhang, J. Campbell, M. Heeney, J.R. Carlyle, S. Ganguly.
K. Ozato, H. Vasavada, and J.W. Chamberlain. 1997. A 150-base pair 5' region of the
MHC class 1 HLA-B7 gene is sufficient to direct tissue-specific expression and locus
control region activity: The alpha site determines efficient expression and in vivo
occupancy at mu1 tiple cis-active si tes throughout this region J. Imrnunol. 159: 49 1 3-4929.
3. J.R. Carlyle, A.M. Michie, T. Nakano, C. Furlonger, C.J. Paige, M.J. Lenardo. and J.C.
Ziïfiiga-Pflücker. 1997. Identification of a novel developmental stage marking lineage
commitment of progenitor thymocytes. J. Exp. Med. 1 86: 1 73- 182.
4. J.R. Carlyle, A.M. Michie, S.K. Cho, and J.C. ZiIfiiga-Pflücker. 1998. Naturai killer cell
development and function precede ap T cell differentiation in mouse fetal thymic ontogeny . J. Immunol. 160: 744-753.
5. A.M. Michie, J.R. Carlyle, and J.C. ZSi6iga-Pflücker. 1998. Early intrathyrnic precursor
cells acquire a C D ~ ' ' ~ phenotype. J. Immunol. 160: 1735- 174 1 .
6. J.R. Carlyle and J.C. Zuiiiga-Pflücker. 1998. Requirement for the thymus in ap T lymphocyte lineage commitment. Immunify 9: 187- 197.
7. J.R. Carlyle and J.C. Ziiiiiga-Pflücker. 1998. Lineage commitment and differentiation of
T and NK lymphocytes in the fetal rnouse. Imrnunol. Rev. 165: 63-74.
8. J.R. Carlyle and J.C. Ziifiiga-Pflücker. 1998. Regulation of NKl . 1 expression during
lineage cornmitment of progeniror thymocytes. J. Immunol. 1 6 1 : 6544-655 1 .
9. A.M. Michie, J.R. Carlyle, and J.C. ZUfiiga-Pflücker. 1998. Phenotypic characterization
xiv
of thymic l ymphoid progenitor cells. Proc. 10th Intl. Congress Immunol. 14 1 - 146.
10. J.R. Carlyle, A. Martin, A. Mehra. L. Attisano, F.W. Tsui, and J.C. Zliiiiga-Pflücker. 1999. Mouse NKR-P I B, a novel NK 1 . 1 antigen with inhibitory function. J. Immunol.
162: In press.
1 1 . S.K. Cho, T.D. Webber, J.R. Carlyle. T. Nakano, S.M. Lewis, and J.C. ZiiRiga-Pflücker. 1999. Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Submitted.
12. S.K. Cho, J.R. Carlyle. and J.C. Ztifiiga-Pflücker. 1999. Direct generation of T lymphocytes from embtyonic stem cells in vitro. In preparation.
Ab:
Ag:
AGM:
B: B6:
BrdU:
CLP : dG:
DN:
DP: FACS:
FL:
FT: FTLP: FTNK:
Froc: HSC:
HSA:
ICAM:
IL: KAR: KIR:
Lin:
LD:
mA b:
MHC:
NK:
PAS:
R:
RAG:
SCF:
SCID:
Antibody
Antigen
Aorta-gonad-mesonephros region
B lymphocyte
C57B116 strain (mouse)
Bromo-deoxyuridine
Common lymphoid progenitor
2-deoxy guanosine
Double-negative
Double-positive
Fluorescence-activated ce1 l sorter
Fetal liver
Fetal thymus
Fetal thymic lymphoid progenitor
Fetal T/NK progenitor
Fetal thymic organ culture
Hematopoietic stem cell
Heat stable antigen
Intercellular adhesion rnotecule
Interleukin
Killer-ce1 l activatory receptor
Killer-cell inhibitory receptor
Lineage (hematopoietic) differentiation markers Lymphoid dendntic ce1 l
Monoclonal antibody
Major histocompati bility
Natural kilIer cell
Para-aortic splanchnopleura region
Receptor
Recombination-activating gene
Stem ce1 l factor
Severe combined immunodeficiency
xvi
Sw:
T: TCR: TLP: TN: VLA:
Swiss.NIH strain (mouse) T lymphocyte T ceIl receptor Thyrnic lymphoid progenitor Triple-negative Very laie activation anUgen
xvii
SELECTED CD NOMENCLATURE
CDI: Non-polymorphic MHC class 1-related antigens (a-e gene products): p,m- - associated.
LFA-2 integrin; binds CD48, CD58 (LFA-3). T3 cornplex; y,6, ê, 6 subunits; associates with TCR O@ and y6 subunits.
L3T4; binds class II MHC.
Ly- 1 ; binds CD72.
Lyt-2/Lyt-3 ( a / p subunits); binds class 1 MHC. Mac-1; CR3; a& integrin; binds CD54, iC3b, fibronectin.
FcyRIIJAI. low/intemediate affinity Fc receptor for IgG (mIgG2b>2az l s>3).
B4; B li neage signal transduction molecule.
HSA; gpi-lin ked; binds CD62P.
IL-2Ra; binds IL-2 associated with CD122lCD132.
Mucosialin; binds CD62L.
S7, leukosialin. sialophorin; binds CD54.
Pgp- 1 ; binds cell matrix proteins (e.g.. hyaiuronate. collagen, fibronectin).
Ly-5, LCA.
B220; CD45 isoforrn.
ICAM- 1 ; binds CD 1 1 ai1 8 (LFA- 1 ), other adhesion molecules.
VEA- 1, EAM- 1 . Thy- 1 ; gpi-linked.
NK cell receptor. associates with NKG2 subunits.
Fas, APO- 1 ; binds CD95L. Fas ligand.
c-kit; Steel. stem-cell. rnast-cell growth factor receptor.
IL-2115RP; associated with CD 132. binds IL-2 and IL- 15,. IL-7Ra; associated wi th CD 1 32, binds IL-7.
IL-2/4/7/9/ 15Rx common y subunit; yc.
VE-cadherin; homotypic binding.
NKR-P 1 (A-C gene products); NK 1.1 antigens (NKR-P 1 WC); protein ligands
unknown.
xviii
ERUDITION
Dans les champs de l'observation le Iiasard ne favorise que les esprits préparés.
In the fields of observation, chance favors only the mind that is prepared.
Louis Pasteur Address at the University of Lille
xix
INTRODUCTION
Understanding how molecular signals in developing tissues induce lineage comrnitment and
differentiation of stem cells is a fundamental question of developmental biology. *In the immune
system, the thymus provides a mode1 system to study the mechanisms controlling tissue-specific
differentiation events and lineage comrnitment pathways. The thymus is formed when circulating
hematopoietic stem cells (HSCs) colonize the mdimentary thymic stroma by approximately day 1 1 -
12 of gestation in the mouse, providing the necessary elements for the commitment and
differentiation of precursor cells into mature and functional T cells ( 1-3). Upon entry into the
thyrnic microenvironment. multipotent precursors rapidly commit to one of a few hematopoietic
lineages present within the adult thymus (1 -5). However, it remains unclear when and how
commitment and lymphocyte lineage restriction occur. The work outlined here attempts to
phenotypically and functionally dissect the early ceIl fate determination events that occur within the
mouse fetal thymus, in which the ordered appearance of various ceIl lineages and developmental
stages occurs on sequential days of fetal ontogeny.
Origin and formation of the thymic microenvironment
The thymus is formed during embryonic development from an involution of the third
pharyngeal pouch by day 10- 1 1 of mouse gestation (6). In addition to cells of hematopoietic
origin. the thymic anlage includes epithelial and mesenchymal tissues, both of which are required
for functional thymopoiesis (7,8). Moreover, fibroblast lineage cells permeate the thymus
framework and recent evidence indicates they may be required for induction of mesenchymal
maturation during early thymic organogenesis (8,9).
It is believed that the early thymic rudiment is first colonized by definitive HSCs by day 1 1
12 of gestation (10). These precursors travel via the fetal circulation and originate from early
1
embryonic and fetal sites of primary hematopoiesis (1 1-1 7). In addition to providing a continuous
source of thymocyte precursors, the contribution of HSCs in completing the thymic architecture
includes dendritic cells and macrophages, which play an important role in thymocyte selection (8).
Furthemore, there is growing evidence that formation of a mature and functional thymic
microenvironment requires reciprocal and synergistic interactions between the rudimentary thymic
stroma and cells of hematopoietic origin (6-9, 18. 19).
The architectural organization of the thymus is such that colonizing hematopoietic
precursors am ve in the subcapsular/outer cortex region, where the majonty of earl y th y mocy te
differentiation takes place (2.3, 10.20). As these cells continue to differentiate and undergo early
selection events and CD4/CD8 lineage cornmitment, they progress from the ccrtex region to the
conicomedullary junction (20). Late selection events take place in the medulla. such that the exit of
mature and functional T cells into the circulation occun near the core of the thymus (20).
Origin of thymus-colonizing hematopoietic precursors
There is recent evidence that hematopoietic stem cells (HSCs) and vmcular endothelia! cells
are derived from common precursors known as hemangioblasts (2 1 ). Originating from the
embryonic mesodemi, these endothelial precursors to HSCs express the fetal liver kinase- 1 (flk- 1 )
receptor, VE-cadherin (CD 144). and CD34, but they Iack expression of CD45, the leukocyte
common antigen (LCA) expressed by al1 non-erythroid hematopoietic cells (2 1-26). Cells with ihis
phenotype are capable of giving rise to both primitive and definitive hematopoietic precursors (23,
37). the latter of which serve as precursors to T lineage cells in the thymus (14, 16).
The first wave of definitive HSC aciivity arises by day 9- IO of gestation in the rnouse
embryo proper, prior to the appearance of these cells in the yolk sac (12- 14, 16,26). These
definitive HSCs derive from the dorsal aortic region of the developing embryo known as the para-
aortic splanchnopleura (PAS) ( 13, 16.26), which subsequently gives rise to the
2
aorta-gonad-mesonephros (AGM) region ( 12, 14). Given the early vascular endothelial nature of
the dorsal aortic region of the embryo, it can be inferred that al1 hematopoietic activity ultimately
derives from this area common to the AGM and PAS (26). This provides a logical ongin for the
dissemination of HSCs into both the embryonic yolk sac by day 9- I O and the fetal circulation by
day 10- 1 1 ( 1 2- 16,26). It is at this timc that HSC activity colonizes the fetal liver, where it
predominates during fetal development ( 14, 15.26). Thereafter, between approximately day 1 7 of
gestation and birth, the fetal marrow becomes established and provides the foundation for neonatal
and adult hematopoietic activity ( l5,28).
It is generally believed that fetal liver-denved hematopoietic precursors migrate through the
circulation to colonize the early thymic rudiment by day 1 1 of mouse gestation (10). Hcwever.
recent findings indicate that it may be the same first wave of definitive HSC activity in the
embryonic circulation that is responsible for colonizing both the fetal liver and thymic rudiment
( 15). This may explain early findings suggesting that fetal thymopoiesis occurs in two temporally
and functionally distinct waves (10,29,30). Nonetheless, both fetal and adult thymopoiesis depend
on a continual source of incoming hematopoietic precursors (2). Thus, it is likely that both the fetal
liver and adult bone marrow provide primary reservoirs for the circulating hematopoietic precursors
that seed the thymus ( 1 1, 14, 15, 17,28,30).
After the entry of hematopoietic precursors into the fetal thymus, thymopoiesis proceeds in
an ordered fashion such that sequential stages of thymocyte differentiation occur on different days
of fetal life (3). This is in contrast to the situation in the adult thymus, where al1 stages of
development are present simultaneously and a dynamic equilibrium is created between incoming
precursors and emigrating T cells (2.3). Nevertheless, much progress has been made recently
regarding an understanding of the key developmental stages that are constantly recapitulated
throughout fetal and adult thymopoiesis, as outlined below.
Early t hymocyte differen tiation events
Upon entry into the thymus, multipoient hematopoietic cells undergo a series of T lineage
differentiation events prior to the appearance of C W D 8 immature double-positive (DP) and
mature single-positive (SP) T cells. These early stages of thymocyte differentiation occur among
the CD3/CD4/CD8 triple-negative (TN) fraction of thymocytes, which corresponds to 15% of total
adult thymocytes, but contains the entire population of fetal thymocytes prior to day 16 of gestation
(3). The characterization of several developmentally-regulated of surface rnarkers, including
CD 1 17 (c-kit), CD44 (Pgp- 1 ), and CD25 (IL-2Ra). has allowed a convenient classification of
thymocyte subsets with distinct functional characteristics ( 1 -3, 8 ,3 1-35).
Figure 1 shows a representative analysis of surface expression of CD25 versus CD 1 17 on
day 15 fetal thymocytes. This analysis allows for the now classical breakdown of TN thymocyte
subsets (3), as outlined in Figure 2. Recent insights delineating fetal mouse thymocyte subsets with
distinct lineage potential are discussed in further detail below.
Thymic lymphoid progenitors (TLPs): ~ ~ 1 1 7 + / ~ ~ 4 4 + / ~ ~ 2 5 '
TLPs represent the most immature hematopoietic precursors common to the fetal and adult
thymus (2.3). As the name suggests, they display enhanced lymphoid lineage precursor potential
and are capable of giving rise to B, T. iiatural killer (NK), and lymphoid dendritic (LD) cells (2, 3).
They are typically characterized by high-level expression of CD 1 17 and CD44 and a lack of CD25
expression, a phenotype which constitutes 55% of the TN thymocyte population (Figures 1 & 2)
(2, 3,36). Additionally, despite their residence in the thymus, TLPs display low to negative
expression of a number of markers found on differentiating thymocytes, including CD5 (Ly- 1).
CD24 (HSA), and CD90 (Thy- 1 ) (3).
In many respects, TLPs are reminiscent of HSCs (37. 38). They display the stem ce11
4
dl5 FT (Total)
Figure 1. Classical breakdown of TN thyrnocytes. Flow cytomeuic analysis of' CD25 versus CD 1 17 expression on total day 15 fetal thymocytcs; CD44 expression is approxirnaied by that of CD 1 17 on TN cclls. Quadrants delincate the TLP (lowcr right), pro-T (upper right), early pre-T (upper Icft). and lare pre-T stages (lower left), as outlined in ihe text.
TLP CD 1 17+/CD#+/CD25 -
4 IL- l/TNF-induction of CD35 + T lincage cornmitment
Pro-T O IL-7-mcdiatcd proliferrition CD 1 17+lCD4rl+lCD25+
4 TCRD rcmngcrncnt onser
Early Pre-T CD 1 17-/CD44-/CD25+
CD~-/+/cD~-/+/cD~-
Dou bie-positive (DP)
C D ~ + / C D B + I C D ~ ~ ~ / ~ ~ ~
Single-positive CSP)
C D ~ + I C D ~ - / C D ~ ~ ~
- - - - - d m - - -
O-t U nproductivc TCRP remanpcmcnt
I
O ~ck-mcdiaicd signal
$ & TCRa rearnnpcrncnt onaci Immature Single-Positive (ISP)
O Unproductivc O - t TCRa rcmrngemcnt/ Fiilcd positivc sclection/ Ncgative selciion
Export to Pcripliery
Figure 2. Simplified scheme of mouse thymoeyte differentiation. The relative sizes of the thymocyte symbols rcflect their relative proliferativc status. Each arrow depicts an itrevcrsible step in thymocytc differcntiation. Crosses (f) rcpresent events leading to prograrnmed ce11 dcath. See tcxt for funher details. Adapted h m (3).
antigen, Sca- 1 (Ly-6A), and lack surface expression of dl hematopoietic lineage markers (Lin- ).
including CD45R (B220), CD I 1 b118 (Mac- 1 ), Gr- 1 (Ly-6G), and TER- 1 19 (2,3.37,38).
However, they differ from HSCs in some important respects (3). Notably. TLPs lack the capacity
to serve as precursors to cells of the myeloid, erythroid, and megakaryocytic lineages in vivo (2-5).
In addition, they give rise to T lineage cells faster than HSCs (39). In the adult mouse. TLPs also
display a cI14'OW phenotype, indicating they rnay not be strictly "triple-negative" (2.39); however.
recent data from our laboratory indicates that this phenotype may be passively acquired rather than
autonomously expressed (40). TLPs are more metabolically active than HSCs, as indicated by
brighter staining with the mitochondrial dye, rhodamine- 123 (37.4 1.42). They are also more
dynamic in terms of their proliferation. with about 10% actively taking up BrdU during ü ? hour
pulse (43.44). In keeping with this, TLPs express the a-chain of the IL-7R (CD 127). which rnay
be induced prior to their mobilization into the circulation and homing into the thymus (15-47). This
is supponed by recent work indicating that functionally similar CD l27+ precursors. termed
common lymphoid progenitors (CLPs). are found in adult bone marrow, and rnay exist at other
primary sites of hematopoiesis (46). Taken together, the above characteristics suggest that TLPs
differ from hematopoietic stem cells in that they are more responsive to activation and/or
proliferation signais.
Pro-T cells: C D ~ 1 7 + / ~ ~ 4 4 ' / ~ ~ 2 5 '
An early event in fetal thyrnocyte development is the thymic induction of CD25 expression
(5). High-level expression of this marker on CD 1 1 7 + / ~ ~ 4 4 + TN thymocytes identifies the pro-T
ceil subset (4.5). w hich like the TLPs accounts for S5% of TN thymocytes (Figures 1 & 2) (2,3,
36). Although committed to the T cell lineage, pro-T cells maintain their T cell antigen receptor
(TCR) gene loci in the gennline configuration (43,44,48-50). Thus, they retain the ability to give
rise to both a@ and @ T cells (4). In addition, this has prompted some investigators to suggest that
7
these cells may serve as precursors to NK cells andlor CD& LD cells (5 1-53). Nonetheless.
further experiments using sensitive in vitro assays for these lineages are required to determine
whether high-level CD25 expression strictly correlates with full commitment to the T lineage.
Within the thymic microenvironment, pro-T cells undergo extensive prolifention. a
response mediated prirnady by exposure to the cytokine, IL-7 (4.43). This serves to greatly
increase iheir numbers pnor to the induction of unique somatic rearrangements of their TCR genes
(3). In addition, pro-T cells have been shown to display a phenotype characteristic of
antigen-activated mature T celis (54). This includes an upregulated expression of
activation-induced transcription factors (such as NF-KB. NF-AT, and AP- 1). adhesion molecules
(including the ICAMs and VLAs), and CD25 itself (3, 54-56). There is evidence that the signal
required for this TCR-independent activation event involves the cytokines TNF-a and IL- l a. as
coculture of TLPs with exogenous TNF-am- la augments CD25 expression, and antibodies to
these cytokines block CD25 expression in fetal thymic lobes (3.5). However. these are not the
only signals required for commitment to the T lymphocyte lineage. Recent evidence indicates that
thymic epithelium is required for full commitrnent to the ap T lymphocyte lineage (7. 8. 57.58).
Early pre-T cells: C D ~ 1 7 ï ~ ~ 4 4 ' / ~ ~ 2 5 +
Pre-T cells may be divided into early and late stages based upon CD25 expression ( 1. 3.59,
60). Together, early and late pre-T cells comprise over 90% of TN thymocytes, with each subset
containing roughly half this proportion (Figures 1 & 2) (2, 3, 36). Early pre-T cells maintain
expression of CD25, but downregulate expression of precunor markers, such as CD 1 17 and CD44
(3). In addition, these cells are characterized by high-level expression of CD24 and CD90 (2,3).
Cells at this stage in development initiate and complete V@)J rearrangements at their TCRP,
y. and 6 loci (48. 50). As a result of ongoing genetic recombination events, there is a transient arrest
in proliferation at the early pre-T ce11 stage (3,43,48-50,6 1 ). Moreover, due to the formation of
8
non-productive TCR gene rearrangements, w hich result in programmed ceIl death, about 70% of
these cells do not progress further in development (3,43,48). Thus. the early pre-T stage also
marks the developmental block in thymocyte differentiation seen in both RAG-deficient and SCID
mice, which cannot initiate or complete V(D)J rearrangements, respectively (3.43.62-64). On the
other hand, expression of a productive TCRP chain, together with the monomorphic pre-Ta subunit.
leads to the formation of the pre-TCR complex and allows these cells to undergo P-selection (6 1.
65-67). Early pre-T cells that successfully navigate pselection re-enter the ce11 cycle and undergo a
second burst of proliferation in progression to the late pre-T ceIl stage (2,3,36,43.48. 50.61 ).
It is postulated that the early pre-T stage represents a likely branch point for the divergence
of the ap and $3 T ceIl lineages (2,3. 36). Recent evidence implicates the transmembrane receptor
Notch in regulating lineage cornmitment to the a0 versus y6 T ce11 lineage (68). Notch signal iing
prornotes the differentiation of ap over y6 T lymphocytes, even in the absence of a productive P
chain (36, 69.70). Yet in addition, signals derived from a productive pre-TCR (in combination with
the CD3 complex and the p56'C\yrosine kinase), also transduce a selective signal resuiting in the
promotion of ap over y6 T ceIl development (3.36.69-7 1 ). It is presumably this same signal from
the pre-TCR complex that prevents further V(D)J recombination and promotes allelic exclusion at
the TCRP locus pnor to advancement to the iate pre-T cell stage (2.3, 36,70).
Late pre-T cells: CD1 177CD44'/CD25-
Late pre-T cells have already undergone p-selection, which leads to the loss of CD25
expression and subsequen t di fferentiation to the CD4/CDS DP stage of th ymocyte development
(Figures 1 & 2) (1 -3,36,70). This stage is also marked by the renewal of brisk proliferation,
allowing significant expansion of the -30% minority of thymocytes that make it this far (3,43, 61).
Expansion of these thymocytes is important for the generation of combinatonal diversity in the
formation of a broad repertoire of ap TCRs (3). In this regard, remangement and expression of
9
the TCRa loci begins at or soon after the Iate pre-T ce11 stage (2,3. 36,70). Production of a variety
of newly-synthesized TCRa chains results in the displacement of the monomorphic pre-Ta chah
and the formation of clonotypic ap TCR complexes. At the same time, these cells progress to the
immature single-positive (ISP) and large-sized DP stages of thymocyte differentiation, in
preparation for repertoire selection and CD4/CD8 lineage cornmitment events (Figure 2) (2.3.36.
70).
Although signalling through the pre-TCR is required for the normal progression of
thyrnocytes from the TN to DP stages, a number of TCR-independent mechanisms have also been
demonstrated to elicit the same phenotypic differentiation (2,3,36,70). These mechanisms include
sublethal ?irradiation (72-75), genetic ablation of p53 (76-79), overexpression of bcl-2 (80-84).
anti-CD3 antibody-mediated stimuiation (85,86), and transgenic expression of constitutively-active
Lck or MAP kinase (87). While it is clear thai some of these treatments promote thymocyte
differentiation by mimicking the signals induced by the pre-TCR complex, others appear to rescue a
small number of thymocytes from programrned ceIl death. Thus. molecular blockade of thymocyte
apoptosis could allow transient development to the DP stage in the absence of the normal
proli ferative burst. In this regard, it is postulated that pre-TCR signals function to induce both
survival and proliferation in developing thyrnocytes, rather than to pmmote the initiation of a
differentiation protocol direct1 y (3. 36,70).
Late thymocyte differentiation events
Upon differentiation to the DP stage. thyrnocytes begin to express low levels of ab
TCRKD3 complexes on their cell surfaces (3 1,36,70,88). In conjunction with the CD4/CD8
coreceptors and various adhesion molecu les, the TCRs allow for the speci fic cognate in teraction of
developing thymocytes with the class 1 and II major histocompatibility complex (MHC) gene
products and associated peptide antigens on the surface of thymic stroma1 cells (8,3 1, 88-90). It is
10
lo/int these interactions that dictate the fate of CD~+/CDS+/CD~ DP thymocytes during their
selection, lineage commitment, and functional maturation en route to becoming mature
C D ~ + / C D ~ - / C D ~ ~ ' and C D ~ + / C D ~ - / C D ~ ~ ' SP T cells (8,3 1. 88-90). Figure 2 outlines these late
thymocyte differentiation events that determine the specificity and function of cytotoxic and helper
T lymphocytes, discussed briefly below.
The immature DP to mature SP transition during T ceil development
DP ihymocytes can be subdivided according to their expression levels of TCRM3D3 as well
as their size, proliferation, and activation status (8.3 1,88,89). DP cells not yet expressing
detectable levels of TCR are predominantly large-sized, cycling blasts and represent a minority (8.
3 1,89). Large DP blasts are the immediate progeny of late TN cells, as evidenced by the finding
that late pre-T cells spontaneously progress to this stage ovemight in the absence of exogenous
growth factors (9 1 ). Small-sized, post-mitotic DP cells expressing low to intermediate ievels of
TCR begin to undergo the processes of positive and negative selection (8, 3 1 , 88-90). These cells
can be subdivided according to expression of the activation markers CD5 and CD69, which are
upregulated in response to TCR ligation during selection (8,89). Coreceptor-skewed (CRS) DP
cells that express high levels of TCR complete selection and undergo lineage commitment to the
CD4 and CD8 SP T cell fates; here, they acquire the functionality that later determines their role in
the immune response (8,3 1 .89).
Positive and negative thy mocyte selection
Th ymocyte selection represents the process of educating immature th ymoc y tes to recognize
specific foreign antigens in the context of self MHC molecules, while at the same time ensuring that
they are tolerant to self antigens (8 ,3 1,88-90). Selection may be broken down into two conceptual
I l
events leading to three possible outcornes. only one of which allows for thymocyte survival.
Positive selection involves the process of actively recruiting only those thymocytes with TCRs
capable of recognizing self class 1 and II MHC molecules (88-90). Thus, failed positive selection
or neglect entails the default targeting for destruction (by programmed ceIl death) of thymocytes
bearing TCRs incompatible with self MHC molecules. On the other hand. negative selection is the
process of deleting or inactivating TCR-bearing cells with specificity for potentially autoreactive self
antigens (88-90,92). Cells with specificities not reactive or only weakly cross-reactive w ith self
antigens escape negative selection and mature into functional T cells capable of recognizing foreign
antigenic peptides in the context of self MHC with high affinity (88-90.92). Weakly autoreactive T
cells that escape central tolerance may still be eliminated or inactivated by fai lsafe periphenl
tolerance mechanisms (92).
Although numerous models have been offered to explain the cellular and molecular basis of
positive and negative selection, it is clear that the ovenll avidity of the TCR and associated
coreceptors for MHC/peptide complexes plays an integral role in detemining the outcome of
selection (88-90). This concept provides the foundation of the affinitylavidity model of thymocyte
selection (88-90). Thus. DP cells displaying TCRs with insufficient basal affinity for either self
class IAI MHC, even in light of the added avidity afforded by the CD4lCD8 coreceptors, do not
undergo positive or negative selection and are thought to undergo programmed ceIl death due to
neglect or failed positive selection. Mean while. thy mocytes expressing TCRs capable of
recognizing self MHCIpeptide with sufficient affinity, but incapable of strongly binding to MHC-
associated self antigens, undergo positive selection and survive to mature into SP T cells. Finally,
DP cells that possess TCRs with too high an affinity for self iMHC1antigen are actively deleted and
undergo apoptosis to prevent autoreactivity. Despite numerous variations and modifications of this
model, both quditative and quantitative, the overall concept of avidity continues to permeate the
literature and seems to offer the most sound explanation surrounding thymocyte selection in
general (88-90). Thus, although TCR affinity is likely not the only factor involved in detemiining
12
the outcome of thymocyte selection (88,90), it is clearly the single most important factor -- after all,
the TCR molecule is what distinguishes one T ceIl from another. The successful TCR must
possess sufficient intrinsic affinity to be able to bind and recognize foreign antigen in the context of
self MHC in vivo, and its affinity for foreign antigen must be above and beyond that for self
antigen/MHC.
CD4 versus CD8 thymocyte lineage commitment
Lineage commitment of immature thymocytes to the CD4/CD8 SP fates involves the
determination of the class of MHC restriction and the functional capacity that a given thymocyte
wi Il acquire as it becomes either a C D ~ + or C D ~ + mature SP T cell (93,94). DP ce1 ls that
downreguiate CD8 expression represent class II-restricted CM+ T cells, the majority of which
display helper T cell function (88, 89). On the other hand, those that downregulate CD4 expression
represent class 1-restricted CDS+ T cells, most of which exhibit cytotoxic T cell function. This
rnodality is due to the intrinsic affinity afforded by the CD4 versus CD8 coreceptor for ciass II
versus class 1 MHC, respectively, in detemining the overail avidity of the TCWcoreceptor to
MHUantigen interaction (88-90). Conceptually. this duality also makes sense in terrns of the
nature of the MHC-restricted antigen and functional role of the coreceptor-beanng T ceil involved
in the immune response. For instance. as class 1 MHC presents mostly intracellular antigens. the
class 1 restriction of cytotoxic CDS+ T cells allows these cells to directly recognize and destroy
infected cells and tumour cells that express foreign or aberrant proteins (88, 89). Likewise. as class
II MHC is responsibie for presenting extracellular and soluble antigens, C D ~ + helper T cells are
functionally suited to providing cytokines and cell contacts that aid B cells in the production of
specific antibodies, which in tum are capable of coating and neutralizing foreign microbes and
toxins. Thus, the self-tolerant and MHC-restncted mature T cells that emerge display a coreceptor
profile that suits their functional characteristics, class of MHC restriction, and cooperative role in
13
the execution of a successful immune response.
The signals that govem CD4 and CD8 lineage commitment are still not completeiy
understood. There has been much controversy recently in support of both stochastic and instructive
models (93-95). The stochastic model predicts that the signals that govem the dwnregulation of
either CD4 or CD8 are random yet mutually exclusive (93-95). Thus, a given thymocyte bearing a
class II-restricted TCR could commit to either the CD4 or CD8 lineages, such that the former fate
would receive survival signais and the latter fate would undergo programrned cell death as a result
of neglect. In contrast, the instructive model dictates that the lineage decision is detemined by the
specificity of the TCR and the restricting MHC (93-95). In this case. a thymocyte displaying a
class II-restricted TCR would receive a signal to downregulate CD8 expression and maintain that of
CD4. while this signal would be distinct from that received by thymocytes expressing class 1-
restricted TCRs. Thus, the TCWcoreceptor signal is unique for each of the lineage pathways and
"instructs" the fates of selecting thymocytes. Although the role of the TCR in determining the
lineage is clear under the instructive model, it appears to be subservient under the stochastic model,
and it has long rernained unclear exactly what fom of higher signal could mediate lineage
commitment prior to the TCWcoreceptor to MHC/peptide interaction. However. recent findings
suggest that, akin to ap versus y6 T lineage commitment (68,69). the CD4 versus CD8 ce11 fate may
be deterrnined by signals derived from the Notch transmembrane receptor (93,94). Thus. the
receipt of a Notch signal favours commitment to the CD8 over the CD4 lineage. even in the absence
of class 1 MHC (93, 94). Nonetheless. this finding does not rule out the-possibility that the
KWcoreceptor pair may provide two distinct signals depending on the choice of the lineage (95).
Models of lymphocyte lineage commitment and differentiation in the fetal mouse
Lineage cornmitment can be defined by the irrevocable determination of a ceIl fate that
retains precursor potential for certain but not al1 ceIl types. Any analysis of lineage commitment
14
requires two fundamental pieces of evidence. First, a precursor ceIl population must be identified
that contains precursors for two or more cell lineages. Ideally, the demonstration of the ability to
give rise to multiple lineages should be demonstrated at the single ce11 level from cells within a given
population. This ensures that the lineage potential observed in the progeny does not result from the
inclusion of two distinct but phenotypically similar unipotent progenitors. Second. it must be
demonstrated that the identified population is not capable of giving nse to other closely related cell
lineages, to avoid rnisinterpretation of a ce11 with multipotent or stem cell origin. Thus. the cell
population should be phenotypically distinguishable from other populations with broader precursor
potential. This is important because every cell in a multicellular organism ultimately ürises from a
single totipotent cell with a fixed genome. and indeed the foundation of the embryonic stem cell
technology depends on this fact. Taken together. these distinctions require correlation of
phenotypic and functional data in order to establish such precursor-product relationships.
To date, one of the most widely studied models for lineage commitment and ce11 fate
detemination is the mammalian hematopoietic system. More recen tly, the uniqueness of the thymic
microenvironment has prompted much research into the role of this organ in commitment of
immigrating precursors. Yet despite intensive investigations, the phenotype and precursor potential
of thymus-colonizing precursors remain controversial. as outlined below.
The earliest precursor population to colonize the fetal thymus appears to contain rnultipotent
hematopoietic potential, including that for both the lymphoid and myeloid lineages (96.97). These
celis reside in the fetal thymic rudiment by day 12 of gestation and are phenotypically and
functionally similar to hematopoietic stem cells (1 8.97). However, between days 12- 14 of
gestation, recoverable myeloid potential within the thymocyte precursor population rapidly
diminishes (96). such that after day 13 of gestation and throughout adult life, only lymphoid
potential can be rescued from intrathymic precursors (3,5.98). Taken together, these fïndings
suggest that the characteristics of either the thymic microenvironment, or the thymus-colonizing
precurson themselves, change during development.
15
In keeping with this, the day 1 1-1 2 fetal thymic rudiment does not appear to be capable of
supporting complete ab T lymphopoiesis (9, 18). This suggests that there may be a delayed
functional maturation of the thymic microenvironment itself. which may include its ability to
efficiently induce lymphoid lineage cornmimient of incoming precursors. Interestingly. the simple
addition of cells of fibroblast origin to the day 12 thymic rudiment results in the restoration of an
optimal thymic microenvironment that supports the full differentiation of mature ap T cells (9).
Thus. the formation of a functional thymic architecture rnay depend on the early colonization by
cells of both hematopoietic and non-hematopoietic origin (8).
Altematively, there is evidence that supports a requirement for the cell-autonomous
maturation of hematopoietic activity dunng development. For example. full lymphohematopoietic
potential emerges during embryonic development with the maturation of HSC activity from a
primitive to definitive state ( 12, 14, 16). Only cells with definitive hematopoietic precursor potential
are capable of giving rise to T lymphocytes (16,27). Yet the onset of functional thymopoiesis may
not depend simply on sufficient colonization by these definitive HSCs, but perhaps on an
intermediate organ such as the fetal liver to produce even more mature progeny. For example, there
is evidence during both fetal and adult hematopoiesis of extrathymic cells termed prothymocytes,
which possess a restricted lymphoid potential and are capable of giving rise to T lymphocytes more
rapidly than pluripotent HSCs (10, 17,28,3 1,45.99-104). Furthermore, recent evidence that
restricted CLPs exist in adult bone marrow irnplies that similar precursors rnay develop during fetal
ontogeny (45.46). It may be that these more mature precursors with restncted lymphoid potential
are predominantly responsible for mobilization and homing to the thymus later in fetal and adult
life.
In any case, the most immature hematopoietic precursors common io the fetal and adult
thymus appear to be multipotent lymphoid-cornmitted precursors capable of giving rise to the B, T,
NK, and LD cell lineages ( 1 -4, 105, 106). Collectively termed the TLPs, these crlls are
phenotypically and functionally similar to HSCs (1 8,96,97). but they lack myeloid and other
16
hematopoietic potentials (2, 3). This suggests that they represent a collection of common
precursors with restricted lymphoid potential, similar to the proposed clonogenic CLPs in the adult
bone marrow (46). Nevertheless, whether TLPs comprise a homogeneous population of
l y mphoid-restricted precursors or represent a collection of phenotypicall y simi lar
lineage-committed cells is unknown. The work outlined in this thesis re-examines the TLP
population for evidence of lineage-restricted progeny.
As discussed above, the stages outlining the differentiation of thymus-colonizing precursors
along the pathway to the T lymphocyte lineage have ken extensively characterized, both
phenotypically and functionally. However, the contribution of the fetal thymic microenvironment in
driving NK lineage commitment and differentiation remains largely uncharacterized. As T and NK
cells are presumed to share a common intrathymic precursor (35, 106- 1 1 O), and mature and
functional NK cells are present within the adult thymus ( 1 I I ) , this suggests that a pathway for in
situ differentiation of thymocytes to the NK lineage may exist within the early fetal thymus.
Therefore, immature fetal thymocytes were re-examined for evidence of developmental stages
signifying lineage commitment to the NK cell fate. by investigating intrathymic expression of the
relatively uncharacterized mouse NK 1.1 marker ( 1 12- 1 15). In mouse strains that express NK 1 . 1
(NKR-P 1. CD 16 1) ( 1 16, 1 17). this antigen identifies large granular lymphocytes with NK ce11
function (1 1 1. 1 14, 1 18). Through the use of the NKI. 1 marker in combination with other well-
characterized surface molecules, this work aims to resolve the events that take place during TINK
lineage commitment in the early fetal thymus. Ultimately, these investigations have led to the
discovery of a novel NKI. I antigen with distinct function in NK cells, suggesting a role for the
NKR-Pl (CD 161) molecules in the regulation of selfhon-self recognition in the immune system.
IDENTIFICATION OF A NOVEL DEVELOPMENTAL STAGE
MARKING LINEAGE COMMITMENT OF PROGENITOR THYMOCYTES
James R. Carlyle, Alison M. Michie, Caren Furlonger*, Tom Nakano*.
Michael J. Lenardos, Christopher J. Paige*, and Juan Carlos Zuiïiga-Pflücker
Department of Immunology, University of Toronto, Toronto, ON, Canada
*Wellesley Hospi ta1 Research Institute, Toronto, ON, Canada
$Depanment of Molecular Cell Biology, Osaka University, Osaka, Japan
SLaboratory of Immunology, NIAID.Nationa1 Institutes of Health. Bethesda. MD.
(All work shown was performed by J.R. Carlyle)
U.S.A.
Pubiished in The Journal of Experimental Medicine
2 1 July 1997. Volume 186, pp. 173- 182
CHAPTER 1: A Novel Developmental Stage in Mouse Fetal Thymopoiesis
Introduction
Bipotent progenitors for T and NK lymphocytes are thought to exist among early precursor
thymocytes in both mice and humans, yet the phenotype and fùnctional properties of such a
progenitor population remain controversial. Several reports have suggested, but not defined. the
presence of a common intrathymic progenitor for T and NK cells within the TLP population (34.
35, 103, 106-1 10). However, these studies are confounded by the possibility that progeny derived
from putative bipotent populations may have arisen from pre-existing unipotent precursors with
similar phenotypes. In particular, NK cells derived from in vitro culture or intravenous injection of
immature thymocytes may represent the outgrowth of NK lineage-committed or mature NK cells
(34,35, 103, 106-1 10).
To address these questions, we analyzed mouse day 13-15 fetal thymocytes. Thymocytes at
this stage in developnient display enriched precursor activi ty for al1 the lymphoid lineaps, yet
contain no mature ap T or B lymphocytes, and have an overall CD3-/CD4-/CD8- (TN) phenotype
( 1 -5) . In addition, NKI. I + (NK lineage) cells have been previousiy reponed to be absent during
fetal ontogeny (34. 106, 10% 1 19). Nonetheless, to minimize contamination with lineage-committed
precurson. we depleted fetal thymocytes populations of more mature progeny by
antibody/complement-mediated lysis. then sorted the remaining cells for an early precursor
phenotype (CD 1 1 7 + / ~ ~ 9 0 ' 0 / ~ ~ 2 4 ~ ~ ICD25').
Here, we report the identification of a novel developmental stage during fetal thymic
ontogeny that delineates a population of TNK-committed progenitors
(NK 1 . 1 +/CD 1 1 7 + / ~ ~ 9 0 ' ~ / ~ ~ 2 4 ' ~ / ~ ~ 2 5 - ) . Surprisingly, these thyrnocytes are phenoty pically and
functionally distinguishable from the multipotent TLP pool based upon expression of the NK ce11
marker, NK 1 . 1 (1 16, 120). Fetal TLPs lacking NKl. 1 (FI'LPs) maintain multipotency for the B. T.
and NK lineages, whereas those expressing NKl . 1 (fetal thymic NK 1.1+ or FTNK progenitors)
are committed exclusiveiy to the T and NK ce11 fates. and have lost B lymphopoietic potential. We
provide evidence that a restriction point to the T/NK lymphocyte destinies is marked by a
thymus-induced differentiation step.
Resul ts
Identification of NKI. 1 + / ~ ~ 1 1 7 + (FTNK) progenitors in the mouse fetal thymus
Analysis of mouse day 13- 16 fetal thymocytes revealed a small percentage of NK 1.1 +
lymphocytes (-4%) as early as day 13 of gestation (Figure 3). We were surprised to detect
NKl . 1' cells in the fetal thymus, because NKl . I + thymocytes were previously reponed to be
absent early in fetal ontogeny (34, 106, 107, 1 19). However, an earlier report ( 1 13). which
employed a polyclonal anti-NK 1.1 antisemm, did provide evidence of NK 1.1 cells in fetal
development. Perhaps the fact that NKl. 1' cells represent fewer than 2% of total day 15 fetal
thymocytes (Figure 3) may explain why some investigators failed to notice this subset during
thymic ontogeny (34, 106, 107, 1 19). Importantly. though. the identification of NK 1.1' thymocytes
suggests that putative bipotential populations of precursors for T and NK cells may have contained
preexisting NK lineage cells (34. 107. 1 19. 12 1 ). Therefore, we further analyzed fetal thymocytes
for expression of the stem ce1 l factor (SCF) receptor, CD 1 1 7 (c-ki t), which is characteristic of
hematopoietic precursors in the fetal liver. bone marrow, and thymus ( 1-3,38. 106. 122- 125).
CD 1 1 7 is expressed on the majority of day 13- 14 NK 1.1 + thymocytes but on very few NK 1.1 +
thymocytes later in ontogeny or in the adult thymus (Figure 3). Significant expression of NK 1.1
was not detectable among CDI 17' fetal liver (FL) cells (Figure 3), suggesting ihat it may be
induced during or after migration to the thymus. Thus, two populations of NKi . I + thymocytes
could be identified, distinguishable based upon CD 1 1 7 expression. This chapter wi 1 l be confined
to discussion of the NK 1.1 +/CD 1 17+ subset ( 126). whereas NK 1. I+/CD 1 17- cells will be
addressed elsewhere (see Chapter II) (1 27).
The most immature progenitor thymocytes are CD 1 17' cells that have not yet expressed the
Fetal Liver (Day 13)
Day 15 Fetal Thymus Z
Day 13 Fetal Thymus
Day 16 Fetal Thymus
Day 14 Fetal Thymus
Adult Thymus (8wks)
Figure 3. Identification of NK1.1+ cells during mouse fetal thymic ontogeny. Two parameter flow cytorneinc analysis of ccll surfacc expression of NK 1.1 vcrsus CD 1 17 on total fetal thymocytes (days 13, 14, 15. 16 of gestation), fetal livcr cclls (day 13 of gestation). and adult thymocytes (8 weeks old) from timed-pregnant Swiss.NIH micc. NK 1 . I +/CD 1 1 7+ (FTNK) cells predominatc early in thymocy ie differcntiation. while NK 1 . IVCD I 17- (mature NK) cells constitute thc majoriiy after d 14 of gestation.
interleukin-2 receptor a-chain (CD25) and bear low levels of heat-stable antigen (HSA; CD24) ( I -
3). We purified these progenitors by depleting day 14- 15 thymocytes of ~ ~ 2 5 ' and ~ ~ 2 4 ~ ' cells.
Within this immature C D ~ ~ ' ~ / C D ~ S ' thymocyte pool, NKI. 1 expression was evident on a higher
percentage of the cells (10-20%) (Figure 4a). Analysis of these cells for several other ce11 surface
markers revealed that they display a composite phenotype comparable to that of
previously-described early progenitor thymocytes ( 1-3.34, 106, 107). dernonstrating that the TLP
population is not homogenous. Rather, we identified a population with the cell surface phenotype:
NK 1. I +/CD 1 1 ~+/CD~~+/CD~~-/CD~~'*/CD~O'~/CD( l6/3î)+/TN, termed fetal thymic NK 1.1 +
(FTNK) progenitors. These cells display markers characteristic of thymic progenitor cells as well
as the NK 1.1 molecule (NKR-PI C, CD 16 1 C) ( 1 16, 120, f 28) of NK cells. A similar finding was
recently observed for early immature human thymocytes, in which a small subset of immature
C D ~ ~ + / C D 1 17' ihymocytes was shown to express a different member of the NKR-P I gene farnily,
NKR-P 1 A ( 129). Thus. the expression of NKR-PI genes by early thymocytes appears to be a
common feature during mouse and human thymic development.
FTNK progenitors serve as precursors for both T and NK cells
To test whether FTNK progenitors represent a novel population of lymphoid precursor
cells. we isolated FT'NK cells from day 14 fetal thymocytes (the population of CD 1 17+, ~ ~ 9 0 ' ~ .
~ ~ 2 4 " . and CD2S cells that express NK 1 . 1 ) by antibody/cornplement-mediated l ysis followed
by fluorescence-activated ceIl sorting (FACS) (Figure 4a. R2 gate). FTNK cells were tested for
precursor potential by a 24-hour incubation with host fetal thymic lobes, which were depleted of
endogenous lymphocytes with deoxyguanosine (dG), followed by standard fetal thymic organ
culture (FTOC) for 12 days ( 130, 13 1 ). Reconstituted tnyrnic lobes were analyzed by flow
d 14 FT ( ~ ~ 2 4 ~ 0 / 2 5 - ) Figure 1. FTNK progcnitors give rise to both T and NK lymphocytes in FTOC. (a) Fluorcsccrice-aciivaied cell sorting (FACS) of CD24lCD25
CD1 17
Cont rol
antihodylcotiiplerneiii-dcplcted day 14 fetal thymocyk. Regions 1, 2. and 3 (RI, R2, and R3) indicaie the gütcs used for isolating the NKI . I -/CD1 17+ (FTLP, 79.4%), NKI .I+lCDl i7+ ( F ï N K , 8.4%), and NKI .l+/CDI 17- (mature NK, 3.4%) subpopulations, respcctively. (b. c) Flow cytometric analysis of cell suriace cxprcssion of CD4 versus CD8, and NKI. I versus a$ TCR, on cçlls recovered 12 days after FTOC reconsiiiution. Panels show dG-FTOCs without the addition of reconstituiing cells (Conirol) or wiih the addition of 1 x 1 0 ~ d l 4 fetal liver (FL), FïLP, or F ï N K progenitors. These results are represeniative of at least four indcpendent experiinerits.
FL FTLP FI'NK
CDS
ap TCR
cy tometry . dG-depleted ROCS that were not reconstituted with precursors rernained devoid of T
lymphocytes (Figure 4b, Control) ( 130, 13 1 ), whereas non-depleted ROCS typically gave rise to
both immature CWICDS double-positive (DP) and mature CD4 and CD8 single-positive (SP) T
lymphocytes (data not shown) ( 130, 13 1 ). dG-depleted thymic lobes reconstituted wi th fetal liver
(FL) ce115 or fetal TLP cells that lack NKl . 1 expression (FTLPs: NKI. 17CD 1 1 7 + / ~ ~ 2 4 " / ~ ~ 2 5 -
cells: Figure 4a, R 1 gate) resulted in the generation of DP and SP T lymphocytes (Figure 4b) (4. 5).
Sorted FTNK cells (NK 1. ! +/CDI I ~ + / c D ~ ~ ' O / C D ~ S cells; Figure 4a. R2 gate) also had
reconstituting ability, giving rise to DP and mature CD4 and CD8 SP T lymphocytes (Figure 4b).
Thus. both FTNK and FTLP thymocytes display T ceIl precursor potential. Moreover, additional
reconstitution experiments revealed that the precursor potential of both populations titrated to a
similar dilution (230 cells/lobe, data not shown). ruling out the possibility that a minor admixture of
FTLP cells accounts for the reconstitution of thymic lobes by ITNK cells. Furthemore. N K l . I +
fetal thymocytes lacking CD 1 17 expression (Figure 4a, R3 gate), corresponding to a mature NK
phenotype (see Chapter II), failed to reconstitute dG-FïOCs ( 127). in accord with prior evidence
that CD 1 17 expression correlates with precursor activity ( 106, 122- 125).
The progeny of F ïLP as well as FTNK cells rxpressed high levels of ap T ce11 receptors
and expressed IL-2 mRNA after concanavalin A stimulation. indicating a mature T cell phenotype
(Figure 4c; data not shown). Thus. despite bearing the NK 1. I marker, FTNK cells display a ce11
surface phenotype similar to TLPs and serve as precursors to conventional T cells. In addition.
both FTLP and FTNK ceils gave rise to NK 1. I +/TcR~P- as well as a few NK 1.1 + / T c R ~ ~ +
thymocytes. with the former population representing conventional NK cells and the latter probably
corresponding to the recently-described CD 1 -restricted and IL-4-producing subset of T cells ( 132,
133). Thus. FTNK as well as FIZP thymocytes contain cells with T and NK precursor potential.
FTNK progenitors give rise to NK cells but not B lymphoid or myeloid lineage cells
We and other investigators have proposed that TLPs display a multipotent lymphoid
precursor potential, which includes the ability to give rise to the T, B. and NK lineages. but not to
myeloid lineage cells (1-5,39, 105, 106). We applied an in vitro mode1 system to test if the FTLP
or FTNK subsets of TLPs possess B-lymphoid or myeloid differentiation potential. Sorted
NK 1. 1 -/CD 1 1 7+ day 14 FL cells cocultured with the bone marrow-derived stroma1 ce11 line. OP9
( 134, 135), predominantly differentiated into functional B cells, as determined by IgM surface
expression on ~ 2 2 0 ' (CD45R) cells and IgM secretion after induction with LPS and IL-7 (Figure
5a; data not shown) ( 1 36, 137). FL cells cocul~red with OP9 also gave rise to a myeloid, Mac- l +
(CD I 1 b), population (Figure 5 2 ) . A small population of NKI . 1' cells was also detected from FL
cells cocultured on OP9 (Figure Sb). Thus, the OP9 cell hie supports in vitro B, myeloid. and NK
cell differentiation (134. 135), while TCRap-bearing T lymphocytes were not detected (Figure 5;
data not shown).
We next tested the differentiation potential of FïLP thymocytes after coculture with OP9
cells. As reponed for TLPs in vivo (4,5,39, 105. 106), day 14 FTLPs showed a potent ability to
give rise to functional B lymphocytes in vitro (Figure 5a). expressed membrane and secreted IgM,
and gave rise to a small percentage of NKl . 1 + lymphocytes (Figure 5b). However, unlike FL cells.
FTLPs lack myeloid potential, as demonstrated by their inübility to differentiate into CD 1 lb' cells
(Figure 5c). These findings support the notion that the earliest fetal thymic progenitor population
contains multipotent lymphoid-restricted precursors (2-5.39, 105, 106). However, our faiture to
detect myeloid lineage cells derived from day 14 FI'LPs is not consistent with work from groups
that used day 12 fetal thymocytes as a source of progenitor cells (96,97). The discrepancy between
these results may be due to the irnmaturity of the day 12 fetal thymic microenvironment, as the full
FTNK
FSC
Figure 5. FTNK progenitors give rise to NK cells but faii to generate B and myeloid cells upon OP9 coculture. Flow cytornetric analysis of cell surfacc expression of (a) CD45R (8220) versus igM, (b) NK1.1 versus CD90 (Thy-1). and (c) CD1 1 b (Mac- 1 ) versus forward scatter (FSC) on sorted d 14 FL, FïLP, and F ï N K cells coculiured with OP9 bone mmow stroma1 cells. Cells were cocultured on confluent OP9 monolayers in the presence of IL-3, IL-6, IL-7 and SCF for 1 1 days, then stimulated with LPS and IL-7 for an additional 4 days prior to analysis.
capacity of the fetal thymus to support thymopoiesis does not develop until day 13- 14 ( 18).
Restriction of myeloid potential may be a very rapid event upon enhy of a multipotent precursor
into a mature thymic microenvironment, but may not occur efficiently in the day 1'2 fetal thymic
rudiment.
Both FIZP and FTNK progenitors displayed T and NK lineage precursor potential in
FTOC reconstitution assays (Figures 4b & 4c); however, no detectable B cell precursor potential
was evident when FTNK cells were cocultured with OP9, as shown by the lack of C D ~ S R + cells
expressing surface or secreted forms of IgM (Figure 5a; data not shown). As expected, FTNKs
also lacked myeloid potential, as demonstrated by the absence of CD 1 1 bf cells upon cocu1 ture with
OP9 cells (Figure Sc). Despite the inability of FTNK progenitors to serve as precursors for B cells
after OP9 coculture, these cells showed a strong precursor potential for NKI. I + lymphocytes
(Figure 5b). Thus, the inability to give rise to B cells is not due to an incapacity of F ï N K cells to
grow on OP9; rather. it demonstrates a T and NK lineage cornmitment by FTNK progenitors.
which in the absence of a proper thymic microenvironment results in the generation of NK cells
( 1 19). Parallel assays in which sorted FTNK cells were used for both FTOC reconstitution and
OP9 coculture resulted in the generation of T and NK cells in R O C , but failed to give rise to B
cells in OP9 coculture. Furthemore, the absence of B lymphocytes in the FïNWOP9 cocultures,
at up to 4,000 cells in culture, demonstrates that their ability to give rise to T and NK cells in the
thymus does not come from an admixture of FTLP cells, which are clearl y capable of B
lymphopoiesis in OP9 cocuitures, with as few as 30 cells in culture (data not shown). Thus, our
results identify a novel population of CD 1 17+ fetal thymocytes thai express the NK 1.1 surface
marker and serve as committed bipotent precursors for mature ab T lymphocytes and NK cells,
while CD1 17+ thymocytes lacking NK 1.1 expression can act as multipotent precursors for the T, B,
and NK lymphocyte lineages.
FTNK cells represent an early developmental stage in thymocyte differentiation
During early development, thymic progenitors transiently express various markers
associated with activated mature T cells (54, 138) and NK cells (34, 107). These include
lymphokine receptors such as CD25, several adhesion molecules such as intercellular adhesion
molecules (ICAMs), very late activation antigens (VLAs). and CD44, and other function-associated
molecules such as CD 16/32 ( 1-3.54. 107, 138). It would appear that the NK 1 .1 + phenotype also
corresponds to a temporary stage that T lymphocytes pass through on their way to maturity. To
test this. we purified day 14 FïLP and R cells by FACS and followed their developmental
progression to determine whethe: these isolated precursors could give rise to FTNK cells in
reconstituted dG-treated fetal thymic lobes. Indeed, both FL and FTLP precursors directly give rise
to substantial populations of FTNK cells within 48 hrs after transfer into FT'OCs (Figure 6).
FTLPs appear to differentiate and reach the F ï N K stage with faster kinetics than FL-derived
progenitors (Figure 6). This finding is in agreement with previous reports showing that fetai
th ymus-derived precursors show faster reconstitution kinetics than fetal liver-derived progenitors (3.
60). The appearance of FTNK cells peaks by day 4 after thymic reconstitution with FTLPs (Figure
6; 83%). and declines by day 6. It is ai these later time points that F ïNK cells gain CD25
expression (Figure 7b; 37% of CD 1 17+ gated cells coexpress NK 1.1 + / ~ ~ 2 5 + ) , and later lose
NK 1.1 expression as irrevocable commitment io the T ce11 lineage occurs and the
C D ~ ~ + / C D I I ~ + M K I . 1' stage is reached (Figure 7b) (4,5). Again, the temporal kinetics of ~ ~ 2 5 +
expression by FTNK cells and loss of CD 1 17 expression by developing thymocytes is more
accelerated in dG-FTOCs reconstituted with FTLPs than with FL-derived progenitors (Figures 7a
& 7b). Figure 7b also shows that by day 6 of reconstitution both precursor cell types give rise to
NK 1.1 +/CD] 177CD25- celis, presumably pre-NK cells, while few NKl . 1 -/CD 1 1 T/cDZ+ (pre-T
FTLP
Day O
Day 2
Day 4
Day 6
I . ".*T . . "-1 ' - ..'".1 ' .-..'-Y
Figure 6. Temporal generation of FTNK cells from multipotent FTLP and FL precursors in ETOC. dG-ROCS werc reconstituted with NK1.l -/CD 1 17+ FTLP and FL-derived precursors for the indicated times then analyzed by flow cytomctry for expression of NK 1 . 1 versus CD 1 1 7. Reconstitutions were performed usine 3x 104 soned cells pcr lobe (3 lobesitirne point).
FTLP Control
FL Control
FTLP --> FTOC
FL --> m O C
Conirol FTOC
Figure 7. i T L P progenitors predominantiy differentiate into T-lineagc cells in =OC. Threc-paramcter flow cytomeiric analysis of ceIl surfacc expression of CD1 17, NKI.1, and CD25 on sorted dl4 FTLP and FL- derivcd precursors either (a) beforc. or (b) 6 days aftcr transfer into dG- n o C s . Panels show relative ceIl number (RCN) versus CD1 17 expression on total cells. and NKI . 1 versus CD25 cxpression gated on CD1 17* (R 1) and CD1 17- (R2) ceil populations, rcspectively.
cells) are detected. These cells developed at later time points (data not shown). Finally. Figure 7b
shows that nonreconstituted dG-FTOCs (Control ROC) remain devoid of lymphocytes. Thus,
when purified Fi'LPs. which make up 4% of total day 14 fetal thymocytes. or FL cells are
reintroduced to the thymic microenviroment a synchronized progression through the FTN K stage is
clearly observed (Figures 6 & 7). which occurs pnor to the CD 1 1 7 + / ~ ~ 2 5 + stage of T ce11
development.
To further delineate this developmental stage, we punfied FI'NK progenitors by FACS and
analyzed changes in their phenotype using defined in vitro culture conditions that promote
commitment to the NK lineage (OP9 cocultures: Figure 5). Isolated FTNK progenitors cultured in
medium containing the cytokines IL-3. IL-6. IL-7. and SCF to maintain viability (4). retain their
phenotype after short-terni (48h) culture (Figure 8, Control). In contrast. a similar exposure of
purified FTNKs to the OP9 bone marrow-derived stroma1 ce11 line stimulates commitment to the
NK ceIl lineage. as indicated by the rapid loss of CD1 17 expression with the retention of NK1.l
expression (Figure 8.OP9). On the other hand, during reconstitution of dG-FTOCs, not only is
CD 1 17 expression retained on FI'NK cells but CD25 expression is induced (Figure 7b). In
contrast, FTNK cells cultured in isolation or on OP9 cells remained NKl . 1 + and did not propress
to the ~ ~ 2 5 + stage of thymocyte developrnent (Figure 8).
These findings suggest that the F I N K stage represents a developmental crossroad during
thymocyte differentiation, whereby the majority of precursors in the thymus are guided to enter the
T lineage pathway and express CD25 (Figure 7), while a minor fraction enter the NK lineage, lose
CD1 17 expression and fail to express CD25 (Figures 7 & 8). The generation of small nurnbers of
mature NK cells in FT'OCs (Figure 4c) and during thymic ontogeny in vivo (Figure 3) rnay result
from the occasional or rare failure of the local thymic microenvironment to efficiently support T
lineage commitment and di fferentiation. In such instances, the thymus is still capable of supporting
Figure 8. FTNK progenitors rapidly differentiate into NK-lineage cells upon OP9 coculture. Thrce-pliranieter flow cytomciric analysis of cet1 surface expression of (a) NKI . I versus CD25, (b) NK 1.1 versus CD 1 17, aiid (c) CD25 vcrsus CD 1 1 7 on sorted, culturcd FT NK progenitors. FTNK progenitors were gcnerated in vitro as in Figure 6 (FTLP, driy 2). then culturcd for an 2 days in medium plus cytokines (IL-3, IL-6, IL-7, SCF), citlier alone (Control) or on conflueni nionolriycrs of OP9 hone ninrrow sirortiril cells.
Control
OP9
NK ceil maturation. Stroma1 influences, thyrnic or otherwise, appear to be necessary or at least
sufficient for NK ce11 maturation, as cells at the bipotent FRVK stage fail to mature into NK cells
when cultured without stroma1 cells in the presence of IL-3, IL-6, IL-7, and SCF for up to 8 d in
vitro (data not shown). Cytokines, such as IL-12, IL- 15, and perhaps IL-2, may play a role in the
differentiation to the NK lineage (1 15, 121, 139), while it appears that the cytokines TNF-a and
IL- la are required for T lineage cornmitment and differentiation (5). The identification of the
Fl?rlK stage of thymocyte development marks an important step towards the elucidation of the
molecular signals responsible for mediating cornmitment to T and NK lineages.
Discussion
Our results demonstrate the existence of a stage in early thymocyte differentiation dunhg
which progenitors~ransiently express some hallmarks of NK lymphocytes; a stage that defines
commitment towards the T/NK lineages with the concomitant loss of B lineage potential( 126).
Thymocytes at this stage are phenotypically similar to the TLP population. but can be distinguished
from TLP cells and mature NK cells based upon expression of NK 1 . 1 and CD 1 1 7. respective1 y.
Hence, some or al1 of the previously reported NK cells derived from intravenous injections of
populations of TLP thymocytes (34, 106, 107) are likely due to the differentiation and outgrowth of
the FTNK papulation (126) or from pre-existing mature NK cells (see Chapter 11) ( 177). F ï N K
cells that retain their NK phenotype but lose CD1 17 expression would also lose their übility to
differentiate into T cells and therefore possess an NK-restricted potential. Whi le this remains to be
demonstrated at the clonal level, we propose that NK 1 . 1 - FTLPs represent the earliest thymic
progenitors that are multipotent for the B, T, and NK lineages and rapidly give rise to FTNK
precursor cells, which represent restricted bipotent T/NK precursor cells.
The FTNK stage. as shown in Figure 9. may represent one of the earliest phenotypic
changes that occurs after hematopoietic progenitors enter the thymus. Our data suggest that the
expression of NK 1.1 by early precursor thymocytes denotes their loss of B ce11 potential and,
therefore. commitment to the T andor NK lineages. The stage shown in brackets represents a
transition stage (Figure 7; NK 1. !+/CD 1 1 7 + / ~ ~ 2 5 + ) that would be indicative of precursor
thymocytes progressing to the pro-T ceIl stage (1-5), but may still display some NK cell potential
(4.52). Our findings are the first to delineate and characterize a separate phenotypic stage for
progenitor thymocytes that possess a T/NK-restricted precursor function, which is distinguishable
from the multipotent TLP population ( 126) and free of pre-existing mature NK cells (see
HSC I +
Ly mphoid Myeloid
Etc.
Ex-Thymic Lineage Potential
Th y iiiiis
--+ NK Thyriius-induccd
Coiiirnitriieni S tagcs ------ ------ f Pre-N K
I I I I
I FTLP 1
I I
I I I I
I I I I I I I
--* T I I
I I I I I I I 1 t Pro-T Earl y Late I
\--- T--------' Pre-T Pre-T
I
1 # +
NK t NK
B
Figure 9. Model of early lineage cornmitment and differentiation events in the mouse fetal thynrus. Upon entry into the fetal thyrnus, ~iiultipotcnt progenitors rapidly commit to the lyiiiphoid lineages (FTLP stage), restricting other heniaiopoietic potentials including that of the mycloid lineage. Soon afier ihyiiiic immigration, a thymus-induced differentiation siep marked by cxprcssion of NK 1 . I (FïNK stage) signifies cominiiment to the T and NK lineages, with the conconiitani loss of B lyriiphoid potential. K N K progenitors which undergo a second thymus- induced conirtiitment step, marked by cxprcssion of CD25, lose NKI. I and coniinit io the T cell lincage (Pro-T stage). FTNK cclls wliich do not undergo the second thymus-induced differcntiatiori event lose CD 1 17 cxprcssion and hccoiiic N K lincnge-conitniite prccursors (Pre- NK strige).
Chapter II) (127). Thus, Figure 9 provides a new paradigm for T ce11 development and defines a
novel thymocyte developmental stage within the full context of thymocyte differentiation.
Our findings allow us to redefine which fetal thymocytes possess a lymphoid-restricted
mu1 tipotent reconstitution potential (Figure 9). In the adult thymus, progenitor cells w ith a similar
reconstitution potential have been characterized by their C D ~ ~ + / C D 1 1 7 + / ~ ~ 4 ' ~ phenotype. These
C D ~ " precursors have been shown to serve as progenitors for not only T, NK, and B cells, but also
for a novel subset of thymus-derived dendntic cells (2,39,51, 105). However, dendntic cell
potential is not restricted to the C D ~ " stage but is maintained up to the C D ~ ~ + / C D ~ 1 7 + / ~ ~ 2 5 +
stage of thymocyte development (5 1, 105). Thus, we predict that FI'NK as well as FïLP cells
would also serve as progenitors for thymic dendritic cells, aç both the FTNK and FTLP
developmental stages occur before the induction of CD25 expression (Figures 7 & 9). Moreover, it
appears that the FTNK stage is not restricted to fetal thymocyte development. as FACS-purified
C D ~ " progenitor cells from adult mouse thymus also contain a subset of NKI. 1' cells; these cells
comprise 4% of C D ~ " progenitors and display a C D ~ ~ + / C D 1 1 ~ + / c D ~ " / N K ~ . 1 + phenotype (data
not shown). Thus, the expression of NKR-PI genes by early immature thymocytes appears to be a
common feature during both fetal and adult development in the mouse as well as the human thymus
( 129).
Taken together, our identification of FT'NK cells in the thymus and the recent description of
mature NK 1.1' ap T cells indicate that T and NK lymphocytes may be closely linked from their
earliest differentiation steps and throughout their maturation. Identification of the restriction point
in which B ceIl potentiality is lost, whereaî that for the T and NK cell fates is maintained, will
facilitate the molecular characterization of thymic signals that control this event.
CHAPTER II:
NATURAL KILLER CELL DEVELOPMENT AND FUNCTION
PRECEDE ap T CELL DIFFERENTIATION IN MOUSE FETAL THYMIC ONTOGENY
James R. Carlyle, Alison M. Michie, Sarah K. Cho, and Juan Carlos Ziifiiga-Pflücker
Department of Immunology, University of Toronto, Toronto. ON. Canada
(Cytotoxicity assays were performed by J.C. Zufiiga-Pflücker.
All other work was performed by J.R. Carlyle)
Published in The Journal of lmmunology
15 January 1998. Volume 160, pp. 744-753
CHAPTER II: Intrathymic NK Cell Differentiation in the Feîal Mouse
Introduction
Soon after the immigration of fetal liver-derived hematopoietic precursors into the fetal
thymus, a wave of thymocyte differentiation is established, marked by the ordered appearance of
various developmental stages along the pathway to mature T cells (1-3). In fetal thymic ontogeny.
the development of a defined subset of y6 T cells precedes that of conventional ab T cells (29).
However, the developmental appearance of mature NK cells remains unknown within the context of
fetal thymopoiesis, and functional NK cells are thought to be absent in mouse fetal ontogeny.
Natural killer (NK) cells rnediate MHC-unrestricted cytolysis of virus-infected cells and
turnor cells ( 1 1 1 , 140- 142). In the adult mouse, NK cells are bone marrow-derived lymphocytes
that mature predorninantly in extrathymic locations ( 1 1 1). Whereas the efficient development of
mature peripheral ap and y6 T cells is thymus-dependent, the development of NK cells is thymus-
independent, and these cells are present at normal to elevated levels in athymic nude mice ( 1 43. 144)
as well as in mice defective in the ability to reamnge genes encoding the Ag receptors (SCID and
RAG-deficient mice) (62, 63, 145, 146). Nonetheless, in addition to peripheral sites for NK
lyrnphopoiesis, NK cell development can occur within the thymus, and these cells are ihought to
share a common intrathymic progenitor with T lymphocytes within the TLP population (2-5, 106,
107. 1-6). Previous studies have provided evidence for the existence of a bipotent thymic
progenitor for T and NK celis (35, 107, 109. 121. 126). However, these reports failed to outline the
earliest stages of NK cell development during fetal ontogeny. Moreover, these studies did not
address the possibility that NK cells derived frorn intravenous injection or in vitro culture of
precursor thymocyte populations may represent the outgrowth of a preexisting subset of marure
NK cells.
To investigaie these questions. we re-analyzed day 13- 16 mouse fetal thymocytes for
40
evidence of hinctional NK cells. Although NK 1.1 + cells were previously reported to be absent
during fetal ontogeny (34, 106, 107). this is cleady not the case (see Chapter 1) (1 26). Moreover, in
addition to Our identification of NK 1.1 +/CD 1 17+ T/NK precursors, a second population of NK 1.1 +
thymocytes was identified that possessed a phenotype charactenstic of NK lineage-committed cells.
lacking expression of CD 1 17.
Here, we report that thymocytes with a mature NK cell phenotype (NK1 .I+/cDI 17-) are
evident as early as day 13- 14 of gestation, approximately 2-3 days before the appearance of
CD~+/CDS+ double-positive (DP) cells. NKI. I + / C D ~ 17- fetal thymic NK cells express genes
üssociated with NK ce11 effector function and display MHC-unrestricted cytolytic activity directly
ex vivo, without a requirement for expoîure to exogenous cytokines. Strikingly. despite the above
functional characteristics and lack of CD 1 17 surface expression, fetal thymic NK cells possess a
composite phenotype similar to early precursor thymocytes. Moreover, their capacity for sustained
growth both in vitro and in vivo confounds previous assessments of NK lineage precursor function.
Thus, mature NK cells may have been inadvertently included in previous attempts io identify
multipotent and bipotent precursor thymocytes. We provide evidence that the early fetal thymus is
fully capable of supporting NK lineage development and that NK ce11 maturation and function
precede ap T cell differentiation in mouse fetal ontogeny.
Results
Mature NK cells ( N K I . ~ + / c D ~ ~ ~ - ) develop early in mouse fetal thymic ontogeny
To outline the developmental appearance of the NK ce11 lineage in fetal thymic ontogeny. we
analyzed expression of NK 1.1 and CD 1 17 on day 13- 16 fetal thymocytes and fetal liver-derived
hematopoietic cells. As mentioned previously, NK 1.1 + cells were reported to be absent in fetal
thymic ontogeny (34, 106, 107, 1 19), although earlier investigations had suggested thnt this was not
the case ( 1 13, 1 14). Our findings demonstrated that a significant percentage of total thymocytes
display detectable NK 1.1 expression as early as day 13 of gestation (Figure 3; see Chapter 1). when
NK 1.1 +/CD! 17+ cells represent the majority of NK 1.1 + cells in the fetal thymus ( 126. 127).
However, approximately one day after the NK 1.1 +/CD 1 17+ stage is first observed, between days
1 3- 14 of gestation, there is an emergence of NK 1.1+ thymocytes lacking expression of CD 1 17
(Figure 3), corresponding to a mature NK ce11 phenotype. By days 14- 15, the NK 1 .1 +/CD 1 17-
population predominates and the percentage of cells coexpressing NK 1.1 and CD 1 17 diminishes
thereafter (Figure 3). Significant expression of NKI . 1 was not detectable on day 13- 16 fetal liver
cells (Figure 3; data not shown) (127), suggesting that efficient generation of NKl . I + cells in fetal
ontogeny occurs dunng or after migration of hematopoietic precursors from the fetal liver to the
thymus.
Fetal thymic NK cells resern ble early precursor thymocytes
During experiments aimed at purifying F M K and FIZP progenitors, we noticed that
NK1. I + cells were frequently enriched among populations of precursor thymocytes (see below).
CD24 (HSA) and CD25 (IL-2Ra) are two markers frequently used to discriminate later T lineage
differentiation stages from the TLPs, which are C D ~ ~ ' O / C D ~ S . Figure 10 shows day 15 fetal
42
Day 15 FT Day 15 FL Figure 10. Fetal thymic NK cells phenotypically resemble early precursor thymocytes. (a ) Flow cytometric analysis of c r l l surface expression of CD1 17 versus CD24 on d 15 fetal thymocytes (FT) and fetal liver (FL) cells before and after CD24/CD25 an t ibody /complemen t - mediated depletion. (b) Analysis of CD24WCD25- FT and FL cells for expression of various lymphocyte differentirition inarkers versus CD I 17.
Total
CD2410/25 -
Day 15 FT (CD24I0/25-)
Day I S E (CD24Io/25-)
Day 15 FL (CD2410/25-)
Day 15 FT (CD2410/35-)
thymocytes and fetal liver cells before and after anti-CD24 (J 1 ld.2) and anti-CD25 (7D4)
depletion. Day 15 fetal thymocytes were chosen because these cells contain no mature ap T or B
lymphocytes (3), possess an overall CD3'/CD4/CDI triple-negative (TN) phenotype (48.60). and
contain a significant population of fetal thymic NK cells (NK 1. CD 1 17'; Figure 3) ( 127).
Post-depletion analysis verified that these populations were r98% depleted of both ~ ~ 2 4 ~ ' and
~ ~ 2 5 + cells (M 1 /69, anti-CD24: 3C7, anti-CD25; Figure 1 Oa; data not shown); C D ~ ~ " / C D Z -
cells represented -4% of total day 15 fetal thymocytes. Although CD24KD25-depletion
dramatically entiches for CD 1 17+ precursor cells among both fetal thymocytes and fetal liver cells
(Figure 1 Oa), a significant increase in CD 1 17- cells is also observed among depleted fetal
thymocytes but not fetal liver cells (Figure 1 Oa; CD 1 lT/CDW thymocytes). Therefore. depleted
ceIl populations were analyzed further for expression of various lymphocyte differentiation markers
(Figure 1 Ob).
Early investigations into thymocyte differentiation reveated that precursor potential resides
in a triple-negative (TN) fraction, a designation which originaliy included a CDS'"' (Ly- 1 )
phenotype ( 1 47). Recent reports have determined that the earliest precursor thymocytes are CD~",
while fetal liver-derived hematopoietic precursors possess a CDS phenotype ( 148). Analysis of
C D ~ ~ " / C D ~ S fetal thyrnocy tes and fetal l i ver cells for expression of CD5 con firmed that among
the CD 1 17' populations, which have been shown to possess multi-lineage precursor potential. fetal
thymocytes express higher levels of CD5 than fetal liver cells (Figure lob). However, a significant
population of CDS cells lacking expression of both CD 1 1 7 and CD24 was present among fetal
thymocytes, but absent in fetal liver cells (Figure lob; data not shown). Remarkably, this
CDSICD 1 17'1CD24' thyrnocyte population accounted for approximately 30-40% of
CD24/CD25-depleted day 15 fetal thymocytes (Figure lob).
Previous studies have shown that earl y progenitor thymocytes express CD44 (Pgp- 1 ),
CD 16/32 (FcyRIIVII), and low levels of CD90 (Thy- 1 ) ( 106, 107). Although the roles for these
markers in lymphocyte development remain to be elucidated, they provide useful tools for the
44
developmental staging of distinct precursor thymocytes: CD44 is present on precursors up to and
including the pro-T ceIl stage, when CD25 is upregulated and cornmitment to the T 1 ineage occurs
(1,4, 5-60); high level expression of CD16132 has k e n associated with a putative bipotent T/NK
precursor stage. with diminishing expression after the pro-T cell stage (34. 100, 107); and the
earliest precursor thymocytes bear low levels of CD90 until the pro-T ce11 stage. when high level
expression of CD90 is attained (1). Further analysis of CD24/CD25-depleted fetal thyrnocytes
revealed that a significant population of CD1 17- cells. absent in fetal liver ce11 preparations.
expressed both CD44 and CD 16/32 (Figure I Ob). Analysis of CD90 expression demonstrated that
these CD 1 17- thyrnocytes were predominûntly C D ~ O ~ ' , while CD 1 17' precursors were ~ ~ 9 0 ' ~ to
CD90- among fetai thymocyte and fetal liver cells, respectively (Figure lob).
Another marker which has recently been correlated to a subset of early fetal thymocytes is
CD 122 (IL-2RP) ( 12 1, 149). CD 122 has been suggested to be expressed on a population of
putative bipotent TINK precursors, as well as on the earliest thymic immigrant ceils ( 12 1. 149). A
subset of fetal liver cells, including the earliest committed B lineage precursors (Fraction A,
C D ~ ~ R + / C D ~ ~ + / C D ~ ~ - ) ( 1 50). also expresses CD 122 ( 149). Flow cytometric analysis of
CD24KD25-depleted cells revealed that the majority of CD 1 17- thymocytes expressed CD1 22,
while expression of CD122 was virtually absent on CD1 17' thymocytes and fetal liver cell
preparations (Figure lob). Because CD 122 is also present on al1 mature resting NK cells in the
dduit mouse. and the overall phenotype of the CD 1 17- thymocyte population resembied that of NK
cells, we analyzed these cells for expression of NK 1.1 . As shown in ~ i ~ u r e lob, the majority
(s80Q) of CD 1 17' cells among CD241CD25-depleted thymocytes expressed NK 1.1, while
significant expression of NK 1 . 1 on depleted feral liver cells could not be detected. These
NK 1.1 +/CD 1 17- fetal thymocytes also express the novel pan-NK ce11 marker, DX5 ( 15 1 ). while
fetal liver cells and CD1 17+ fetal thymocytes, including NK1. 1 + / ~ ~ 1 1 7 + FîNK progenitors, lack
expression of DX5 (data not shown, see Chapter III) (127, 152). Further characterization of the
overall phenotype of these cells was performed by multiparameter flow cytometnc analysis on
45
sorted NK 1. I + day 15 fetal thymocytes. A detailed summary of the composite phenotype of the
NKl . 1 +/CDI 17- fetal thymic NK ce11 population is outlined in Table 1, in comparison to
NK 1.1 +/CD 1 1 7+ TINK-committed FTNK progenitors, and NK 1.1 -/CD 1 1 7+ FïLPs ( 126, 1 27).
Taken together, these results show that cells with a mature NK phenotype (NK I . l + /
CD 1 1 7-) are highl y enriched among precursor-phenotype thymocyte populations in w hich CD 1 1 7
expression is not determined (Figure 10). Thus, the fetal thymus contains a subset of mature NK
lymphocytes that displays some markers typical of precursor thymocytes, such as CD44 and
CD 16/32, yet lacks expression of nurnerous differentiation markers, including CD3. CD4. CD8
(i.e., TN), CD5, CD24, CD25, and other lineage (Lin) markers, such as B220. Mac- 1. Gr- 1 . and
TER- 1 19. To determine whether these fetal thymic NK cells were indeed functionally mature, we
charactenzed their properties further.
Fetal thymic NK cells express genes associated with NK cell effector function
The NK 1 . 1 molecule (NKR-P I C) is a member of the NKR-P 1 (CD 16 1 ) gene family ( 1 16,
120). and foms part of a proposed NK receptor gene complex that identifies TN lymphocytes with
NK ce11 function ( 1 14, 1 18. 153, 154). To determine whether the mature phenotype of
NK 1 . 1 +/CD I 17- fetal thymocytes correlated with NK cell function, we assessed the expression of
various genes associated with NK cell effector function by performing RT-PCR on RNA isolated
from total and CD24fCD25-depleted (NK-ennched) day 1 5 fetal thymocytes and fetal liver cells
(Figure 1 1). As a positive control. RNA was also isolated from CD24ICD25-depleted adult
RAG? thy muses, which lack thymocy tes beyond the early pre-T ceIl stage (CD#'/CD~~+), yet
contain normal to elevated numbers of mature NK cells ( 146).
Consistent with the finding that celis with a mature NK phenotype are virtually absent
among fetal liver suspensions (Figures 3 & IO), no significant expression of these genes could be
detected by RT-PCR on RNA isolated from day 15 fetal liver cells (Figure 1 1 a, FL). In contrast,
46
Table 1. Phenotypic characterization of fetal thymocyte subsets grouped according to expression of NK 1.1 and CD 1 17%
- FTLPb FTNK Mature NK
Marker NK1.l-/CD117+ NKl.l+/CD117+ NK1 .l+/CDI 17-
NK1.l (NKR-PIC) CD117 (c-kit) CD44 (Pgp- 1 ) CD25 (IL-2Ra) CD2 (LFA-2) CD5 (Ly- 1 ) CD3e CD4 CD8 CD 1 6/32 (FcyRIlTII) CD24 (HSA) CD90 (Thy-1) CD 1 22 (IL-211 5RB) MHC Class 1 aPTCR y8TCR Lin DXS"
- low -
- -
low low -
high - -
low -
- - +
low +/Iow
- high
- -
low -
- +
IOWA +/- +
high -
.- . . . . - -- -- - -- -
Cell surface expression was determined by flow cytometric analysis of day 13 (FTNK enriched) or day 15 (mature NK enriched) FT after CD24KD25-depletion and sorting for celis with or without NKl.1 expression.
Abbreviations: FT'LP, fetal thymic lymphoid progenitor (BfiYNK multipotential); FI'NK, fetal thymic NK 1.1 + progenitor (TNK bipotential): mature NK, mature NK cell; low, low but significant staining detected; high. high level staining; Lin, lineage commitment markers, other than those rnentioned above, in cocktail (B220. Mac- 1, Gr- 1 , Ter- 1 19).
Multiple designations indicate heterogeneous expression on cell populations. DX5 is a novel pan-NK ceIl marker which binds an unknown antigen on NK cells from mice
of ail strains tested to date ( 15 1).
p-actin
NKR-Pl A
NKR-P1B
NKR-PIC
Ly -49A
Ly -49C
Perfonn
CD95L
FL fl Total Tocal (Sw) (Sw) -- i c.
mm
Figum 1 1. NKenriched tetaf thymocytes ex- chancterisüc gene prwiucts of functioiial Nii cells. RT-PCR d y s i s for the expression of NK cc11 effector function-associated genes on RNA isotated from (a) total d l 5 Swiss.NIH (Sw) FT and FL cells, and (b) CD24CD25depleted d l 5 Sw and CS7W6 (B6) Fï and adult RAG-2-'- thymocytes (AT).
among total day 15 fetal thymocytes, iow level expression of NK-related genes could be detected
(Figure 1 1 a, FT), including products of the NKR-Pl gene family (NKR-Pl A; NKR-Pl B: and
NKR-PIC, NK1.l) ( 1 16. 120), the Ly49 gene family (Ly-49A; Ly-49C) (1 53. 155). Fas ligand
(CD95L) ( 156. 157). and the cytolytic pore-fonning molecule, perfonn ( 158- 160). In addition, it
has been previously demonstrated that immature fetal thymocytes express the ce11 death-associated
proteasekaspase ( 16 1 ), granzyme B, at an early stage in ontogeny ( 102). To further c haracterize
expression of these NK-related gene products, day 15 thymocyte suspensions from two unrelated
(albeit NK 1.1 -expressing) strains of mice, Swiss.NIH (Sw) and C57B116 (B6). were isoiated and
enriched for mature NK cells by depleting for CD24lCD25 prior to RNA isolation (Figure I 1 b).
Consistent with the degree of NK cell enrichment observed phenotypically in Figures 3 and 1 0.
cxpression of NK-related genes was dramatically enhanced by CD24/CD25 depletion (Figure I 1 a
versus 1 1 b, respectively, Fï). As expected. CD24KD25-depleted adult RAG-2-" thymocytes
(Figure I I b, AT) and splenocytes (data not shown) also express these NK function-associated
genes. Among CD24lCD25-depleted fetal liver cells, only background expression was detectable
for any of the NK-related gene products tested (data not shown); the inability to phenotypically
identify significant numbers of NK cells among fetal liver suspensions limits further attempts to
enrich for such cells or their immediate precursors. without employing in vitro culture techniques.
Nevertheless, ci- 15 fetal thymocytes express numerous gene products typically associated with
NK cell effector function.
RT-PCR analysis of NK-enriched ceIl populations. although not quantitative nor conclusive,
revealed a number of significant findings. In comparison to NKR-Pl A and NKR-P 1 C levels,
NKR-P 1 B expression was quite high among Sw fetal thymocytes, while remaining very low in fetal
86 and adult RAG-~-'- thymocytes (Figure 1 I b). This difference appears to be stnin-specific,
because the same trend was observed for adult spleen cells from each of the strains (data not
shown). These data suggest that strain-specific expression of NKR-Pl gene family members may
be controlled at the transcriptional level. In contrast. expression of Ly-49 gene family members
49
appears to be developmentally regulated in the thymus. Expression levels of both Ly-49A and Ly-
49C, in cornparison to the other genes tested, were higher in adult (RAG-2"') thymocytes than in
fetal (Sw and B6) thymocytes (Figure 1 lb). This does not appear to be due to strain-specific
differences, because Ly-49AK expression were comparable in al1 three strains among adult
splenocytes (data not shown). Thus, it appears that in the fetal thymus, expression of Ly-49 genes
may be developmentally delayed, while NKR-Pl molecules appear quite early in ontogeny. This
may have important functional consequences, as these two families of molecules are postulated to
possess opposing regulatory roles in NK cell effector function, with Ly-49 molecules acting as
inhibitory receptors ( 153, 155. 162), and NKR-PI molecules acting as positive modulators of NK
ce11 activity and lytic function (1 54, 162); although, recent investigations have demonstrated that
there are exceptions to this simplified view of NK cell receptors (see Chapter V).
Freshly-isolated fetal thymic NK cells display MHC-unrestricted cytotoxicity
To determine whether fetal thymic NK cells are functional, we tested the ability of freshly-
sorted NK 1.1 + (CD 1 1 77 fetal thymocy tes to perfonn MHC-unrestricted cytol ysis of "~r-labelled
Y AC- 1 target cells (Figure 1 2). Thymocytes obtained at day 15 of fetal gestation were sorted by
FACS for a CD~' /CD~O+ phenotype, with or without NKl. 1 expression. As shown in Figure 12,
freshl y-soned NK 1. l + day 1 5 fetal thymocytes, wi thout a requirement for exposure to cytokines
such as I M - y , IL-2. IL- 12. or IL- 15. were capable of lysing NK-sensitive YAC- 1 target cells, but
failed to lyse the NK-insensitive EL-4 cell line. As expected, freshly-isolated NKl . I + thymocytes
from adult RAG-~"- rnice also lysed YAC- I targets, while failing to lyse EL-4 cells (Figure 12). In
contrast, fetal thymocytes lacking expression of the NK1. 1 marker failed to lyse either target, as did
total (unsorted) fetal thymocytes (Figure 12). The latter observation is consistent with previous
attempts to detect NK cell function in freshly-isolated fetal thymocytes (1 07, 1 13, 1 14, 1 19); in the
absence of purification, the high frequency of NK1.1' thymocytes could inhibit NK cell cytotoxic
50
d NU 1. I + Adult RAG-Z-I- Thymocy~cs
NU 1. I + Day 15 Fctal Thymocytcs
NK1.I-Day l5FctdThymocytcs
Total Day 15 Fctnl Thymocytes
E:T Ratio
Figure 12. Fetal thymic NK cells mediate MHC-unrestricted cytotoxicity ex vivo. Frcshly-isolated 86 d 15 fetal thymocytes and adult RAG-2-1- thymocytcs were sorted for CD3-ICD90c cells, wiih or without NK 1 . I expression, and subscquently tcsted for cytotoxicity apainst NK-scnsitivc YAC-1 celis or NK-inscnsitive EL-4 cclls. Cytotoxicity was tested in the abscncc of exogenous cytokines.
function. Importantly, we show that freshly-isolated fetal thyrnic NK cells possess cytolytic
function at a developmental stage prior to the appearance of C D ~ + / C D ~ + DP cells in fetal thymic
ontogeny. Thus, functional NK ce11 development precedes ap T ce11 differentiation in mouse fetal
thymic ontogeny.
In vivo adoptive transfer of precursor-phenotype thymocytes
The early developmental maturity of fetal thymic NK cells (Figuos 1 1 & 12). combined
with their close phenotypic resemblance to early progenitor thymocytes (Figure 10 & Table 1).
implies that previous descriptions of purported multipotent, bipotent TMK. or unipotent NK lineage
"precursor" thymocytes, in particular those involving populations not defined according to CD 1 17
or NK 1 . 1 expression, may have inadvertently included pre-existing mature NK ce!ls. To address
this issue directly, we used in vivo adoptive transfers. C D ~ ~ + / C D ~ S - cells, which contain
multipotent precursors for the T. B, and NK lineages, were sorted from CD24ICD25-depleted day
15 fetal thymocytes (Swiss mice. H-2'). and subdivided according to NK 1 . 1 expression (Figure
13a). Tliree weeks after iniravenous injection into sublethally-imdiated (750 cGy) RAG-~- / -
(H-2h) host rnice. tissues were exarnined for evidence of donor-derived (H-zK~+) progeny. As
shown in Figure 13, both the NK1.1+ and NKI .l' subsets of C D ~ ~ + / C D ~ S ' thymocytes were
capable of giving rise to donor-derived splenic NK cells (Figure 1 3b. NK 1 . 1 +/Il-Xq+). However,
only the multipotent NK 1.1 - subset (CD 1 17+. Figure lob) was capable o f generating B cells. as
determined by CD45R expression on NK 1.1 - donor-derived progeny (Figure 1 3b,
C D ~ S R + / H - ~ K ~ + ) . These donor-derived C D ~ ~ R + cells also expressed surface IgM (data not
shown). Cells from nonreconstituted (Control) mice showed no background staining for donor
class I ( H - ~ K ~ + ) expression (Figure 13b). The reconstitution potentiai of the NK1.1- subset is not
limited only to the B and NK ceIl lineages because these cells are also capable of giving rise to T
cells in R O C reconstitutions (Figure 4; see Chapter 1) ( 1 26). However, we could find no evidence
52
Control
H-2Kq (Donor)
Figure 13. Precursor-phenotype fetal thymocytes sorted according to NK1.1 expression show distinct reconstitution potential in vivo. (a) FACS soning of CD24lCD25-depleted d 1 5 Sw (H-29) fetal thymocytes for NK 1.1 -/CD444 (R 1 , CD 1 17+. 27%) and NKl.l+/CD44+ (R2, CD1 17-. 12%) cells. Cells (1x105) werc injccted intravenously into subleihally-imdiated (750 cGy) adult RAG-2-1- mice (H-2b). (b) Spleen cells from reconstituted or nonreconstituted (Control) mice werc analyzed 3 weeks after adoptive transfer by flow cytometric analysis of NKI . l versus H-2Kq and CD45R versus H-2Kq.
of T lineage reconstitution iri the thymus upon in vivo adoptive transfer of either subset. This
observation is consistent with previous studies assessing the precursor potential of fetal thymocytes
upon adoptive transfer into adult host rnice (5,98). It has been suggested that this may be due to a
developmental stage difference; fetal thymocytes exhibit a reduction in thymic reconsitution
potential compared with their analogous "cD~~o" adult counterpart upon intravenous adoptive
transfer and rnay have difficulty homing to the adult thymic microenvironment (98, 100. 107).
Therefore, we employed an in vitro FTOC reconstitution assay for T and NK ce11 potential.
Additionally, for detecting B and NK ce11 potential, we employed a sensitive in vitro coculture assay
using the bone marrow-derived stroma1 ce11 line, OP9 ( 126).
Fetal thymic NK cells are capable of sustained ggrwth in vitro
To address the growth potential of fetal thymic NK cells in a T lineage assay. we assessed
their ability to reconstitute dG-depleted FT'OCs. CD24lCD25-depleted day 15 fetal thymocytes
were sorted for NKl . I7CD 1 1 7+ (mLP) and NK 1.1 +/CD 1 17- (mature NK) cells. and 1 -3x 1 o3 donor cells were used for R O C reconstitution. As previously demonstrated (Figure 4: see Chapter
1) ( 1 26), sorted NK 1. I7CD 1 17+ (FTLP) cells give rise to CD4/CD8 DP and mature CD4 and CD8
SP T cells (Figure 14a). In addition, FïLP cells were capable of generating both mature T cells. as
determined by high-level expression of aPTCR on NKl . 1- cells (Figure 14a. ~PTCR'MK 1.1 -), as
well as a few NK cells (Figure 14a, aPTCR*/NK 1.1+) ( 126). In contrast, NK 1. ]+/CD 1 17- (mature
NK) celis remained double-negative for both CD4 and CD8. and exclusive1 y gave rise to an
outgrowth of NK cells (Figure 14a). These in vitro-cultured NK cells are large granular
lymphocytes, and the low level staining observed for aPTCR is due to increased background
staining, as we failed to detect DJP rearrangements in their genomic DNA by PCR (data not
shown). Ce11 yields from FTOCs indicated that fetal thymic NK cells have the capacity to expand
at least IO-fold in this assay. depending on the length of the culture period.
54
CDS
Figure 14. Fetal thyniic NK cells display sustained growth in vitro. CD24fCD25-depletcd dl.5 fctal thy niocy tes werc soned for NK 1 . I -/CD 1 1 7+ and NK I . I +/CD 1 1 7- cells, then cultured in parallel uiidcr dilfereritial condiiions. (a) dG-FTOCs werc rcconstiiuied with 1-3x103 cells of ench subset, as indicatcd, and cultured for 13 days prior io llow cyioriictric analysis for CD4 vcrsus CD8 and NKI.! versus aPTCR expression. (b) In parallel wiih FTOC reconsiitutioris, 1 - 3 x 1 0 3 cells wcre coculturcd for I I days on confluerit riionolnpcrs of OP9 bonc iiiarrow stronial cells in the presciicc of cyfokincs (IL-3, IL-6, IL-7, and SCF). Cells were [tien siiniulaicd witti LPS alid 11,-7 for an addirioiiol 6 dûys prior to tlow çy toinciric. aiiiilysis Cor NK 1. I vcrsus CI190 aiid NK 1 . I versus CD45R.
To examine the growth potential of fetal thymic NK cells further in a B and NK ceIl assay,
we seeded sorted cells ont0 OP9 bone marrow-derived stroma1 cells. In parallel with FTOC
reconstitutions, 1 -3x 1 o3 sorted NK 1. I -/CD 1 17+ (FTLP) and NK 1.1 +/CD 1 17' (mature NK) cells
were cocultured with confluent OP9 cells. As previously shown (Figure 5; see Chapter 1) (126).
sorted NK 1.1-/CD 1 17' (FïLP) cells gave rise to mature B cells. indicated by expression of surface
IgM on C D ~ ~ R ' cells (Figure 14b, CD~SR+II~M+). and a few NK cells (Figure 14b.
NK 1.1 +ICD~O+'-). However, NKI .lC/cD1 17- (mature NK) cells remained negative for both
CD45R and IgM, and again gave rise to an outgrowth of mature NK cells. the majonty of which
expressed CD90 (Figure 14b. NKl. I+/cD~o+''); these NK cells also stained positive for the DX5
mAb (data not shown). Although we have not quantitated the extent of growth potential exhibited
by fetal thymic NK cells in this aîsay (at least 50-fold), they continue to divide and can be
maintained for at least 1-2 months in vitro. However, they must be continually passed onto fresh
OP9 cells because the OP9 cells are lysed due to the cytolytic activity of the NK cells. In addition.
the growth of fetal thymic NK cells is enhanced with exogenous IL-2. Therefore, the reported
existence of NK ceIl precursors in the fetal thymus that can grow out in vitro in the presence of IL-
2 was most likely due to an expansion of this pre-existing subset of mature NK cells ( 1 19. 12 1.
163).
The data shown in Figures 13 & 14, together with our previous evidence (see Chapter 1)
( 126). suggests that the NK 1. I ' (CD 1 17+) subset of C D ~ ~ + / C D ~ S thymocytes represents
multipotent lymphoid-restricted precursors, while the NK 1. l + populatioi conrains mature NK cells
(CD1 17') which are capable of sustained growth after adoptive transfer in vivo or in vitro. Thus,
there is a population of pre-existing NK 1.1 + cells among precursor-phenotype fetal thymocytes that
contains mature and functional NK cells capable of confounding lineage potential assays if not
properly distinguished from multipotent CD I 1 ~ + / c D ~ ~ + / c D ~ S / N K ~ . 1- precursors. In light of
these findings, the purported discovery of bipotent T/NK precursors requires reassessment.
Discussion
These data provide the first evidence of the developrnent of mature and functional NK cells
in mouse fetal ontogeny. The fact that NK cell maturation initially occurs within the early fetal
thymus. together with the recent description of NKl.l+ afi T cells (1 33) and the NKI . I +/CD 1 17'
(FTNK) bipotent T/NK progenitor stage of thymocyte development (see Chapter 1) ( 1-6). further
reinforces the developmental and lineage relationships between T and NK cells. Moreover. we
show that NK cells are phenotypically present in the fetal thymus by day 13 of gestation. prior to
the onset of V(D)J remangement of the TCRP locus, and that NK ceIl function is detectable by day
1 5, prior to the appearance of C D ~ + / C D ~ + DP cells in fetal thymic ontogeny . This indicates that
NK ceIl development precedes that of up T lymphocytes. Although an analogous subset of NK
cells ( c D ~ ~ + / c D ~ ' ) has been observed in the human fetal thymus (108, 164). the earliest stages of
NK cell development were not outlined. and it remains unknown during human fetal thymic
ontogeny whether NK cells are present and/or functional prior to ap T ce11 differentiation. Thus,
these data are the first to show that the maturation of functional NK cells, Iike that of the canonical
vy3+ y6 T cells (29), precedes ap T ceIl development.
The identification of fetal thymic NK cells. together with the inability to detect significant
NK 1 . 1 expression in the fetal liver (Figures 3, 10, & 1 1 ), suggests that fetal NK ce11 differentiation
may be thymus-dependent until the establishment of periphenl sites for NK lymphopoiesis. This
could explain why NK cells do not reach significant levels in the circulation until the neonatal stage
( 1 13. 1 14, 1 18, l65), when hematopoietic function shifts from the fetal liver to the neonatal/adult
bone marrow, a tissue that is known to be capable of supporting NK lineage maturation ( 101, 166).
It may be that the bone marrow is primarily responsible for peripheral NK cell production, whereas
the fetal liver may be incapable of supporting NK lineage differentiation, possibly due to the
absence of particular cytokines or stroma1 microenvironments ( 1 15, 1 67). Although the fetal
thymus is capable of supporting complete NK ce11 maturation, thymus-derived NK cells may be
57
locally involved in regulation of thymopoiesis (168) and may not reach the periphery. Consistent
with this, we have failed to detect significant niimbers of NK cells in the fetal blood and spleen until
day 16 of gestation (see Chapter III) (152). Nonetheless. mature NK cells differentiate early dunng
fetal thymic ontogeny (Figure 3), exhibit gene expression patterns a consistent with NK cell
effector function (Figure 1 1 ). and display MHC-unrestncted cytotoxicity ex vivo (Figure 12).
without a requirement for exposure to exogenous cytokines.
Importantly, the close phenotypic resemblance of fetal thyrnic NK cells to early precursor
thymocytes implies that previous descriptions of purported NK precursor and bipotent T/NK
precursor potentials may have been due to contamination with these preexisting mature NK cells.
Indeed, Our in vivo transfer experiments provide direct evidence that the NK 1. I ' subset of
C D ~ ~ + / C D ~ S fetal thymocytes (which also expresses CD 16/32) can reconstitute NK cells upon
adoptive transfer. Therefore, previous reports employing CD44 CD1 6/32. and/or CD 132 to
identify progenitor thymocytes, in panicular where characterization of NK 1.1 andor CD 1 17 are
lacking. may have inadvertently included mature NK cells within a putative precursor population.
Fetal thymic NK cells are capable of sustained outgrowth, both in vitro and in vivo, potentially
obscuring bona fide multipotent, bipotent. and unipotent NK lineage precursor activity . Hence,
investigations that failed to exclude NK cells prior to assessing NK lineage potential(34. 106. 107.
1 1 9, 1 2 1. 1 63), including the purported discovery of bipoten t T/NK precursors. must now be
re-evaluated in light of these observations. Indeed. the early developmental expression of NK 1.1
and other members of the NKR-PI gene family ( 1 13, 1 14, 126, 127, 169) suggest that such natural
killer ce11 molecules should be included as lineage (Lin) differentiation markers for future
hematopoietic precursor evaluations, both intrathymic and extrathymic. Our identification of mature
and functional NK cells in fetal ontogeny sheds new light on Our understanding of NK lineage
development and function and could aid in the derivation of longer-lived mouse NK cell lines.
REQUIREMENT FOR THE THYMUS
IN ap T LYMPHOCYTE LINEAGE COMMITMENT
James R. Carlyle and Juan Carlos Zhiiiga-Pflücker
Department of Immunology , University of Toronto, Toronto. ON. Canada
(Al1 work was performed by J.R. Carlyle)
Published in Immunity
20 August 1 998. Volume 9, pp. 1 87- 1 97
CHAPTER III: Requirement for the Fetal Thymus in T Lineage Commitment
Introduction
Fetal liver-derived hematopoietic precursors migrate through the circulation to first colonize
the early thymic rudiment by day 1 1 of mouse gestation (10). However, the phenotype and
precursor potential of thymus-colonizing precursors remain controversial. The most immature
precurson common to both the fetal and adult thymus, the TLPs, appear to be a homogenous
population of multipotent lymphoid-committed precursors capable of giving rise to the B. T. NK.
and lymphoid dendritic (LD) cell lineages ( 1-3, 105. 106, 126). H~we-ier, in the day ! 2 fetal
thymus, prior to the establishment of an intact thymic microenvironment capable of supporting
complete ap T iymphopoiesis ( 18), myeloid lineage potential is recoverable, and these precursors
seem to be phenotypically and functionally similar to hematopoietic stem cells (96,97). Thus.
aithough thymus-colonizing precursors may be heterogeneous, entry into the thymic
microenvironment, even by experimental means. ultimately leads to the induction of commitment to
the T lineage (37,58).
Nonetheless. the role of the thymic microenvironment as a unique site for the induction of
commitment to the conventional ap T lineage remains in question. In particular, the characterization
of CD~O+/CD 1 17'0 "prothymocytes" in mouse fetal blood suggests that ap T lineüge-committed
precursors may colonize the thymus during fetal ontogeny ( 100). ~orebver, CD~O+/CD 1 17" fetal
blood cells were reported to occur at a normal frequency in athymic nude (nu/nu) mice, which fail to
develop intact thymic and peripheral epithelium due to a defect in expression of the winged-helix
nude (whn) gene (143, 144). This was taken to suggest that commitment to the ap T ceIl lineage
can precede thymus colonization (34, 100). However, NK lineage potential was not addressed in
these reports, and the close lineage relationship between T and NK cells might be taken to indicate
that these fetal blood cells may not be exclusively T lineagetommitted pnor to entry into the
60
thymus.
To address the requirements for commitment to the T and NK lymphocyte lineages. we
examined day 13-16 fetal blood and spleen cells from normal and athymic nude mice. Here. we
report that the majority (80-90%) of fetal blood "prothymocytes" (previousl y defined as
CD~O'ICD 1 1 ~'O/CD~' ( 100)) possess a phenotype identical to R N K progenitor cells (see Chapter
1) ( 1 26). Important1 y, these circulating precursors display NK 1.1 (NKR-P 1 /CD 1 6 1 ). lack markers
associated with T lineage commitment. maintain a germline TCRP locus, and are capable of
efficiently generating both T and NK cells. Furthemore. NKl . 1 +/CD 1 17" fetal blood precursors
are present from the onset of thymopoiesis during fetal ontogeny and in the circulation of athymic
nude rnice. These results provide further evidence of the existence of a developmental stage
common to both T and NK cells during fetal hematopoiesis, which defines a lymphocyte lineage
commitment pathway independent of thymic epithelium. Moreover, these findings support a unique
role for the thymus in the induction of full commitment to the ap T lineage during fetal
developmen t.
Results
Identification of ~ ~ 1 . 1 ' cells in the mouse fetal circulation
To out1 ine the developmental appearance of TMK 1 ineage precursors in the circulation
during fetal ontogeny, we analyzed day 13- 16 fetal blood and spleen cells for NKl . I expression. A
significant percentage of NKl. 1' cells was observed in the fetal blood, spleen, and thymus by day
13 of gestation, although NKI. I expression could not be detected to a significant extent on fetal
liver cells (Figure 15). The paucity of NKl. 1' cells in the fetal liver suggests that efficient
generation of NK 1 .1 + cells in fetal ontogeny occurs during or after emigration from the fetal l i ver
into the circulation. Notably, NKI. l + fetal blood and spleen cells express CD 1 17. albeit at low
levels. similar to the NK 1. I '/CD 1 17' cell population in the fetal thymus (Figure 15: top panels).
This suggests that these circulating NKI. l C cells are either early NK lineage precursors or an
analogous population to F ï N K progenitors, which are capable of giving rise to both T and NK
cells ( 126). However. further analysis for CD90 (Figure 15, middle and bottom panels) and CD3
expression (data not shown) revealed that the majority of NKl. l + cells in the fetal blood and spleen
display a phenotype similar to previously-described fetal blood "prothymocytes"
(CD~O+/CD I 1 7 I 0 / c ~ 3 - ) ( 100). As differential expression of the NK 1.1 molecule would be
inconsistent with previous work defining CD1 1 ~'O/CD~O+/CD~- fetal blood cells as a hornogenous
population of ab T lineage-committed prothymocytes (100). we analyzed these cells funher for
various lineage markers.
Circulating fetal NKI.I+ cells resemble fetal blood ''prothymocytes"
CD24 is a marker frequently used to discriminate a number of lineage-committed
hematopoietic differentiation stages in the fetal thymus and liver from early multipotent precursors,
62
d 13 Fetal Splceri d 13 Fetal Thyiniis
Figure 15. Identification of NKi.1+ cells in the rnouse fetal circulation. Total d l 3 Sw fetal tiver, blood, spleen cells, and thyrnocytes were analyzed by three-parmeter flow cytoinetry for cell surface expression of NK 1.1, CD90, and CD 1 17.
which are ~ ~ 2 4 " ( 1 1, 127). As thymus-colonizing precursors uprcgulate CD24 expression to
high ievels after cornmimient to the T lineage (pro-T stage) (5,3 1 ), we analyzed total day 15 fetal
blood and spleen cells for expression of CD24. Although the majority of fetal blood and spleen
cells express high levels of CD24 (Figure 16a), among the CD 1 1 7 ' 0 / ~ ~ 9 0 + / ~ ~ 3 * population. only
low level expression of CD24 could be detected (data not shown). Therefore. to enrich the
CD1 1 7 1 0 / ~ ~ 9 0 + / ~ ~ 3 - population for funher analysis, we depleted day 15 fetal blood and spleen
cell suspensions of ~ ~ 2 4 ~ ' cells by antibody/complement-mediated lysiç (a-CD24, J 1 l d.2). as
previously described ( 11, 1 27). Figure 16a shows CD24 (a-CD24, M 1 /69) versus CD 1 17
expression on day 15 fetal blood, spleen cells, and thymocytes before (Total) and after CD24
depletion ( ~ ~ 2 4 " ) . Fetal thymocytes were included as a reference population for the expression
of various markers on committed pro-T cells and NK cells (127).
Figure 16b shows flow cytometric analysis of expression of lymphocyte differentiation
markers on ~ ~ 2 4 " fetal blood, spleen cells, and thyrnocytes. Expression of each marker was
plotted versus CD 1 1 7 expression, both with and without gating on ~ ~ 9 0 ' cells (Figure 16b, CD90
versus CD1 17, top panels; dark shading, C D ~ O + gated: light shading. ungated). As previously
reported, fetal blood contains a substantial population of C D ~ O + cells with low level expression of
CD 1 17 (Figure 16b) (100). Examination of fetal spleen reveals that these cells closely resemble
fetal blood among the ~ ~ 2 4 " population, and most likely represent part of a common but
non-circulating pool, with the exception that the fetal spleen contains additional populations of cells
probably undergoing in situ hematopoietic differentiation (Figure 16a, C D ~ ~ ~ ' / C D 1 17" cells). In
contrast, al though fetal thy mocytes contain a signi fican t population of CD~O+/CD 1 17" cells, an
additional ~ ~ 9 0 ~ 1 ~ ~ 1 1 7 ' population is evident. which contains functional NK cells ( 1 27).
Subsequent panels include gating on C D ~ O + cells as indicated.
As demonstrated in Figures 16a and 16b, the CD~O+ICD 1 17'' population is significantly
enriched by CD24 depletion, as are the multipotent precursors and mature NK cells in the fetal
thymus (see below). Analysis of markers associated with T lineage differentiation in the thymus,
64
dl5 Feiol Spleen d 15 Feu1 Thymus Figure 16. NK1.1+ fetal blood and spleen cells resemble fetal blood "prothymocytes". (a) Day 15 fetal blood (FE!), spleen cells (FS). and thymocytes (Fï) wcre analyzed for expression of CD24 versus CD1 17. Panels show cells before (Total) and afier (CD2410) anti body/complement-mediâted de pletion of cells expressing high levcls of CD24. (b) CD2410 FB, FS, and FT cells were analyzed by three-parameter flow cytametry for expression of lymphocyte differentiation markers. Panels show cells ungated (light shading) and gated on CD90+ cells (dark shading); pcrcentagcs in each quadrant for ungated (plain typc) and CD90+-gated (bold type) cclls arc shown.
CD3e. CD44 and CD25, shows that CD~O+/CD 1 17" fetal blood and spleen populations display a
CD3Ë /cD~~+/cD~s ' phenotype. This phenotype corresponds in the fetal thymus to the
CD4/CD8 double-negative (DN 1) population (1,44,60), the subset that contains the earliest
multipotent thymic precursors (TLPs) as well as mature NK cells ( 127). As thymus-denved pro-T
cells (DN II) express CD25 and upregulate CD24 expression to high levels upon commitment to
the T lineage (3-5,44), CD~O+/CD 1 17" fetal blood and spleen cells differ from their thy mic
counterpart in that they possess lower levels of CD24 and lack expression of CD25 (Figures 16a &
16b). Thus, the circulating ~ ~ 9 0 ' cells exhibit a phenotype more consistent with uncommitted
precursors or committed NK lineage cells, rather than pro-T cells.
As CD90 is also expressed on the majority of thymic and peripheral NK cells ( I 1 1 ). we
investigated the possibility that ~ ~ 9 0 ' fetal blood and spleen cells might include committed T/NK
progenitors and/or NK lineage cells. To this end, we analyzed expression of NK 1 . 1 ( 126. 127).
CD 16/32 (107. 170). CD 122 ( 12 1 , 149), and the recentiy-described pan-NK cell marker, DX5
( 15 1 ) (Figure 16c). Analysis of NK 1.1 and CD 16/32 reveals that the majority (80-90%) of day 15
CD~O+/CD 1 1 7 " / ~ ~ 3 - fetal blood and spleen cells express both NK 1.1 and CD 16/32 (Figure
16c). As lineage-committed TMK progenitors and mature NK cells in the fetal thymus also
express these markers ( 126, 127), this suggests that circulating ~ ~ 9 0 ' cells have a phenotype
consistent with the loss of precursor multipotency. that is, loss of B lymphoid and myeloid potential
( 100). Furthemore, almost half of CD~O+ fetal blood and spleen cells express CD 122 (Figure
1 6c). a molecule necessary for NK lineage differentiation ( 1 7 1 ) and reported to be expressed on
bipotent TMK precursors in the fetal thymus ( 12 1 , 149). Alihough the regulated expression of
DX5 during lymphocyte lineage commitment remains unknown, DX5 is expressed by al1 mature
NK cells ( 15 1 ). Analysis of DX5 expression revealed that the majority (290%) of C D ~ O + fetal
blood and spleen cells lack expression of DX5 (Figure 16c). In contrast, a significant population of
fetal thyrnocytes. which contains mature and functional NK cells ( l27), coexpresses NKI . 1 ,
CD 1 6/32, CD 122, and DX5, and lacks CD 1 17 expression (Figure 16c). Thus, NK 1 . 1 ' fetal blood
66
and spleen cells are not mature NK cells, and at least the majonty do not obviously appear to be NK
lineage-committed. Rather, they exhibit a phenotype rerniniscent of NK 1.1 +/CD I 17+ FïNK
progenitors in the fetal thymus (Figure 16c; FI', NK 1.1 versus CD 1 17, upper right quadrant) ( 126).
These findings also support Our previous work demonstrating that NK cell differentiation first
occurs in the thymus dunng fetal ontogeny (see Chapter II) (127), and that mature CD 1 1 ~'/Dxs+
NK cells appear to be absent from the early fetal circulation.
~ ~ 1 . 1 + fetal blodspleen cells express genes associated with lymphoid lineage
commi tment
To characterize expression of lymphocyte differentiation genes in circulating fetal NK 1 .1 +
cells, we employed RT-PCR analysis (Figure 17). Day 15 fetal liver cells and thymocytes were
included as controls, as these populations contain defined subsets of uncommitted and lymphocyte
limage-cornrnitted progenitors. As shown in Figure 17, soned NK 1. I+/CD~O+/CD 1 17" fetal
blood and spleen cells express transcripts for the Ikaros family of genes (172), consistent with their
lymphoid lineage-committed phenotype. These cells also express the T lineage-specific
transcription factors GATA-3 and TCF- 1 ( 173, 174). We also detected gemline transcripts for
TCR Cp, which is known to be expressed on lymphoid lineage precursors ( 1 75). As previously
reported for soned CD~O+ fetal blood cells (176), low level expression of the pre-Ta gene was also
detected among sorted NK I - 1 ' fetal blood and spleen cells. However. these cells lacked expression
of C D ~ E , which is expressed in the earliest T lineage-committed subset in the fetal thymus at day 13
of gestation (97, 148, 177). Expression of RAG-2, a lymphoid-specific gene expressed at the
CD I 1 7 + / ~ ~ 2 5 + pro-T stage in the fetal thymus pnor to the initiation of TCRP rearrangement, was
also lacking in these cells (49). However, low-level expression of RAG- 1 was detected, suggesting
that RAG- 1 and RAG-2 are differentially regulated in NKI . l+ fetal blood and spleen cells.
Nevertheless, RAG- 1 expression in the absence of RAG-2 expression implies that these cells are
67
Ikaros
GATA-3
TCF- 1
TCR Cg
Pre-Ta
CD3 c
RAG- 1
RAG-2
Lck
IL-7Ra
IL- 15Ra
NKR-PI A
NKR-Pl B
NKR-Pl C
Perfori n
CD95L
Figum 17. NK1.1+ fetai Mod and spleen ceih ex- p e s cuisociated with lympttucyte linmg&! cornmitment. Total RNA from NKI. l+lCD9û+/CD I lgo ( ~ ~ 2 4 ' ~ ) Sw dl5 fetal M d and spleen cells (NK1. l 4 FBIS) was analy-ted for expression of genes associateci with lymphocyte differentiation by RT-PCR. Total (unsorted) d 15 Fï and FL cells were included as controls.
not yet capable of undergoing site-specific DNA recombination (see below) (62,63).
Lck, a protein tyrosine kinase expressed early in lymphocyte differentiation (97) was
detected on sorted NKl. 1' fetal biood and spleen cells. These cells also express high levels of
IL-7Ra, as previously reported for ~ ~ 9 0 ~ fetal blood prothymocytes (34, 100). Expression of
IL- 1 SRa, reported to be present on CD1 17+/Lin'/~ca-2+ lymphoid lineage-comrnitted progenitors
in adult bone marrow ( 178). was also detected in sorted NKI . 1 + fetal blood and spleen cells. albeit
at lower levels than those observed for IL-7Ra. Importantly, we detected expression of genes
associated with NK lineage differentiation and function (l27), including the NKR-PI family
members, perforin, and CD95L, indicating that NK lineage-specific markers other than NK1.1 are
also present in these cells. This suggests that circulating C D ~ O + cells may not be exclusively
committed to the T cell iineage, as previously proposed (100).
~ ~ 1 . 1 ' fetal blood/spleen cells rnaintain their TCRP locus in the germline configuration
C D ~ O + fetal blood ce1 1s have been reported to include celis undergoing TCRP
rearrûngement, an indication that at least a small subset of these cells is fully T lineage-committed.
However, due to the paucity of CDZ+ cells in the fetal blood and spleen, and because previous
reports could not distinguish whether the DJB rearrangements detected were due to the inclusion of
a small number of C D ~ O + recent thymic emigrant cells, we further analyzed NK 1 . 1 + fetal blood and
spleen cells for DJ rearrangements at the TCRP locus.
NK 1. I+/CD~O+/CD 1 17'0 fetal blood and spleen cells are present early in fetal ontogeny
(Figure 15), prior to the onset of DJP rearrangement in the thymus ai approximately day 13 of
gestation (58. 179). As these circulating cells appear to be at a developmental stage pior to full T
lineage cornmitment (Figures 16 and 17), we hypothesized that the detection of DJP rearrangements
in the circulation would only occur after the appearance of such rearrangements among fetal
thymocytes during ontogeny. Moreover, although CD~O+/CD 1 1 ~'O/CD~- progenitors have been
69
reported to be present at similar levels in athymic nude (nuhu) mice (100). the DJp reamngement
status of cells isolated from these mice was not directly investigated. To address these issues, we
used PCR to examine the extent of DJP remangement in DNA isolated from sorted C D ~ O + and
NK 1. I + (CD 1 1 ~ ' O / C D ~ - / D X S - ) fetal bloodfspleen cells at various days of gestation. Figure 1 8
shows the flow cytometric profiles of NKI. I versus CD90 expression on ~ ~ 2 4 " day 15 fetal
blood, spleen cells. and thymocytes (Fig. 18a). and on total day 15 athymic nude fetal blood cells
(Fig. 18b). In contrast to previously pub!ished results, we could find no evidence of DJP
rearrangements in DNA samples isolated from either the ~ ~ 9 0 ' or the NK 1 . 1 + subset. even by day
16 of fetal ontogeny (Figure 18c). Furthemore. DIB rearrangements were absent among C D ~ O +
cells isolated from the circulation of fetal athymic nude (nuhu) mice (Figures 18b & 1812).
Importantly, this was not due to a Iack of sensitivity in our assay, as mixing experiments involving
titration of d 15 fetal thymus DNA into RAG-2" adult thymus DNA dernonçtrated that
rearrangements were detectable to lower than 1 % ( 1/243) of the level found among total d 15 fetal
thymocytes (Figure 18d). One possible explanation for the discrepancy between our results and
those previously published (34. 100) might be that the C D ~ O + population of cells contains srnall
numbers of thymic emigrant cells that may have been removed by the CD24 depletion ernployed in
our protocols. In any case, the N K l . l + precursor subset in the fetal blood and spleen. which
accounts for 80-90% of CD~O+/CD I 1 7 ' * / ~ ~ 3 - cells (Figure 1 8a). has not yet undergone
rearrangement of the TCRp locus.
Circulating fetal NKI.I+ cells are capable of giving rise to T and NK cells in FTOC
CD~O+/CDI 1 ~ ' o / c D ~ - fetal blood cells were previously shown to be capable of generating
T lymphocytes in vivo. both intrathymically and intravenously (34, 100). However, the T lineage
precursor potential of the NK l . l + subset, which accounts for 80-90% of these cells. remains
undetennined. To assess T lineage precursor potential, we sorted CD24-depleted day 15 fetal
70
1 { ,../..; - . . .;i:.cf:5 .. $&p ..!,.. : . ... .- .. . . . . ' :.-.* .. .. . .:;*,-.-.- " - . . .. . . . . -
. L.! -- . , . . . . .
nuhu d 15 FI3 (Total)
mainWn thdr TCRg lod 6 the geimiiae coaiïgurrtlon. (a) CD24-depletal Sw d l 5 FB, FS, and FT celis and (b) toial d l 5
k FB cells f m athymic nude (ndnu) mice were
t Pa a d d y z d by flw cytometry f a expression of
NK 1.1 versus CDW. Quadrants indicaîe s c ? o i & & s F ~ o ~ . + ~ ~ . + ~ divisions used for sorting NKl .I+ (upper
quadmts) and C m (right quadrants) cells for DNA analysis. (c) Genomic DNA was prepared from cell populations as indicatcd and anrilyisd for DJg rcarrangcrnents by PCR. Amplified DNA was Southern Motted and visuali7d using an -800 bp intemal gcnomic probe spanning the JP2 regim. Panels show PCR analysis ni toiai dl415 kcal thymocytcs
1 . . (FT) and adult RAG-2-1- lhymocytes (AT), as cmud samples, and day 1516 combiriod FE and FS cclls (FBJS) frorn ntumal and athyrnic nu& (nulnu, day 16) micc, as indicaled. (d) T d dl5 FT DNA was titraicd into RAG-2-1- AT DNA prior 10 PCR amplification (ratios rire expresscd as amount of di5 Fï DNA : inhl DNA). PCR amplificd DNA was analylxxl in parailel with (c).
blood for NKl. I+/CDI 1 7 1 0 / ~ ~ 3 - / ~ ~ 5 ' cells (Figure 19a: R2, 38%), and assessed their ability to
reconstitute dû-depleted lTOCs derived from RAG-~-'- rnice. Sorted
NK 1. I -/CD I 1 7 h i / ~ ~ 3 7 D ~ 5 - fetal blood (Figure 19a: R I l 39%) and fetal liver cells (not shown)
were included as controls. as high level expression of CD1 17 has been shown to correlate with
multipotent hematopoietic precursor potential in the fetal blood, thymus, and liver (17. 100. 106.
122). As shown in Figure 19b. sorted NK1.1+ fetal blood cells, as well as control NK 1.1- fetal
blood and liver cells, gave rise to both CD4lCD8 immature DP and mature SP T lymphocytes. A
subset of these cells represented mature T cells as demonstrated by high level expression of ap
TCR (Figure 19c). Interestingly, each of these sorted precursor populations gave rise to a small
iiumber of NKI. 1 %'CR ap- cells, which expressed DX5 (data not shown), suggesting that they
also possessed NK lineage precursor potential . To address this possi bility direct1 y , and
simultaneously examine the B lymphoid precursor poteniial of these cells, we employed a sensitive
in vitro coculture assay.
Circulating fetal N K I . ~ + cells give rise to NK cells but not B cells upon OP9 coculture
As the phenotype and T lineage potential of NK1. I + fetal blood and spleen cells resembled
that of fetal thymic NKl . I + (FTNK) progenitors. we assessed their ability to generate NK and B
lineage cells upon coculture with the bone marrow-derived stroma1 cell line, OP9 (126. 134). For
these üssays. sorted NK 1.1 +/CD I 1 7 ' 0 / ~ ~ 3 7 D ~ 5 - and NK 1.17CD I 1 7 h ' / ~ ~ 3 - / ~ ~ 5 - fetal blood
cells were used in parallel with FTOC reconstitutions (Figure 19). Figure 20a shows that each of
these populations of cells was capable of giving rise to NKI . 1 % ~ 1 9 - NK lineage cells in vitro.
As demonstrated previously (126, 127). the majority of these cells expressed CD90 (Figure 20b).
Additionally. a subset of these cells represented mature NK lineage cells, as indicated by their
DXS+/CD~-/CD 1 17- phenotype (Figure 20b; data not shown). Moreover, while NK 1.1- precursors
were capable of giving nse to B lymphocytes, as indicated by CD 19 expression on NK 1.1 - cells,
CD1 17
Control
Figure 19. NK1.1+ fetal hlood cells generate T and NK cells in FTOC. (a) CD24-deplcicd dl5 FB cells wcre sorled according to expression of NKI .I and CD1 17 (and for CD3-IDX5- cells) by FACS. Regions 1 and 2 (RI, R2) indicrite the gütes used for isolaiing NKI .l-ICD117hi ( R l , 39%) and NK 1 .]+/CD1 1710 (R2, 38%) cclls. (h,c) Day 15 RAG-2-1- dG-FTOCs were reconstituied with ihe indicated cells and analyzcd alter 12d for exprcssion of (b) CD4 versus CD8 and (c) NK 1.1 versus ap TCR by flow cyiometry. Panels show RAG-2-1- dG-FTOCs wirhout the addition of reconstituting cells (Control), or with the addition of 1x103 CD1 17hi FL cells or sortcd cells, as indicated. Boili FB subsets titraicd to similar ce11 numbers (>30/lohe) for successful reoonstitiitions. Results for FS cells (not shown) are siniilrir to those of FI3 cells.
ap TCR
Figure 20. NKl.I+ fetal hlood cells give risc to NK but not B-lineage cells upon OP9 coculture. In paraIlcl with FïOC reconstiiution assays (Figure 19), sorted FL and FB crlls ( l x 103) cells were cocultured on conflucni OP9 rnonolnyers in itie presence of IL-3, IL-6, IL-7, and SCF (50 ngfnil of cach cyiokinc) for 7d. Cocultured cells wcre thcn recovered and plaied on fresh OP9 riionolayers in the prcsencc of IL-7 and IL-? for an addiiional 5d prior to llow cytornetric analysis for expression of (a) NKI.1 versus CD19 and (b) DX5 versus CD90. Resulis for sorted fctd splccri cclls (no! sliown) arc similrir io those of fetal hlood.
the NK 1. I + precursors from the fetal blood failed to generate B lymphocytes (Figure 20a). This
suggesü that NK1. I + fetal blood and spleen precursors have an identical precursor potential to the
phenotypically analogous fetal thymic FïNK population. However, CD~O+/CD 1 1 7I0/c~3' fetal
blood cells, of which the NK1.1' subset constitutes the vast majority, were previously suggested to
be T lineage-comrnitted "prothymocytes". Although NK lineage precursor potential was not
addressed in these reports, coculture experiments with the bone marrow stroma1 cell line. PA6. were
performed which gave rise to slow-growing CD45R7IgM- cells in vitro, the majority of which
expressed CD90 ( 1 OO), a similar phenotype to the NK lineage cells generated in our assays (Figure
20). We postdate that these slow-growing cells were indeed NK lineage cells. and offer the
alternate hypothesis that CD~O+/CDI 1 7 " / ~ ~ 3 ' fetal blood (and spleen) cells represent cornrnitted
progenitors for T and NK cells, rather than T lineage-committed "prothymocytes".
Discussion
The mouse fetal thymus is thought to be colonized by at least two populations of circulating
hematopoietic progenitors, multipotent progenitors and prothymocytes, the latter of which contains
T lineage-committed precursors (34). This notion served to suggest that the thymus. or at least
thymic epithelium. is not required for full commitment of hematopoietic precursors to the aP T
lymphocyte lineage. Here. we provide evidence supporting a requirement for the thymic
microenvironment in the induction of irrevocable af3 T lineage commitment. These data show that
the previousl y -described fetal blood prothy mocytes. w hich can be subdivided on the basis of
NK 1 . 1 expression. lack markers associated with T lineage commitment, and are capable of giving
rise to both T and NK lymphocytes in vitro. Thus. the vast majority of these cells display a cellular
and molecular phenotype similar to that of bipotent T/NK progenitors present in the fetal thymus
( 1 26).
The striking implication of these findings is that full commitment to the ap T lineage during
fetal development requires n thymic inductive event. This report also bridges a gap between human
and mouse T and NK ceIl differentiation, as the NK 1.1+ fetal murine precursors appear to be
analogous to bipotent T/NK progenitors descnbed in the fetal human circulation ( 104, 164). Taken
together. these results suggest aB T lineage comrnitment of hematopoietic precursors in the fetal
circulation of mice and humans does not to precede thymus colonization. I
One of the hallmarks of irreversible T lineage commitment is the initiation of
rearrangements at the TCR P gene loci (44.48,49). Previously, the detection of TCR DJP
rearrangements was used to establish whether T lineage committed cells are present in the fetal
blood prior to thymic colonization. These reports relied on the detection low leveis of DJP
rearrangements by PCR. which were estimated to represent -5% of soned CD~O+ fetal blood
prothymocytes (34, 100). Furthemore, these analyses were carried out with fetal blood at day 15
of gestation, a stage in ontogeny after the onset of DJP rearrangements in the thymus. Thus, it
76
remains possible that the low level of DJP rearrangements detected in the fetal circulation may be
the product of a small number of contarninating recent thymic emigrant cells that had committed to
the T lineage under thymic influence prior to entering the circulation. This possibility is supponed
by the presence of a very small number of C D ~ ~ + / C D ~ O + cells, pro-T-like. evident by day 15 in
~ ~ 2 4 ' ' fetal blood and spleen cells (Figure 16b). Furthemore, whereas we were unable to detect
DJp rearrangements anywhere in the fetal circulation even by day 16, such rearrangements are
clearly detectable among total fetal thymocytes at day 14 of gestation. It remains possible that the
absence of detectable rearrangements in our samples may be due to the CD24 depletion employed
in our protocol, as DJp-rearranged day 15 pre-T, DN III, fetal thymocytes are ~ ~ 2 4 ~ ' (44). To
address whether DJP rearrangements among fetal blood cells might have been derived from recent
thymic emigrants, and because a6 T lineage-committed (DJp-rearranged) prothymocytes have not
been directly demonstrated in athymic nude (nuhu) mice, we examined C D ~ O + fetal blood cells
from these mice (Figure 18a). Consistent with their athyrnic phenotype, we could find no evidence
of DJP rearrangements in ~ ~ 9 0 ' fetal blood cells from nude mice (Figure 18b). These data
suggest that the thymus, or at least intact epithelium (143, 144), is required for full cornmitment to
the ap T lineage, and initiation of TCRP gene rearrangement (58). Consequently. the detection of T
lineage committed-progenitors in the fetal circulation may result from a low level of thymocyte
emigration after the establishment of thymopoiesis ( 1 80). Such emigrating cells may be
responsible for the establishment of thymic-derived gut-associated T cells, which can be blocked by
nronatal irradiation and thymectomy (1 8 1 - 183).
Alternatively, as NK 1. I +/CD~O+/CD 1 17" fetal blood cells bear a similar phenotype to the
recently-described T cell progenitors present in intestinal cryptopatches ( 1 84, 185), these cells may
be responsible for the colonization of the intestinal cryptopatches, which give rise to intestinal
intraepithelial T lymphocytes. Although the chanctenzation of the intestinal cryptopatch precursors
revealed that these cells may represent T lineage-committed progenitors (1 85), their NK cell
precursor potential was not assessed. We predict, based on their phenotypic similarity, that these
77
cells possess precursor potential for both the T and NK cell lineages. and represent an analogous
population to TfNK progenitors present in the fetal blood, spleen, and thymus.
These data provide evidence for the existence of common cornrnitted progenitors for T and
NK cells in mouse fetal circulation. The finding that such lineage cornmitment occurs in the
absence of a thymus and in nude mice suggests that intact epithelium is not required for
commitment to this stage during T and NK lineage differentiation. In contrat, the thymus or at
least thymic epithelium appear to be necessary for full commitment to the ap T lineage. as
demonstrated by the absence of TCRP rearrangements and CD3c, RAG-2, and CD25 expression in
the circulating progenitors of fetal normal and athymic nude mice. Furthemore, full NK lineage
commiiment and differentiation is not apparent among these cells, as indicated by their
CD 1 1 7 + / ~ ~ 5 - phenotype and expression of several T lineage-associated genes (Figure 17).
Interestingly. NK ce11 differentiation to a functional DW+ stage appears to be thymus-induced
during the earliest stages of fetal ontogeny, prior to establishment of the bone marrow as a site for
hematopoiesis and NK ce11 development. These data support Our previous work demonstrating that
functional NK cells differentiate first in the fetal thymus dunng ontogeny ( 1 S7), and that such cells
are absent from the carly fetal circulation (Figure 16c). Importantly. these results also support a
role for the thymus as a unique tissue in fetal development, as it is not only capable of inducing
commitment to the bipotent and unipotent T and NK lineages, but it is also capable of
autonomously supporting the full differentiation and functional maturation of both T and NK
lymphocytes from uncommitted precursors.
Model of T and NK lineage commitment events in the fetal mouse
The identification of a common phenotype that defines bipotent precursors for T and NK
lineage cells in the fetal thymus. blood, and spleen, represents a significant advance in the
understanding of lymphocyte lineage commitment. Furthemore, the demonstration that bipotent
78
T/NK ceII progenitors are phenotypically distinct from subsets of unipotent T and NK lineage-
comrnitted progeny, strongly suggests that these cells may serve as cornmon precursors at the
single-ce11 level. We are currently investigating whether a single ce11 with the NKl . 1 ' phenotype
can simultaneously give rise to both T and NK cells using micromanipulation and retroviral tagging
strategies. The fact that this bipotent stage is not dependent on intact epithelium should facilitate the
identification of the rnolecular signals that control lymphocyte lineage commitment to the T and NK
cell fates.
Figure 2 1 shows a scheme of T and NK lineage differentiation. Multipotent hematopoietic
progenitors enter into the circulation from the fetal liver dunng early ontogeny, along with various
lineage-committed progenitors. A subset of multipotent cells gives rise to NK 1 . 1 + fetal TMK
(FTNK) lineoge-committed progenitors. However, the cellular and molecular signals responsible
for this commitment event, and whether it is induced in situ or stochastically reached upon
emigration from the fetal liver, al1 remain unknown. Nonetheless. both multipotent and bipotent
cells in the circulation are capable of seeding other hematopoietic organs, including the fetal
marrow, spleen, thymus, and possibly intestinal cryptopatches. FTNK progenitors may enter the
thymus directly, or rnay be induced to commit to this stage from multipotent progenitors under the
influence of thymic stroma. Upon entry inro the fetal thymus, multipotent progenitors rapidly
commit to the lymphoid lineages (fetal TLP, or FïLP, stage), restricting other hematopoietic
potentials including that of the myeloid lineage. Thymus-i nduced differentiation rnay also commit
mui tipoten t precursors to the FTNK stage. FTLP andor FïNK progenitors which undego further
commitrnent steps, marked by expression of CD35 and loss NKI. 1 , commit to the T cell lineage
(pro-T stage). FT'NK cells which do not undergo the second thymus-induced differentiation event
lose CD 1 17 expression and become NK lineage-committed precursors, perhaps by default
(pre-NK stages). Taken together, this mode1 of T and NK ceIl differentiation underscores the
finding that T/NK lineage commitment can take place pnor to thymic colonization; however, full T
lineage commitment requires the influence of the thymic microenvironment.
79
Fetal Liver - - Multilineage
Multi- + -- @ @ CD90+ --+ T lineage CD25
NK
Fetal Thymus
Pre-NK
CD90+ I
I I 1 CD24+ I I I I I I Pro-T t d
Figure 21. Mode1 for T and NK lineage cornmitment events in the fetd mouse. Proposed schcme for lymphocyte lineage cornmitmeiit to the T and NK cell fates in the fctd mouse. Multipotent hcrnatopoietic progenitors enter into the circulation from the fetal liver during earfy ontogeny, alonp with various lineage-committed progcnitors. A subset of multipotent cells gives risc to NKl. I + fetal T/NK (FTNK) progenitors. Both multipotcnt and oligopotent cells in the circulation are capable of seeding other hematopoietic organs, including the fetal marrow, spleen, thymus, and possibly intestinal cryptopatches. FTNK progenitors may enter the thymus directly, or may be induced to commit to this stage from multipotent progenitors under the influence of thymic stroma. Taken together, this mode1 of T and NK ce11 differentiation underscores the finding that T/NK lineage cornmitment can take place pnor to thymic colonization; however, full T lineage cornmitmeni requires the influence of the thymic microenvironment. See text and Figure 9 for further details.
CHAPTER IV:
REGULATION OF NK1.1 EXPRESSION
DURING LINEAGE COMMITMENT OF PROGENITOR THYMOCYTES:
A DEFAULT PATHWAY FOR THYMOCYTE LINEAGE COMMITMENT
James R. Carlyle and Juan Carlos Ziiiiiga-Pflücker
Department of Immunology, University of Toronto, Toronto, ON. Canada
(AI1 work was perfonned by J.R.Carlyle)
Published in The Journal of Immunology
15 December 1998, Volume 16 1 , pp. 6544-655 1.
CHAPTER IV: A Default Pathway for Thymocyte Lineage Cornmitment
Introduction
The NKl . I+/CD 1 17' stage in fetal ontogeny delineates committed progenicors for T and
NK lymphocytes. Termed fetal T/NK (FTNK) progenitors, cells at this stage are phenotypically
similar to TLPs, and may have k e n previously included among the TLP population (3). F ï N K
progenitors were onginally identified in the fetal thymus as early as day 13 of gestation. but were
found to be absent in the fetal liver ( 1 26). Nonetheless, upon transfer into FTOC. both NK 1 . 1 -
TLPs and multipotent fetal liver-derived hematopoietic precursors could rapidly differentiate to the
NK 1 . 1 + stage ( 126). This suggested that expression of NK 1.1 marks a thymus-induced lineage
commitment event. However. identification of an identical population of FT'NK precursors in the
fetal blood and spleen of both normal and athymic nude (ndnu) mice demonstrates that the thymus,
or at least intact epithelium. are not strictly required for differentiation to this stage.
In an effort to further elucidate the requirements for progression to the F ï N K stage. we
cultured fetal TLPs and fetal liver-derived hematopoietic precursors with exogenous cytokine
combinations in vitro. Surprisingly, a substantial su bset of fetal TLPs, but not fetal-l iver derived
precursors, is capable of upregulating NK 1 . I expression spontaneously upon short-term in vitro
culture. Spontaneous upregulation of NKl . 1 surface expression is minimally affected by
transcriptional blockade, mitogen-induced activation, or exposure of these cells to exogenous
cytokines or stroma1 cells. These data suggest that efficient induction of NKI. 1 expression on
cultured thymocytes may be predetetmined by exposure to the thyrnic microenvironment in vivo.
Importantly. multipotent CDI 17+ thymocytes subdivided on the basis of NKl . I expression after
short-terni in vitro culture show distinct precursor potential in lymphocyte lineage reconstitution
assays. These findings indicate that the TLP population, although phenotypically homogeneous,
contains functionally heterogeneous subsets of lineage-committed precursors. Evidence is provided
82
indicating that spontaneous progression from the FIZP to the FïNK stage refiects a thymus-
induced differentiation signal, and may represent a default pathway for thymocyte differentiation in
the absence of conthued thymic influence. Thus, although not necessary. exposure to the thymic
microenvironment is sufficient for the efficient induction of commitment of multipotent
hematopoietic precursors to the NK 1 . 1 + stage.
Results
A subset of fetal TLPs spontaneously upreplates NK1.l expression ex vivo
To investigate the effects of cytokines on the survival and differentiation of early precursor
thymocytes, we cultured sorted precursors under conditions shown to maintain precursor
multipotency in vitro (4). We have previously demonstrated that sorted F ï N K (NK 1.1 +/CD 1 17')
progenitors cultured under these conditions do not undergo further differentiation in vitro. unless
cocultured w ith bone marrow-derived stroma1 cells to induce cornmitment to the NK lineage (Figure
8; see Chapter 1) (126). However, the effects of culture on the TLP population remain unknown.
Figure 22 shows NK 1.1 versus CD 1 17 expression on sorted NK 1.1 -/CD 1 1 7' fetal
thymocytes (TLPs) and fetal liver precursors before and after in vitro culture in the presence of
exogenous IL-3, IL-6, IL-7, and SCF (4). Strikingly, almost half of sorted fetal thymocytes
spontaneously upregulate NK 1 . 1 expression after 48 hours under these conditions (Figure 22, Fi').
This effect is common to two NKl .1 -expressing strains of mice, as revealed by comparison of
Swiss.NIH and C57B1/6 thymocytes (Figure 22. Sw versus B6, respectively). In contrast, soned
fetal liver precursors from each of these strains remain predominantly NK1.1- upon similar culture
(Figure 22, FL). Nevertheless, we previously demonstrated that a subset of FL precursors does
upregulate NK 1 . 1 expression after exposure to thymic stroma in FTOC (Figures 6 & 7; see
Chapter 1) (126). Taken together, these findings suggest that differentiation to the NKI. I+/CDI 17+
FTNK stage can be induced by either prior or continued exposure to the fetal thymic
microenvironment. Thus, expression of NKl. 1 in vitro may represent a delayed progression to a
developmental stage that reflecü the receipt of a differentiation signal in vivo. Altematively, this
phenornenon rnight represent a default pathway of T/NK lymphocyte differentiation, which may be
programmed to occur in the absence of continued thymic influence. As upregulaiion of NKl. 1
expression on TLPs was substantial within 48 hours culture ex vivo, we investigated the kinetics
84
48h in vitro (Sw)
48h in vitro (B6)
Figure 22. FTLP progenitor thymocytes spontaneously upregulate NK1.1 expression ex vivo. (a) Flow cytometric analysis of cell surface expression of NK 1 . 1 versus CD 1 17 immediately after sorting Sw 614-15 NKI.1-/CD117+ FTLP and FL cells. (b) Cell surface expression of NKI . 1 vscrsus CD 1 17 on sorted NK 1.1 -/CD1 17+ FI'LP and FL cells from Sw and B6 mice after 48 h culture in media plus cytokines (IL-3, IL-6, IL-7, SCF).
further in an attempt to delineate between these possibilities.
The temporal requirements for NKI . I expression on precursor thymocytes were assessed
under conditions identical to those in Figure 22. As shown in Figure 23, surface expression of
NK 1.1 on sorted TLPs becomes evident as early as 16 hours after removal from the thymus.
Furthemore, the majority of thymocytes upregulate NK 1.1 surface expression within 48 hours,
with only a rnoderate increase after this time-point. Importantly, the maintenance of a subset of
precursors with a TLP phenotype, even after 6 days in vitro, indicates that not al1 thymocytes
undergo differentiation in vitro. Similarly, upregulation of NK1.l does not occur at any time-point
on fetal liver-derived precursors, indicating that this effect is specific to only a subset of fetal
thymocytes. Taken together, these rapid and specific kinetics of differentiation may indicate that
on1 y a subset of fetal thymocyte precursors have received an irreversible signal to differentiate. and
that the effect of this signal is manifest in phenotype over the short-term culture period.
Alternatively. due to the distinct lineage potential of the precursor populations involved ( 126). the
conditions of culture might be directly affecting the fetal thymocytes in a differential rnanner from
fetal liver precursors. To address this, we cultured these two precursor subsets under various
culture conditions. ranging from minimal survival requirements to a bone marrow-deri ved stroma1
coculture environment strongly supportive of B and NK lymphoid differentiation ( 126. 127).
Spontaneous upregulation of NKl.1 is predetermined by exposure to fetal thymic stroma
Although the combination of IL-3. IL-6, IL-7, and SCF has been shown previously to
augment proliferation of the earliest CD I 17+ thymic precursors and maintain their precursor
potential in vitro, the effect of these cytokines on the ovenll phenotype was not determined (4).
Nonetheless, in agreement with Our observations, these studies indicated that SCF, the ligand for
CD1 17, appears to be necessary for the survival of progenitor thymocytes when cultured in
isolation (4, 1 24). Therefore. we tested whether spontaneous upregulation of NK 1.1 occuc the
86
Figure 23. Temporal regulation of NK1.1 expression on sorîed FI'LP and FL pragenilors during short-term in vitro culture. Sorted ITLP and FL cells (Sw) were culiured ris in Figure 72 for thc indiçnted intervals prior io analysis of N K 1 .1 vs. CD 1 17 expression hy flow cytometry.
presence of SCF alone. Figure 24 shows that there is little difference in NKl. 1 upregulation on
cultured fetal thymocytes and fetal liver precursors whether cultured in SCF alone (Figure 24. SCF)
or in the presence of SCF plus IL-3, IL-6, and I L 7 (Figure 24, SCF + IL-3,6,7). Thus. as
previously shown, these cytokines do not appear to have a differentiation-inducing effect on these
precursors in vitro.
We next tested the effect of coculture of precursor thymocytes with the OP9 bone marrow-
derived stroma1 ce11 line ( 1 34). which efficient1 y induces and supports B and NK lineage
differentiation ( 126, 127). In particular, OP9 coculture induces rapid (48 h) CD 1 17
downregulation on FTNK precursors (Figure 8: see Chapter 1). and induces predominant B lineage
differentiation of FTLPs (Figure 5; see Chapter 1) (126, 127). However, OP9 had no significant
effect on spontaneous NK 1 . I upregulation, and only a minor effect on CD 1 17 expression. when
cocultured with either precursor subset (Figure 24. SCF + stroma, SCF + IL-3,6,7 + stroma). A
similar resul t was observed upon coculture of these precursors with fi broblasts (data not shown).
Thus, i t appears that spontaneous upregulation of NKl. 1 expression reflects the induction of a ceIl
fate predetermined by exposure to fetal thymic stroma in vivo. Therefore, we examined the
transcriptional regulation of NKl . I expression on fresh and cultured thymocytes.
Initiation of NK1.1 expression in a subset of fetal TLPs occurs in vivo
Spontaneous upregulation of NK 1 . I on precursor thymocytes appears to be a
predetemined cell fate that occurs within the thymic microenvironment during the course of normal
lineage cornmitment. However, initiation of NK1.I expression might occur in vitro simply as a
consequence of removing these cells from the thymus and culturing them in isolation. In the latter
case, NK 1 . 1 expression rnight refiect the induction of a default pathway of differentiation. To
distinguish between these possibilities, we examined NKI . 1 expression at the transcriptional levd,
using the RNA polymerase II inhibitor, a-amanitin, to block nascent transcription of mRNA in
88
SCF
SCF + IL-3,6,7
SCF + Stroma
SCF + IL-3,6,7 + Stroma
Figure 24. Spontaneous upregulation of NKl.l on FTLPs is not affected by exposure to exogenous cytokines or stromal cells. Flow cytometric analysis of sorted FTLP and FL cells (Sw) cultured for 48 h with or without cytokines (IL-3, IL-6, IL-7) and/or OP9 bone marrow- derived stromal cells, as indicated. SCF was added to al1 cultures in order to maintain viabiliiy.
vitro. As a control, a-amanitin treatment of ConA-stimulated splenocytes blocked the induction of
CD25 surface expression (data not shown). Figure 25a shows fetal thymocytes and fetal liver
precursors cultured for 24 hours without (Figure 25a. 24h Control) and with pharmacological
blockade of transcription (Figure 25a. 24h + a-amanitin). A 24 hour time-point was used due to
toxicity resulting from global transcriptional repression for longer time periods. Although
a-amanitin treatment reduced both the fraction of NKI . l+ thymocytes and NK 1.1 expression ievels
significantly (approximately 50%. Figure 25a), it failed to completely block this induction.
sugpsting that at least a subset of thymocytes contained preexisting transcripts for NKR-P 1 C
(CD 16 1 ). the ligand for NK 1 . 1 ( 1 16). To investigate this further, we employed RT-PCR on fresh
thymocytes ex vivo.
Figure 25b shows analysis of NKR-Pl gene expression on RNA isolated from fresh day
15 fetal thymocytes and fetal liver cells (127). RT-PCR analysis reveals that sorted
NK 1.1 ïCD1 17+ TLPs (Figure 25b, TLP) express pre-existing transcripts for members of the
NKR-P I family ( 1 20). albeit at reduced levels cornpared to the sorted NK 1.1 +/CD 1 17' mature NK
cell cohort (Figure 25b. NK). Imponantly. control PCR reactions done in the absence of reverse
transcriptase (Figure 25b. Con), and RT-PCR of soned NK 1. I -/CD 1 17+ fetal liver cells (Figure
2%. FL) did not yield significant product for NKR-PI genes. These data suggest that surface
NK 1 . 1 -/CD 1 1 7+ fetal thymocy tes and fetal liver cells differentially regulate endogenous expression
of the NKR-PI genes. a finding that supports their phenotype change during short-term in vitro
culture. Thus, although this does not rule out the possibility that NK 1. I expression in vitro may
additionally reflect a delayed in vivo signal or default in vitro signal. at least some of the surface
staining for NKl. 1 observed upon culture of thymocytes is likely the result of direct translation of
pre-existing mRNA transcripts (Figure 25b). The presence of NKR-P 1 C transcripts among
freshly sorted NKl. 1- thymocytes and the failure of transcriptional blockade to completely inhibit
NK 1. I expression suggest that upregulation of NK 1.1 on precursor thymocytes a ce11 fate
predetermined by exposure to thymic stroma in vivo.
90
NKR-PIA p l r] NKR-PIC (-;;II F I
4% in vitro --> Re-~ort NK 1 . 1 - --> 72h
Figure 25. Spontunnnar upreguhtlon of NKI.1 on WLPs is minlnully .Ilccted by transcriptional Moekade or mitogtn-indud activation. (a) Flow cytoineiric analysis of sortcd FTLP and FL cclls (Sw) cultwed with SCF plus cytokines (IL-3.lL-6, IL-7) for 34 h with or without the RNA polgrnerase II inhibitor, a-amanitin, as indicaicd (b) R T X R for NKR-Pl genes on RNA prepared from s o n n l NK 1 . 1 - fccal TLP and M< 1 . 1 + mature NK feial thymocyies and NKI.1- FL cclls. The conirol (Con) sample reprcsents FL withcut the addition of reverse tranxriprase prior to PCR. (c) Culiured as in (a, C<mirol). with îhe addition of PMA/ionomycin. (d) FTLP and FL cells wen: culiured for 48 h, thcn re-sortcd for FïLP cells, and further cultured for 72 h (wiih cytokines), prior to analysis.
We next determined whether upregulation of NKl. 1 on precursor thymocytes could be
mimicked by rnitogenic activation of these cells. Pharmacological treatment using phorbol ester and
calcium ionophore induces expression of CD25 to high levels on precursor thymocytes. mimicking
progression to the pro-T ce11 stage (5). A similar effect is induced upon treatment of these cells
with the cytokines, TNF-a and IL-la (5). Interestingly, these treatrnents fail to augment CD25
expression on fetal liver precursors (unpublished otiservations). Therefore, we tested the effect of
these treatments on NKI. I expression in these cells. Treatment of sorted fetal thymocytes and fetal
liver cells with PMA and ionomycin only had a marginal effect on NKI. 1 expression (Figure 25c.
24h + PMAhono; compare Figure 25a. Control). Futthemore, treatment with TNF-a and IL- la
had no significant effect (data not shown). Minimally, these data indicate that CD25 and NK 1.1
expression are regulated by distinct pathways in precursor thymocytes, and that NK 1. I
upregulation is not due to generalized activation, at least that mediated through PKC and ~ a " flux.
The kinetic data presented in Figure 23 suggest that the majority of thymocyte precursors
upregulate expression within 48 hours after removal from the thymus, and that a substantial subset
rernains NK 1 .1 ' in longer culture. This suggests that a subset of fetal thymocyte precursors,
possibly those which have not received a thymic signal. are analogous to fetal liver precursors. To
test this, we cultured fetal thymocytes ex vivo for 48 hours, then resorted the NKl. I - fraction and
retested their propensity for NKI. 1 upregulation in vitro thereafter. As shown in Figure 25d, over
90% of these cells remained NK 1 . 1 - over the next 72 hoiirs (compare Figure 25d to Figure 23).
This lends further support to the notion that upregulation of NKl. I on thymocyte precursors
represents a delayed phenotypic change in response to a signal received intrathymically in vivo.
Spontaneous upregulation of NK1.l represents a differentiation event
Due to the large percentage of NK 1 .1+ precursors that appear after culture, it is possible that
they represent the outgrowth of a small number of differentiating cells. To test this. we sorted
92
thymocytes ex vivo for an NKI. I7CD117+ phenotype and labelled hem in vitro with the vital dye,
CFSE, prior to culture. CFSE is a fluorescent mernbrane-localizing dye partitioned evenl y into
daughter cells upon division, and it allows the visualization of proliferating cells by revealing two-
fold stepwise decreases in green fluorescence detectable by flow cytometry ( 186). Figure 26 shows
fetal thymocytes and fetal liver precursors cultured for 48 hours after CFSE labeiling. Under these
culture conditions, fetal b e r precursors were found to divide more rapidly than the majority of fetal
ihymocytes, as indicated by their lower relative fluorescence (Figure 26, top panel). However. when
cultured thymocytes were gated according to their NK1.l phenotype, NK 1 .1 ' precursors were
found to divide at a rate sirnilar to fetal liver cells, while the NK 1.1' subset divided approximately
one ceIl division less overall (Figure 26. bottom panel). This indicates that the NKI . I + fetal
thymocyte subset does noi outgrow in culture, rather these cells undergo a differentiation event that
appears to slow their proliferation relative to the NK 1 . 1 - fraction.
Spontaneous upregulation of NKl.1 marks lineage commitment to the T/NK Fates
We previously demonstrated that NK 1 . 1 +/CD 1 17+ thymocytes sorted fresh ex vivo are
capable of generating T and NK cells. but lack potential for B lymphocytes in lineage reconstitution
assays ( 1 26). Furthemore. NK 1. I X D 1 17+ TLPs are capable of giving rise to al1 three lineages.
Therefore, to determine whether spontaneous upregulation of NKI. 1 in vitro recapitulated in vivo
lineage commitment, we tested the lymphocyte potential of cultured TLPs subdivided according to
their NKI. 1 phenotype. Figure 27a shows the gates used to sort purified TLPs cultured for 48
hours in vitro. Reconstitution of dG-FïOCs with both the NKl . 1 + (Figure 27a, R 1 ) and NKl . 1-
(Figure 27a, R2) subsets resulted in the generation of both C D ~ ~ ' T cells and NKl . l + NK cells
(Figure 27b). As previously demonstrated ex vivo (Figure 4) ( 1 26), T lineage progeny consisted of
both immature CD41CD8 DP and mature SP conventional T cells, while NK cells lacked
expression of CD3r and TCRap (data not shown). DNA isolated from sorted T lineage cells also
93
CFSE
Figure 26. NK1.1 upregulation represents a differentiation event and is not due to outgrowth of NK1.1+ cells. Sortcd FîLP and FL cclls (Sw) were labelled in vitro with CFSE for 10 min,. cultured for 48 h with cytokines. thcn analyzcd by flow cytomctric analysis for CFSE retcntion. Top panel shows tom1 CD1 17+ FL and FT cclis cultured at 4°C io prcvent cc11 division: middlc panel shows total CD1 17+ FL and FT cclls cultured at 37°C; boitom panel shows CD1 I7+ FT cclls gatcd according to NKI . I surface expression.
dG FTOC
Figure 27. Upregulation of NK1.1 on FTLPs marks the loss of B-lineage potentiat (a) Flow cytometric rinrilysis of soried FI'LPs (Sw) cultured with cytokines for 48 h. then rcsorted according to NK 1.1 expression, and tested for precursor poteiitiat (RI gate, NKI . I + cells, uppcr panels; R2, N K I . 1- cells, lower panels). (b) Flow cytometric analysis of CD3e and NK 1.1 expression on cells recovered from reconstitutcd dG-FïOCs 12 days after the addition of sorted fctal thyniocyte subscts as in (a). (c) Flow cytoinetric analysis of NKI.1 and CD45R expression on çclls recovercd following 13 days in OP9 coculturc. Cells, in paraIlel wiih (b), werc coculturcd on confluent rnonolayers of OP9 cells for 7 days in the presencc of cytokines followed by an additional coculturt: on frcsh OP9 cells in the presence of IL-7 and IL-2, prior to Iiarvesting.
possessed DJ rearrangements at the TCRP loci, while that from NK cells was retained in the
germline TCRp configuration (data not shown). Thus, both populations possess potential for T and
NK cells in FTOC.
However, coculture of the same subsets with the bone marrow-derived stroma1 cell line, OP9
( 134), reveals that they possess distinct reconstitution potential for the B lymphocyte lineage
(Figure 27c). Specifically, similar to their ex vivo counterparts (Figure 5) ( 126), the NK I . 1 +
precursor fraction was capable of generating only NK lineage cells, while the remaining NK 1.1 -
subset gave rise to both NK cells and B lymphocytes on OP9 (Figure 27c). B lineage cells were
identified by their C D ~ ~ R ' N K ~ . 1- phenotype (Figure 27c). expression of CD 19. and detection of
DJ rearrangements at the IgH loci from isolated DNA (data no shown). NK cells derived from
OP9 cocul ture were CD45R-/NK 1.1 + (Figure 27c), a subset also expressed the pan-NK cell
marker, DX5 ( 15 1 , l78), and DNA derived from these cells was retained in the germline IgH
configuration (data not shown). Taken together, these data indicate that the upregulation of NKI. 1
on the surface of fetal TLPs cultured in vitro corresponds io a bona fide lineage commitment event
identical to the FTLP to FTNK transition in vivo (126). Thus, a subset of FTLPs appears to receive
a thymus-derived signal during residency in the thymic microenvironment. which results in their
delayed upregulation of NKl . 1 and commitment to the T/NK ce11 lineages during short-term in
vitro culture.
Discussion
The most immature hematopoietic precursors in the fetal thymus, the TLPs, display
multipotent lymphoid lineage potential and are characterized by high-level expression of CD 1 17
and a lack of expression of lineage differentiation (Lin-) markers (38), including NK 1. i ( 126. 1-7).
Purified fetal thymocytes at the TLP stage in development have been demonstrated previously to be
capable of maintaining precursor multipotency for up to 7 days in vitro in the presence of
exogenous cytokines (4). This technique has been utilized to study the cytokine requirements that
are necessary for the survival and proliferation of early precursor thymocytes. However, the effect
of in vitro culture on the overall phenotype and precursor potential of these precursors as a
population remain unknown. To investipate this further, we examined the effect of short-term in
vitro culture of isolated fetal precursor cells in the presence of exogenous cytokines.
These data indicate that even though the TLPs appear to be phenotypically homogeneous.
they contain a functionally heterogeneous mixture of cells with distinct precursor potential. This
functional heterogeneity is revealed phenotypically in a delayed manner upon short-term culture of
these precursors under conditions that maintain their proliferation and survival in vitro (Figures 22-
26). In particular, the majority of TLPs cultured in isolation. with ai least exogrnous SCF,
efficiently and spontaneously induce NK 1 . 1 expression ex vivo within 48 hours. The subset of
cultured TLPs that remains NK 1.1 - in vitro. retains rnultipotent lymphoid precursor potential for the
T, NK, and B lineages, while the NKI. l 7 subset is restricted to the T and NK lineages (Figure 27).
Thus, spontaneous upregulation of NK 1 . I surface expression in vitro corresponds to a bona fide
lineage cornmitment event analogous to that observed for the phenotypically identical FTNK stage
in vivo.
Spontaneous upregulation of NKI . I in vitro appears to represent a cell fate predetermined
by exposure to the thymic microenvironment. This is supported by the fact that this effect is
specific to fetal thymocyte precursors and does not efficiently occur with fetal liver cells, unless the
97
latter are cocultured with fetal thymic stroma in FïOC ( 1 26). Moreover, induction of NK 1 . 1
expression on th ymoc ytes is minimal1 y affected by transcriptional blockade, mitogen-induced
activation, or exposure to exogenous cytokines of stroma1 cells (Figures 24 & 25). Indeed.
transcripts for the N U - P l (CD1 61 ) farnily members (120), of which NKR-PIC represents a
ligand for the anti-NKI. 1 mAb ( 1 16)- were detected among sorted NKl. 1- TLPs fresli ex vivo.
This suggests that at least a subset of these cells was already destined to express surface NKI . 1 .
Furthemiore, expression of NK1.I in vitro was found to correlate with reduced proliferation.
compared to the remaining NK 1.1- counterpart (Figure 26). This reduced ce11 turnover suggests
that the NK1.1' cells undergo a differentiation event during culture, which is consistent with their
restricted lineage potential.
Taken together, these data suggest that a subset of TLPs has already received a thymus-
induced signal that resulü in lineage cornmitment, while the remaining fraction has not yet received
this signal, despite their residency within the thymus. This signal, in tum, could represent either a
direct commitment event to the FTNK stage or a T lineage commitment event that does not preclude
a defüult developmental pathway for NK ce11 potential. The difference between these two
possibilities may depend on whether the NK 1. I subset was destined to express NK 1.1 in vivo, or
whether this subset would have followed a different developmental pathway if not removed from the
thymic microenvironment. Notably, the phenomenon of spontaneous upregulation in vitro is
specific to NK 1.1 and CD 1 6/32 (data not shown), as induction of CD25 to a similar extent does
not occur (data not shown). Thus. either the duration of this normally transient NKl . 1 + stage is
proportionately pronounced in the absence of continued thymic influence, or this phenomenon
represents a default pathway pnor to the pro-T stage that occurs upon removal from an environment
capable of efficiently inducing T lineage commitmeni. Nonetheless, the interpretation remains that
the rnajority of multipotent TLPs, en route to becoming committed T lineage precursors, undergo
lineage commitment to a stage which does not preclude differentiation to either of the T or NK
lineages. Therefore, the FïNK phenotype seems to represent a tme cellular commitment pathway
98
induced by thymic stroma. However, differentiation to this stage is not thymus-dependent. as
phenotypically and functionally identical precursors are found in the fetal circulation (see Chapter
III) (152, 187). In addition, a small subset (1-2%) of fetal liver precursors also spontaneousiy
upregulates NK1.l surface expression (Figures 22-25), indicating that the thymus is not strictly
required for this event.
These findings characterize a lineage commitment pathway common to T and NK lineage
precursors. Whether this pathway represents the predominant course of T and NK cell precursor
potential in vivo or an alternative, default pathway for T and NK cell development. remain unknown.
However, the ability of precursor thymocytes to spontaneously undergo delayed lineage
commitment in vitro highlights a caveat in assays of precunor activity. in that even a phenotypically
homogeneous population of celis may be functionally heterogeneous. Thus. single-cell assays may
represent the oniy unambiguous means of determining lineage potential. Nonetheless. these
findings caution rhat functional heterogeneity will exist even in single-ceIl assays. Specifically. at
least half of TLPs do not possess B lineage potential.
CHAPTER V:
MOUSE NKFt-PlB, A NOVEL NK1.1 ANTIGEN
WITH INWIBITORY FUNCTION
James R. Carlyle. Alberto Martin, Amn Mehra*,
Liliana Attisano*, Florence W. Tsui, and Juan Carlos Ziifiiga-Ptlücker
Department of Immunology, University of Toronto. Toronto, ON. Canada
*Department of Anatomy and Cell Biology, University of Toronto, Toronto, ON, Canada
(Cytotoxicity assays were performed by J.C. Ziifiiga-Pflücker.
SDS-PAGE was perforrned by A. Mehra and Western blotting was performed by A. Martin.
Al1 other work was perfomed by J.R. Carlyle)
Submitted 9 June 1998
CHAPTER V: Mouse NKR-PlB, a Novel NKl.1 Antigen with lnhibitory Function
Introduction
The nature of how natural killer (NK) cells distinguish between self and non-self and the
molecules that are responsible for this detemination remain largely unknown. Killer-ce11 activatory
receptors (KARs) and killer-ce11 inhibitory receptors (KIRS) expressed on NK cells have been
suggested to allow for this discrimination. Members of the NKR-PI (CD1 6 1 ) family of molecules
are type II transmembrane C-type lectin receptors found on the surface of NK cells and a subset of
T cells ( 128, 1 33, 1 88- 1 90). These molecules have been shown to activate NK ceIl cytotoxici ty
( 1 16, 154, 189, 19 1 ), and thus function as KARs ( 188, 190). The mouse NK 1.1 antigen ( 112).
originally defined as NKR-Pl C (1 16, 120), belongs in this family (128). Recently. it was shown
that NK 1.1 (NKR-Pl C)-mediated activation occurs through association with the FcRy chain ( 128).
which in tum signals through the protein tyrosine kinase, Syk ( 192). Although the NKR-P 1
molecules may recognize carbohydraie ligands on target cells ( 193, 194), cognate protein ligands
for these receptors remain unknown.
KIRS expressed on NK cells prevent activation and lysis of class 1 MHC-bearing cells
( 193, providing a mechanism for the "missing self' mode1 of NK ce11 function ( 1 96- 198). In
mouse NK cells, members of the Ly-49 family of receptors are included in the KIR category ( 1 99).
The ability of these receptors to inhibit NK ceIl function seems to be dictated by the presence in
their cytoplasmic domain of a conserved immunoreceptor tyrosine-based inhibitory motif (ITIM;
INLxYxxLN (200,201)) ( 190,202,203). However, not al1 Ly-49 members are inhibitory in
function, as Ly-49D and Ly-49H, which lack an ITIM, have been shown to activate NK ceIl
function (203,204). In keeping with this, the mouse NKR-P 1 A and NKR-Pl C molecules lack a
consensus ITZM ( 190). On the o t k i hand, the closely related mouse NKR-PIB molecule
possesses a consensus FI. (LxYxxL) in its cytoplasmic domain ( 120, 190). Nonetheless, due to
101
a lack of available specific antibodies, this gene product has remained relatively uncharacterized to
date.
Here we show that the mouse NKR-PlB gene product serves as a ligand for the anti-NK 1.1
mAb, PK 1 36, and thus represents a novel NKI . I antigen ( 1 16, 120). In contrast ro NKR-P 1 C .
NKR-Pl B fails to activate NK cell cytotoxicity. Moreover, in NK cells expressing both the
NKR-P 1 B and NKR-P I C molecules, NK 1. I -mediated redirected lysis of target cells is abrogated.
Taken together. these data indicate that NKR-PlB functions as a KIR ( 190. 195). Indeed. like other
inhibitory receptors expressed by NK cells, NKR-PIB binds the SHZtontaining tyrosine
phosphatase, SHP- 1 (205). in a phosphorylation-dependent manner (1 95,206,207). This provides
a rnolecular mechanism for the inhibition of NK ceIl cytotoxicity through this novel NKI . 1 antigen
and dernonstrates that NKR-P1B is an inhibitory member of the CD 161 family. The existence of
two closely related NKl . 1 antigens with opposite regulatory function suggests a role for NKR-P I
molecules in NK cell-mediated selfhon-self recognition in the immune system.
Results
Expression of NKl.l on CDW+ fetal blood cells is strain-dependent
The molecular cloning of the mouse NKR-PIC gene served to identify it as the NKl . 1
antigen ( 1 16). However, during the course of studying two NKI . 1 -expressing strains of rnice. we
obtained evidence to suggest that NKR-Pl C may not be the only NK 1.1 antigen. Specifically. we
observed that NKI. i is differentially expressed on fetal blood CD~O+/CDI 17" progenitors derived
from two NK 1.1 -expressing mouse strains, Sw and B6 (1 52, 187). Figure 28 shows Sw and B6
day 15 fetal blood cells depleted for ~ ~ 2 4 ~ ' cells by antibody/complement-rnediated lysis (152).
As dernonstrated previously ( 100, l52), a subset of CD 1 17'' fetal blood cells derived frorn both Sw
and 86 rnice expresses CD90 (Figure 28). However, while the v a t majoriiy of CD~O+/CDI 17'0
fetal blood cells from Sw mice display the NKI. 1 marker, the identical subset from 136 mice lacks
NK 1.1 expression (Figure 28). As both subsets express high levels of CD 16/32 and were found to
be functionally identical ( 187), this suggested either that the developmental regulation of NKR-PI C
is strain-specific, even among NK 1.1 expressing strains. or that the anti-NK 1.1 mAb. PK 136.
recognizes a different ligand in each of the strains. To address this issue. we analyzed expression
of NKR-Pl family mernbers by RT-PCR.
Expression of NKR-Pl family members is strain-specific
To investigate the expression of NKR-Pl genes in different strains of mice. we employed
RT-PCR on RNA isolated from spleen cells of adult mice. Figure 29 shows RT-PCR for the
expression of full-length NKR-PIA/B/C transcripts in spleen cells from Sw. B6. and (B6xSw)FI
rnice. Surprisingly, although al1 three strains possessed similar populations of NK 1. l+ splenocytes
(data not shown), the Sw strain lacked transcripts for NKR-PIC (Figure 29). This suggested that
- 103
Figure 28. Expression of NK1.1 on CD90+ fetal blood cells is strain-dependent. Fctal blood cells derived from Sw and B6 mice werc depleted of ~ ~ 2 4 ~ ~ cclls by antibody/complement- mcdiated lysis and analyzed for exprcssion of CD90, NKl.1, and CD I 17 by flow cytometry.
Figure W. Expression oC NKR-Pl family mmibtis b strain- speclfic Tolal RNA was isolated from Sw, B6, and (B6xSw)Fl splenocytes and analyzed by RT-PCR for expression of NKR-Pl gene farnily members.
the NK1.l antigen expressed by Sw NK cells and fetal blood cells might represent the product of a
closely related gene in the NKR-Pl family. As the NKR-P 1 A gene product was previously shown
not to encode an NKl. 1 antigen (1 16), and because Sw mice express abundant mRNA for the
NKR-PlB gene (Figure 29) (127), these data suggested that the NKR-P 1 B molecu le might
represent a novel NKl. 1 antigen in Sw mice. Notably, NKR-P1B shares -96% arnino acid identity
to NKR-PIC in its extracellular C-type lectin domain (120).
Mouse NKR-PlB is a novel NK1.1 antigen
To test whether the NKR-P 1 B molecule might represent a second NK 1.1 antigen. we
cloned the NKR-P IB cDNA from Sw-derived NK cells into a mamrnalian expression vector for
use in transfection experiments. As controls, we also cloned the NKR-Pl A and NKR-P I C cDNAs
from B6-derived NK cells ( 1 16). These constructs were transientiy transfected into Jurkat cells.
To identify transfected cells. we cotransfected a plasmid construct encoding an enhanced
mammalian version of the jellyfish green fluorescent protein (GFP). Figure 30 shows a
representative GFP fluorescence profile of transfected Jurkat cells (Fig. 30a, solid dark line).
Figure 30b shows sequential extracellular versus intracellular staining using the anti-NK1.1 mAb,
pK1 36, after gating on GFP+ Jurkat transfectants. As previously shown (1 16), the B6-denved
NKR-Pl C construct encodes an NK 1 . 1 antigen. while the NKR-P 1 A molecule does not (Fig. 30b.
NKR-P 1 C vs. NKR-P 1 A). Furthemore. as predicted by our previous data (Figures 28 & 29)
( 152, 187), the Sw-derived NKR-Pl B molecule also binds the anti-NK 1.1 antibody (Fig. 30b,
NKR-P I B). Thus, the mouse NKR-PIB molecule represents a second NK 1 . I antigen.
Interestingly, although nucleotide sequencing of each clone confinned their identity, and the
Sw NKR-P 1 B cDNA is indeed identical to the NKR-PI B sequence published previously (120),
analysis of the resulting NKR-Pl B PCR product from 86 mice revealed that it does not correspond
to the previously published sequence (data not shown). Moreover, in transfection experiments, this
1 O6
molecule did not bind the anti-NK1.l mAb and thus does not represent an NKI . I antigen (data not
shown). Using multiple primer pairs for RT-PCR, we have repeatedly failed to detect expression of
an NKR-P 1 B gene product that represents an NK 1.1 antigen in B6 mice. Therefore. it is possible
that this PCR product represents the product of another closely related gene and that B6 mice do
not express detectable message for NKR-PIB. This would be consistent with our previous finding
that NKR-P1B expression is undetectable in fetal B6 mice using altemate PCR primers for the
mouse NKR-Pl genes (127). Notably, altemate PCR primers have also detected a product with the
expected molecular size of NKR-PI C in Sw mice; however, the sequence of this product more
closely resembles NKR-PI A than NKR-PIC, and it does not serve as an N U . 1 antigen upon
transfeciion (data not shown). In any case, it remains likely thai the only NK 1.1 antigen expressed
in 8 6 NK cells represents the product of the NKR-PIC gene, while that expressed in Sw NK cells
represents the product of the NKR-PIB gene. Thus, each NKR-Pl gene appears to be expressed
in a mutually exclusive fashion, at least between these two strains, while (B6xSw)FI NK cells
express the products of both genes (Figure 29: data not shown). Therefore, to assess the function
of each N U - P 1 molecule independently? we tested the capacity for B6, Sw, and FI NK cells to
mediate redirected lysis using the PK 136 mAb.
NKR-P1B functions as a killer-ce11 inhibitory receptor (KIR)
In order to purify NK cells for use in functional assays, splenocytes from each mouse strain
were depleted of C D ~ + , C D ~ + . C D ~ + , ~ D 2 4 + cells, then sorted for DXS+ cells using magnetic
beads. These cells were then expanded in the presence of IL-2 for one week in culture. As shown
in Figure 3 1, the resulting NK cells from each of these strains displayed similar levels of NK1. I
expression (Fig. 3 la). Therefore, to analyze the function of the Sw NK1.I antigen, Sw-derived NK
cells were tested in antibody-induced redirected lysis (AIRL) assays using the anti-NK1.1 mAb,
pK1 36. In AXRL assays, an activating molecule on the surface of an NK ceil augments cytotoxicity
1 O8
Figurp 31. NKR-PIB InhiMts Nli cell hnctioa In antlbody- lnduced rwlirected lysis (AIRL) assays. (a) Purified NK cells were prepared from B6, Sw, and (B6xSw)Fl mice and analyzed for NK1.l surface expression by flow cytometry (a-NK 1.1 t a-moue-IgG, dark line; a-mouse-IgG alone, light line). (b) NK cells were tested for cytotoxicity in AIRL assays usine CD16/32+ (FcyRIII/II+) ml5 targets. Data shown is that of a 30:i effector to mget (E:T) ratio. Percent NK1.1 cytotoxicity is defined as an index& percent speçific lysis observeci with a-NKi.1 rnAb relative to those induced by a- CD16/32 mAb and without added mAb. Error bars indicaîc the standard error of the mean (*SEM) of n=5 independent experiments.
towards FC*+ target cells, such as mouse P8 15 mastocytoma cells, while inhibitory and
non-stimulatory receptors do not significantly affect target ce11 lysis. As a positive control for lysis.
we performed the AIRL assay using the anti-CD16/32 (Fc$IIf/Ii) mAb, 2.462. which activates
NK ce1 1s ( 1 92). Addi tionall y, spontaneous cytotoxicity in the absence of redirecting antibody was
used to determine background levels of natural killing. To simplify these data, the percent specific
lysis values for the NK 1.1 mediated AIRL were indexed relative to the CD 16/32-mediated AIRL
and spontaneous lysis values (a product termed % NKI. 1 cytotoxicity; see Experimental
Procedures). As shown in Figure 3 1 b, NK 1.1 -mediated cytotoxicity was high using Bo-derived
NK cells, confirming an activating role for the B6 NKR-P 1C molecule. In contrast,
NK 1.1 -mediated cytotoxicity was insignificant using Sw-derived NK cells (Fig. 3 1 b). As this
suggested that NKR-P 1 B might function as an inhibitoty receptor, we next assessed
NK 1.1 -mediated AIRL using (B6xSw)Fl NK cells, which express both NK 1.1 antigens. NKR-
P 1 B and NKR-P1C (Figure 29; data not shown). Figure 3 1 b demonstrates that the presence of the
NKR-PIB molecule on (B6xSw)FI NK cells exerts a dominant inhibitory effect over the ability of
NKR-P 1 C to augment AIRL cytotoxicity. Taken together, these data indicate that the NKR-PI B
molecule functions a KIR.
NKR-P1B binds SHP-1 in a phosphorylation-dependent manner
A number of different inhibitory receptors in both human (p58/70 tg-like receptors
(CD 158); NKB 1 ; CD94/NKG2A) and rnouse (Ly-49A/G2) have k e n shown to exert their
inhibitory effects on NK ce11 function by intenupting the early tyrosine phosphorylation pathways
responsible for NK ce11 activation (188). This was first shown at the molecular level by the
demonstration that crosslinking of these inhibitory molecules recruits the SH2-containing tyrosine
phosphatase, SHP-1 (205), in a phosphorylation-dependent manner to the intracellular ITIM (200,
206,208-2 10). Another structurally-related tyrosine phosphatase, SHP-2, was found to function in
110
similar capacity to SHP-1 (210), although SHP-2 may also be involved in transducing activating
signals (2 1 1). On the other hand, SHIP, an unrelated SH2-containing inositol phosphatase
involved in mediating inhibition through the FcvftIIB pathway (212). does not associate with KiRs
(207,2 13). Notably, the mouse NKR-PI B molecule possesses a consensus ITIM (LxY xxL) in its
intracellular domain (1 20, 190). Therefore, to determine the molecular mechanism for inhibition of
cytotoxicity through the NKR-Pl B molecule (Fig. 3 1 b), we tested whether SHP- 1 is recmited to
the ITIM of NKR-PIB upon phosphorylation of the receptor. To achieve this. we utilized an
NK 1.1 + Sw-derived pre-NK ce11 line, MNK- 1, which expresses abundant NKR-P 1 B mRNA but
no NKR-PI C (data not shown). Phosphorylation of the ITIM-based tyrosine was accomplished in
intact cells by disruption of endogenous tyrosine phosphatase activity using pervanadüte stimulation
(2 14). as previously described for the human CD94NKG2A (2 15) and murine Ly-49A and
Ly-49G2 molecules (202.2 16).
As shown in Figure 32, Western blot analysis for SHP- 1 on anti-NK 1.1
immunoprecipitates indicates that the NKR-PlB molecule associates with SHP- 1 upon pervanadate
stimulation (Figure 32a, V05+APaNK 1. I+). This association is phosphorylation-dependent (202.
2 16). as SHP- 1 does not coirnmunoprecipitate with NKR-P 1 B in unstimulated cells (Figure 32a.
VOS-/IPaNKI. I+). Total cell lysate supematant, shown on the right (Figure 32a. S), indicates that
-0.4% of total intracellular SHP- 1 coimmunoprecipitates with the NKR-PIB molecule in response
to pervanadate stimulation. The additional bands observed in the total lysate supematant are due to
anti-SHP-1 cross-reactivity (data not shown) (206). and are absent fromanti-NKI. 1
irnmunoprecipitations. Imponantly, the association of NKR-PI B with SHP- 1 is specific, as
Western blots for SHP-2 (Figure 32b. aSHP-2 blot) and SHIP (Figure 32c, aSHIP blot) revealed
that these phosphatases do not significantly coimmunoprecipitate with NKR-PIB upon pervanadate
stimulation. Moreover, coimmunoprecipitating SHP- I was also detected in pervanadate-stimulated
Sw and (B6xSw)FI NK cells. although SHP-1 could not be detected in anti-NKl . 1
immunoprecipitates from 8 6 NU cells or the NK 1.1 + (NKR-PIC) B6-derived ce11 line, CTLL-2
vos IPaNK 1 . 1
+ SHP- 1
+ SHIP
ffgufe 32. Moutu? NKR-P1B ~ e m r i t s tbc SHtcioRtsiaing tyrodne phqhatase, SHP-1, in a phœphocylritioa-àcpendent manner. An Sw- deRved pre-NK cell iine. MNK-1, was stimulated for 20 min using pervanadate. Cell 1 ysates were immunoprecipitated with a-NKl .1 mA b (PK136). and coprecipitating (a) SHP-1, (b) SHP-2, and [c) SHIP F e i n s were visualized by Western blotting using enhancd chemilurninescence. V05, petvanadate stimulation; IPaNK 1.1. a-NK1.1 immunoprecipitation; S, total cell iysate supernatant.
(data not shown) (190). These data support initial studies demonstrating that crosslinking of
NKR-PIC on B6-denved cells leads to transient increases in intracellular calcium and tyrosine
phosphorylation of downstream substrates (data not shown) (190), while similar responses were
not observed upon NKR-PI B crosslinking using Sw-derived cells (data not shown). Taken
together, these data indicate that the NKR-PIB molecule binds to the SH2-containing tyrosine
phosphatase, SHP- 1. in a phosphorylation-dependent manner, confirming a functional role for the
NKR-P 1 B consensus intracellular ITIM (LxY xxL).
Discussion
A mode1 for NKR-Pl-rnediated signalling events
The finding that NKR-PlB functions as an inhibitory receptor and associates with the
SHP- 1 phosphatase (Fig. 32), while the activating mouse NKR-PI A and NKR-PI C molecules do
not, can be explained in the latter cases by their lack of consensus ITIMs (WILxY xxLN: Fig. 33a).
Both NKR-P I A and NKR-P 1 C possess hydrophilic amino acid residues (R and S. respectively ) at
the Y-2 position relative to their YxxL motif, a position shown to be critical for SHP- 1 binding
(200) . Like the inhibitory Ly-49 family members, NKR-PlB possesses a hydrophobic amino acid
at this position (L for NKR-PIB, VII for Ly-49 molecules). However. unlikc the activating Ly-49
family of molecules, which possess an F residue in substitution of the ITIM-based Y, NKR-Pl A
and NKR-PIC retain the YxxL motif. This may have important implications for their respective
signalling capacities.
Interestingly, both NKR-PlA and NKR-P IC possess a positiveiy-charged arginine residue
near the extracellular region of their transmembrane domains (Fig. 33a). The existence of similar
residues in the activating human CD94MKG2C and murine Ly-49D and Ly-49H molecules has
been shown to be necessary for the association of these molecules with the ITAM-containing DAP-
12 immunoreceptor (203,2 17,2 18). Furthermore, similar residues are important for the association
of the CD3 subunits with the T cell receptor, and the CD79 subunits with the B cell receptor (217).
The finding that NKR-P 1 C, like CD 16 (FcyRIII). functionally associates with the FcRy chain
(FceRiy) ( 128). suggests that this residue may be important for NKR-P 1 -mediated activation.
Similar to DAP- 12 and the CD3 subunits. FcRy possesses a negatively-charged aspartic acid
residue in its transmembrane dornain (2 17). Thus, the absence of a positively-charged amino acid
in the NKR-PIB transmembrane domain supports its inability to activate NK ceIl activity. as
association with FcRy would be undesirable for mediating inhibitory signals.
114
ITIM CxCP
cytoplasmic
mNKR9PI.A SALK niNKR-P1B LALX mNnR-PIC LALK
transmembrane extracellular
i:c" ! Syti
FcRy $$
- C = g l r PTK
Figure 33. Propoeed mdecular mechaniam for NKR-Pl-mediritced slgnding. (a) Sequenœ analysis of the mouse NKR-Pl molecules. Shown are the cytoplasrnic and transmem brane domai ns of the mouse NKR-P 1 A/B/C molecules, adapted from ( 1 20, 190). Amino acid residues of interest are shown in Md-face type, the putative trammembrane domain is boxed, and the ITIM motif of NKR-P1B is underlined. A dash represents the absence of an arnino acid in the sequence. (b) M d e l for NKR-Pl signalling. Shown are the NKR-PIC and NKR-Pli3 receptors and putative associateci signalhg molecules. Plus and minus s i p represent charged amino acid residues in the transmembrane domains; PTKase, protein tyrosine kinase domain; P f k , proiein t yrcsine phosphatase domain; SS, intermolecular disulphide bond; CRD, C-type lectin carbohydrûte recognition domain.
Taken together, these findings and those reported elsewhere (128. 190. 192) suggest a
mode1 for the mechanism of NKR-Pl signalling (Fig.33b). Association of the activating NKR-P I
family members (NKR-P 1 N C ) with the ITAM-containing FcRy chain induces tyrosine
phosphorylation of downstream substrates, including the SHZcontaining tyrosine kinase. Syk.
resulting in the activation of NK cell function. Other motifs present in the intraceltuiar domilins of
these receptors may be responsible for mediating increases in intracellular calcium concentration
and phosphatidylinositol (PI) turnover. On the other hand, phosphorylation of the cytoplasmic
ITIM motif of inhibitory NKR-Pl molecules (NKR-PIB) recruits the SH2-containing tyrosine
phosphatase, SHP- 1. resulting in the disruption of proximal tyrosine phosphorylation pathways and
inhibition of NK cell function. The signalling molecules responsible for inducing phosphorylation
of the ITAM and IT[M motifs remain unknown, although the presence of a conserved CxCP motif
in the NKR-PI molecules (Fig. 33a) suggests that Src-family tyrosine kinases may play a role in
this event (120, 190,219). Further experiments are required to elucidate the details of the molecular
cascades involved in NKR-P 1 signalling.
A potential role for NKR-Pl molecules in selflnon-self recognition
Over twenty years after its original identification ( 1 12), the mouse NK 1.1 antigen is still
incomptetely characterized. Our data demonstrate that the anti-NK 1.1 mAb recognizes the products
of two distinct mouse genes. NKR-PlB and NKR-P I C (Figure 30). Moreover. these two NKR-PI
molecules, which share a cluster of differentiation (CD) designation, CD 16 1 ( 128), possess
opposite roles in the regulation of NK cell function (Figure 3 1 b). Until now, members of the
NKR-Pl family of molecules were thoughr to be responsible for transducing activating signals
( 128, 189). Here. we show that this is not necessarily the case, and that one member, NKR-PlB
( 120. 190), acts as a KZR.
This finding highlights the increasingly similar features that these molecules share with the
116
structurally related mouse Ly-49 farnily of killer-cell lectin-like receptors (KLRs) (1 88, 189, 199).
However, unlike the well-characterized MHC class 1 ligands that regulate specificity of the Ly-49
family of molecules (199), cognate protein ligands for the NKR-PI molecules have yet to be
identified ( 190, 193). If the NKR-P 1 molecules do not recognize specific self molecules. such as
the MHC antigens, the existence of two NKR-Pl receptors with possibly identical ligands yet
opposite regulatory functions presents a potential paradox for NK cell biology. More likely. these
data suggest that the cognate ligands for the NKR-Pl molecules are similar in fonn and function to
the MHC class 1 alleles specifically recognized by members of the Ly-49 family (1 90, 199). or the
related non-classical MHC gene products such as human HLA-E (mouse H-ZQa) recognized by
the heterodimeric CD94MKG2 receptors (2 1 5 ,2 1 8,220-222).
In this regard, there is some emerging evidence that NK 1.1 (NKR-P 1 C) may be involved in
allogeneic target recognition in the NK cell-mediated F 1 -anti-parent "hybrid resistance"
phenornenon ( 190,223). The identification of a negative regulatory member of the NKR-P 1
farnily, NKR-P 1 B, now suggests a role for these molecules in the "missing self' hypothesis ( 1 96.
197). in which the perception of self versus non-self appears to rely on the presence or absence on
the target ce11 surface of MHC or MHC-like gene products ( 198). In a manner analogous to the
recent discovery of MHC class 1-related CD1 ligands recognized by the ap TCR of mouse NK1.1'
natural T cells ( 133), it seems that the variety of receptors and ligands involved in selfhon-self
recognition is sti I l expanding. Interestingly , selective expression in the Sw (this report) and SWJ
mouse strains (Richard G. Miller, personal communication) of NKR-P I B but not NKR-P 1 C may
explain the deficiency in NKI . 1 expression on the surface of ~ ~ T C R ' natural T cells derived from
these mice. This defect has been reported to segregate genetically within the NK gene complex
independently of defects in IL4 production and IgE secretion (224). These findings raise some
important questions as to the role of CD1 6 1 molecules in both mice and humans, and suggest a
previously unrecognized level of specificity in regulation of the immune response.
SUMMARY:
(Adapted from J.R. Carlyle and J-C. ZuRiga-Pflücker,
Lineage cornmitment and differentiation
of T and NK lymphocytes in the fetal mouse,
Immunological Reviews,
October 1998, Volume 165: 63-74)
SUMMARY: T AND NK LINEAGE COMMITMENT IN THE FETAL MOUSE
Lineage cornmitment and differentiation events in the fetal thymus
Thymopoiesis follows an ordered sequence of differentiation events involving the regulated
expression of numerous intracellular and membrane-bound molecules (1 -3,225). Characterization
of several surface molecules has allowed a precise staging of the cellular and molecular events
during thymocyte development. The following phenoiypic stages incorporate some of the recent
findings of early thymocyte differentiation events in the mouse fetal thymus ( 1 87). in which the
ordered appearance of each developmental stage occurs on sequential days during fetal ontogeny:
Thymic lymphoid progenitors (TLPs): ~ ~ 1 1 7 + / ~ ~ 4 4 + / ~ ~ 2 5 7 N ~ l X
The most immature hematopoietic precurson in the thymus, TLPs are capable of giving rise to B, T,
NK, and LD cells (2,3). They are characterized by high levei expression of CD 1 17 and CD44, and
the lack of expression of numerous differentiation markers, including CD25 and NK 1.1 ( 126).
Fetal T/NK progenitors (TNKs): C D ~ ~ ~ + I C D ~ ~ + / C D ~ S ' / N K ~ . ~ +
Phenotypically similar to TLPs, TNK progenitors were previously included among the TLP
population (5, 39,60, 105, 106). As the name suggests, TNK cells serve as precursors to both T
and NK cells, but lack B lymphoid potential(126, 152). These cells maintain expression of CD1 17
and CD44, and remain CD25-, but upregulate expression of NK 1.1.
Pro-T cells: C D ~ 1 7 ' 1 ~ ~ 4 4 + / ~ ~ 2 5 + / N ~ l . l -
Fully committed to the T lineage, pro-T cells possess an expression pattern resembling that of
antigen-activated mature T cells (54, 138). They display CD1 17 and C M , lack NKl . l , possess
high-level expression of CD25, and maintain their TCR gene loci in the germline configuration (43,
119
Pre-T cells: C D ~ 1 7 ~ / ~ ~ 4 4 ' / ~ ~ 2 5 + ' ~ / ~ ~ 1 ~ 1 ~
Pre-T cells have already initiated V-D-J rearrangements at the TCRP loci (48,SO). Expression of a
productive TCRP chain, together with the monomorphic pre-Ta subunit. leads to the formation of
the pre-TCR complex and allows these cells to undergo pselection (61,65,67). Pre-T cells may be
divided into early and late stages based upon CD25 expression ( 1,59,60). Early pre-T cells
maintain expression of CD25. but downregulate CD 1 17 and CD44 Late pre-T cells have already
undergone pselection, which leads to the loss of CD25 expression and differentiation to the
CD~' /CD~+ DP stage of thymocyte development ( 1 ).
Pre-NU cells: ~ ~ 1 1 7 ' / ~ ~ 4 4 + 1 ~ ~ 2 5 ' / ~ ~ 1 - 1 +
This population consists of NK lineage-committed cells, which possess at least some functional
NK ce1 1 activity ( 1 27). Al though pre-NK cells phenotypical ly resemble multipotent precursors
(classically defined as C D ~ ~ + / C D ~ S TN cells), they display NKl . I and lack expression of
CD1 17.
Importantly , al1 of the above subsets reside phenotypically among the CD3-/ClCD4-/CD8- TN
fraction of thyrnocytes, which corresponds to 55% of total aduit thymocytes. but contains the entire
population of fetal thymocytes pnor to day 16 of gestation (3). Each of the above-indicated stages,
schematically outlined in Figure 34, represents a phenotypically distinguishable subset of precursor
cells with distinct functional characteristics, many of which have been descnbed in detail elsewhere
( 1-3.8. 3 1-35). Therefore, recent insights delineating these fetal mouse precursor subsets are
discussed in further detail below.
CD117' 1 CD127+ C M ' 1 CDS' ~ ~ 2 4 ~ ~ 1 CD90+ NKl. 1- / DXS-
1 Early 1 Fetai 1 Thymic 1 Rudiment
Figure 34. Simpllficd scheme outllnlng contrd points In intmthymic T and NK lineage commltment. Indicateà are the relevant phenotypes for each thymocyte subset corresponcûng to known iineage coinmiunent steps. Each thymocyte symbot represents a specific staee in development, with its proposed name and precursor potential indicated therein. Arrows depict irreversible steps in T and NK cell differentiation. This outline represents a compilation of data derived from work perforrned in many laboratories. See text for further details.
Thymic lymphoid progenitors (TLPs)
TLPs represent the most immature hematopoietic precuaors common to the fetal and adult
thymus. Despite their residence in the thymus, TLPs lack expression of a number of markers
expressed by differentiating thymocytes. These include negative surface staining for the TCRs (ap.
y@, CD31CD4fCD8 (TN), CD2 (LFA-Z), CD25 (IL-2Ra). CD 1 22 (IL-21 15RB), and low to
negative expression of CD24 (HSA), CD90 (Thy- 1 ), CD 16/32 (FcyRIMI), CD5 (Ly- 1 ). and the
Lin markers (B220 (CD45R). Mac- l (CD 1 1 b/CD 18). Gr- 1. and Terll9) ( 127). Figure 35a shows
a representative analysis of surface expression of CD25 and CD1 17 on day 15 fetal thymocytes
before and after enrichment for ~ ~ 2 4 " cells. This analysis allows for the now classical breakdown
of TN thymocyte subsets. as shown in Figure 34.
The work outlined in Chapter 1 dernonstrates that the previously-defined TLP population, in
some mouse strains, can be phenotypically and functionally subdivided based upon expression of
NK 1 . 1 (Figure 33b) ( 126). In particular, TLPs lack expression of NK 1.1 (Figure 35b). which
identifies certain members of the NKR-P I (CD 16 1) family of type II transmembrane C-type lectin
receptors ( 1 16. 120, 190,226). This early lymphocyte lineage differentiation marker was
previously shown to be expressed by functional NK cells and a unique subset of mature T cells, but
was relatively uncharacterized during fetal ontogeny ( 1 I 1 , 1 13, 1 14, 133). Nevertheless, the use of
NK 1.1 to phenotypicall y redefine the TLP popiilation raises some new questions as to the role of
NKR-PI family members in lymphocyte lineage cornmitment. Although there are no available
antibodies to mouse NKR-PI A, this molecule has a broader distribution (-25%) on human T cells
than the NKR-PlWC gene products in the mouse (190.227). Collectively, these findings suggest
that there may be a functional role for these molecules during both T and NK ceIl development.
However, even the strict lymphoid lineage distribution of NKR-P 1 family members was recentl y
called into question by the finding that some monocytes and dendritic cells in humans also express
NKR-P 1 A (228). Nonetheless, expression of NKR-P 1 B K in mouse hematopoiesis appears to be
122
Figure 35. Ch~rricterizatiori of fetal tbymocyte subwts and enrichment of eady pmursors by depletion of lineagc-mmmitted pmgeny. (a) Fiow cytornetric analysis of surface expression of CD25 versus CD1 17 on d l 5 Fï More (Total) and after CD24-depletion (CD2do). (b) Flow cytomeuic d y s i s of surface expression of NK1.l versus CD1 17 on d l 4 and dl5 FT alier CDWCD29depletion (CD2Aio15). Colours depictex! in dot plots are intended to reflect the major ihymocyte subset contained within each quadrant, as outlined in Figure 1.
T/NK lymphocyte lineage-restricted ( 126. 127. 152, 190,226).
T/NK progenitors (TNKs)
Recent insight into the developmentally regulated expression of the NK1.l marker has
demonstrated that it may be one of the earliest markers of lymphoid lineage commitment during
fetal development ( 126, 152. 169). The TNK phenotype is not exclusive to fetal development.
however, as a srnall subset of adult C D ~ " TLPs also expresses NKI. 1 ( 1 26). As alluded to above.
NK 1.1 + fetal T/NK progenitors are phenotypically similar to their TLP counterpart (Figure 35).
However, they differ from TLPs with respect to expression of other markee found on natural kilirr
cells ( 127). For instance, fetal TNKs express Iiigher levels of CD 16/32 than TLPs. In fact.
expression of this marker was first used to define bipotent progenitors for T and NK cells in the
thymus, although CD 16/32 is also expressed by unipotent T and NU lineage-committed
progeni tors ( 1 27, 1 70). Another molecule required for NK cell development. CD 1 22 ( 1 7 1 ). is
expressed at low levels on a subset of the fetal TNK population, while surface expression of this
cytokine receptor is undetectabie on TLPs ( 1 27. 1%). In many respects, TNKs possess features in
common with NK lineage precursors. However, they lack DX5 expression and possess little RNA
for NK function-associated molecules ( 1 27, 1 52).
In general, fetal TNK progenitors express higher levels of CD90 and lower levels of CD 1 17
when compared to TLPs ( 1 27, 1 52). Additionall y, they give rise to conventional T cells with faster
kinetics than TLPs or MSCs. although they are more limited in terms of expansion potential and
they have little capacity for self renewal in vitro (unpublished observations) (126). In this regard,
TNKs express higher levels of CD 127 than TLPs ( 152). suggesting they may be more dependent
on IL-7, much like pro-T cells (4,229). Interestingly, TNK progenitors express RNA for a number
of genes associated with T lymphocyte differentiation, including GATA-3, TCF-1, RAG- 1, and
pre-Ta ( 152). As would be expected from their functional capacity , TNK progenitors maintain
124
their TCR loci in the germline configuration (152). Although untested, this implies that they may
also serve as precursors to y6 T cells and LD cells.
Pro-T cells
An early event in fetal thyrnocyte development is the thymic induction of high-level CD25
expression (5, 152), which identifies thymocytes comrnined to the T lymphocyte lineage (Figure
35a) (4.5). Although there is recent evidence that the signal required for this TCR-independent
event involves the cytokines TNF-a and IL- la (5). these are not the only signals required for
commitment to the T lymphocyte lineage. Support for this notion cornes from unpublished
observations that fetal liver-derived hematopoietic precursors do not express CD25 upon coculture
with T N F - A - la alone. This indicates that additional signals received by TLPs during their
residence in the thymus must potentiate their responsiveness to T N F - f i - 1 a. Furthemore. recent
evidence indicates that thymic epithelium is required for full commitment to the ap T lymphocyte
lineage (7, 8,57,58, 152).
Although committed to the T ceIl lineage, pro-T cells do not yet rearrange their gencs
encoding the T ce11 antigen receptors (48,49). Thus, they retain the ability to give rise to both ap
and y6 T cells (4), and may serve as precursors to LD cells andor NK cells (5 1-53). Further
experiments using sensitive in vitro assays for these lineages should determine whether high-level
CD25 expression strictly correlates with full commitment to the T lineage.
Pre-NK / fetal thymic NK cells
Recent insight into the requirements for full commitrnent to the NK iineage haî corne from
studying bipotent T/NK progenitors in the thymus and circulation, and from the observation that the
fetal thymus is also fully capable of supponing NK cell differentiation (4,5,52, 106, 126. 127,
125
152). Indeed, at least some functional NK cells develop in situ within the thymus prior to day 15 of
gestation (1 27). NK lineage-committed cells were defined by their expression of NK 1.1 and lack
of CD1 17 expression among the TN thymocyte population (Figure 35). Moreover. these cells lack
high-level CD24 expression. a fïnding that led to the observation that they phenotypically resemble
uncommitted TLPs (126, 127). For instance, in addition to their TN phenotype, pre-NK cells are
Lin' and reside within the C D ~ ~ + / C D ~ S thymocyte population, an equivocal definition still used in
numerous investigations into the effects of gene-targeted mutations on thymocyte subsets during T
cell development. In addition. they overiap with a number of putative bipotent T/NK precursor
populations. possessing a CD l 6 / 3 2 + / ~ ~ 122+/N~ 1. I + phenotype ( 107, 12 1 , 126, 149. 170). They
also display a heterogeneous but biased range of some T ce11 markers, such as CD2 and CD90
( 1 1 1 , 127). Interestingly, pre-NK cells may even lack expression of the universal NK ce11 rnarker,
DX5, which characterizes mature NK cells with cytotoxic function ( 127, 152. 178). Moreover.
pre-NK cells and even mature D X ~ + fetal thymic NK cells appear to lack expression of the Ly-49
family of genes, and thus both of these cell populations may be functionally distinct from mature
adult splenic NK cells (127. 178).
Despite their restricted lineage potential, pre-NK and mature fetal thymic NK cells possess
iremendous proliferative capacity, particularly under conditions designed to differentiate and expand
NK cells in vitro (127). This finding, especially in light of their precursor-like phenotype,
confounds many previous assessments of NK lineage potential among populations of early
thymocytes (34, 106. 107, 1 19. 12 1. 163). To this end. single-cell assays rnay represent the only
unambiguous rneans of demonstrating bipotential T/NK progenitors.
Intrathymic T and NK lineage differentiation events
In the day 12 fetal thymus, the most immature precursors seem to be phenotypically and
functionally similar to hematopoieiic stem cells (IS), and give rise to both the lymphoid and
126
myeloid lineages (96,97). However, recoverable potential for the myeloid lineage is rapidly lost
and virtually undetectable by day 13- 14 of gestation (see Chapter 1) (96. 126). The discrepancy
between these results on different days of fetal life may be due to a delayed functional maturation of
the thymic microenvironment (9, 18). Both fetal and adult TLPs possess multipotent lymphoid
lineage potential and are capable of generating B, TT NK, and LD cells (1 -3. 105, 106, 126). This
suggests either that induction of commitment to the lymphoid lineages occurs very rapidly once
circulating precursors seed the thymic microenvironment (126) or that these cells enter the thymus
as CLPs (46).
Soon after thymic entry, multipotent TLPs lose B lymphoid potential as commitment to the
fetal TNK stage occurs (see Chapter 1) (126). This stage is highlighted by the upregulation of a
number of NK ce11 markers, including CD 16/32 and NK 1.1 ( 107. 126, 170). Fetal TNK
precursors only represent about 2-3% of total fetal thymocytes (day 13 of gestation). suggesting
that this stage is very transient within the fetal thymic microenvironment in vivo ( 126). However.
antibody/cornplement-mediated depietion of more manire ~ ~ 2 4 ~ ' / ~ ~ 2 5 + thymocytes enriches for
TNK cells to about 10-208 of day 13- 14 fetal CD 1 17+ progenitors (Figure 35b) ( 126). Moreover.
the TNK phenotype becomes much more apparent in vitro. where the majority (250%) of sorted
CD 1 17+ fetal thymocytes upregulate NK 1 . 1 expression within 48 hours (see Chapter IV) ( 126.
230).
The factors required for differentiation to the TNK stage remain unknown. As
demonstrated in Chapter 1. CD 1 17' fetal TLPs generate cells with the TNK phenotype more rapidly
and to a greater extent than CD1 17' fetal liver cells upon R O C reconstitution (126). Moreover, as
shown in Chapter IV. TLPs are capable of spontaneously upregulating NKI. 1 expression when
cultured in isolation, whereas fetal liver cells remain NKI. 1- in similar culture (230). Taken
together, these findings indicate that commitment to the fetal TNK stage can be induced or
predeterminded by exposure to thymic stroma. However, because the TNK stage precedes full T
lineage cornmitment, it remains unlikely that generation of these cells is thymus-dependent. hdeed,
1 27
the identification of phenotypically and functionally identical TNK progenitors in the mouse fetal
circulation indicates that the thymus is not strictly required for differentiation to this stage (see
Chapter III) (1 52).
On the other hand. commitment to the pro-T cell stage does appear to require a thymus, or ai
least intact epithelium (8,58, 152). Evidence supporting this notion cornes from studies using
reaggregate FTOCs, which demonstrate a requirement for thymic epithelium in the induction of T
lineage differentiation and TCR remangement (7.58). Additiondl y. nude mice deficient in the
differentiation of both thymic and peripheral epithelium, fail to induce thymic expression of CD25
(unpublished observations). Moreover, as shown in Chapter III, circulating CD~O+/CD I 17"
"prothymocytes" Iack CD25 expression and do not represent T lineage-committed progenitors
(see below) ( 152). It should be pointed out that the expression of CD25 on CD1 17hi cells in the
fetal thymus suggests that corninitment to the pro-T stage rnay occur directly from TLPs. and not
necessarily from CD 1 17" TNK progenitors (Figures 34 & 35). Nevertheless, this remains
unclear, as TNK cells derived from TLPs in vitro display high level expression of CD1 17 (230).
Moreover, spontaneous upreguiation of CD25 on CD1 17' TLPs occurs to a lesser extent than that
of NK 1 . 1 (230). It is possible that the RVK stage rnay represent a defaul t pathway for T/NK
lineage differentiation from multipotent progenitors, whereas the pro-T stage rnight represent a
prirnary, thymus-dependent pathway for T lineage differentiation. Interestingly, FTOC
reconstitution experiments in Chapter I revealed that a subset of the TLP progeny coexpressed
CD 1 17. NK 1.1 . and CD25 (126). This represents a phenotype that is almost imperceptible in vivo,
but may explain recent data demonstrating NK lineage potential among CDZ+ thymocytes, which
rnay include these ~ ~ 2 5 " cells (52). Recently developed in vitro assays for NK lineage potential
should address these points more clearly.
The signals which govem comrnitment to the NK lineage are largely unknown. Stroma1 cell
coculture experiments, including those with the bone marrow-derived stroma1 ceIl line, OP9 (1 34),
indicate that full NK lineage commitment rnay not occur by default: OP9 promotes the rapid loss
128
of CD1 17 on fetal TNK cells upon short-term (48 h) coculture ( 1 26). and suppons the full
differentiation of NK lineage cells directly from embryonic stem cells in vitro (23 1 ). Moreover, in
vivo evidence of intrathymic NK ce11 differentiation during fetal ontogeny (dl 3- 15), pnor to the
appearance of mature NK cells elsewhere in the fetus, suggests an active role for elements of the
thymic microenvironment in driving NK ce11 development ( 127). One likely candidate cytokine
important for NK lineage differentiation is IL- 15 (1 7 1 ), as recent evidence supports a role for this
cytokine in mediating stroma-independent NK ce11 differentiation in vitro ( 178). IL- 15 shares with
IL-2 the intermediate affini ty receptor subunits CD 122/CD 132 (IL-2RfVy) for i ts cognate receptor
(232,233). and this receptor complex may provide a molecular explanation for the outgrowth of
NK cells from immature precursors cultured in high-dose IL-2 ( 1 19, 12 1 ). Interestingly. the OP9
celi line expresses RNA tnnscnpts for IL45 (unpublished observations), although its production
of functional cytokine remains unknown.
Notably, there are temporal differences in the kinetics of NK lineage differentiation in vitro
compared with in vivo development (unpublished observations) (127). This suggests a role for
additional factors in driving NK ceIl differentiation, some of which may be contact-dependent.
Important1 y, another difference between in vi tro-generated and fresh ex vivo NK cells indudes a
lack of expression of many inhibitory receptors in vitro in both mice and humans (178. 234).
However. expression of these receptors may be developrnentally delayed, as suggested by the low
level expression of Ly-49 gene products in fetal versus adult rnouse NK cells (unpublished
observations) ( 127). Stroma1 cells may provide a crucial role for this aspect of specificity during
NK ceil selection.
Extrathymic T/NK lineage differentiation: Redefining bbprothymocytes"
The thymus requires a continual source of hematopoietic precursors in order to sustain both
fetal and adult thymopoiesis. These precursors are thought to be predominantly multipotent and
129
must first travel via the circulation in order to seed the thymus (34). However. there is evidence that
a subset of hematopoietic precursors present in the fetal liver and adult bone marrow can
reconstitute T lymphopoiesis more rapidly than multipotent progenitors isolated from the same
regions (1 1, 17,28,46). This prompted the identification of CD~O+/CDI 17" fetal blood
"prothymocytes", which were reported to possess a restricted T lymphocyte lineage precursor
potential(34. 100). However, as shown in Chapter III, these cells possess a phenotype sirnilar to
fetal TNK cells. Thus, the majority of fetal blood prothyrnocytes also express both CD 16/32 and
NK 1 . 1 , lack expression of CD25, and do not express transcripts for CD3 ( 152). Indeed. these
circulating precursors are not stnctly T lineage-committed. as they are also capable of giving rise to
NK lineage cells (sIg'ICD37NKl. 1 + / ~ ~ 5 + progeny) (152). Therefore, the previous detection of T
lineage-committed prothymocytes in the fetal blood may have been due to contamination with recent
thymic emigrant cells, as the fetal blood was anal yzed at day 15 of gestation ( 100). two to three days
after the onset of T lineage commitment events in the fetal thymus. These findings support a
unique role for the thymus in the induction of full T lineage commitment, and suggest that
commitment to the ap T lineage does not precede thymus colonization dunng fetal ontogeny.
Figure 36 incorporates these new insights into a developmental scheme of lymphocyte lineage
cornmitment events in the fetal mouse.
As identical populations of TNK progenitors exist in the fetal blood, spleen. thymus, and
marrow ( 152) (unpublished observations), this raises questions about their origin and function.
particularly considering their paucity among fetal liver cells ( 126, 127, 152). It is possible thai
commitment of multipotent precursors to the TNK stage may occur stochastically after exit from
the fetal liver. Altematively. cornmitment may be induced prior to entry into the blood. Notably.
there is a very small but reproducible percentage of TNK progenitors in the fetal liver (0.1-0.2% of
~ ~ 2 4 " cells, which represent 4% of total fetal liver cells), but it is unclear whether they represent
fetal blood contaminants or genuine fetal liver-derived cells. Such a small percentage, though, may
actually represent quite a significant number of cells when considering the large number of cells
130
present in a single fetal liver. In any case, it is clear that commitment to the T/NK precursor stage in
development, although thymus-induced, is not thymus-dependent. Therefore, the signals required
for commitment to this stage may include soluble factors, or stroma1 elements common to the
thymus, spleen, and liver tissues in the developing fetus. This in tum raises the question of whether
this stage is specifically induced or occurs as a result of a default pathway for precursor potential in
the absence of an optimal environment for B and T lymphopoiesis. Interestingly, apart from an
undetemined NK lineage potential and incomplete phenotype with respect to NK 1.1 and CD 1 6/32.
cells similar to TNK precursors are also found in the intestinal cryptopatches in the adul t rnouse
( 184, 185). As these precursors are responsible for the generation of extrathyrnic T cells in the
intraepithelial lymphocyte (EL) cornpartment, this predicts a role for circulating TNK precursors in
establishing gut-associated T cell development.
T versus NK cell fate determination: T by design, NK by default?
Taken together, the stages of fetal lymphocyte developrnent outlined in Figure 34 and
Figure 36 suggest a hierarchy of lineage commitment events following entry into the thymus.
Multipotent precursors lose B lymphoid potential as they upregulate NK 1 . 1 and commit to the
T/NK lineages. then CD25 upregulation or CD 1 17 downregulation follow, as cells uniquely
commit to the T or NK lineages. respectively. However, there is evidence to support that this is not
always the case, and that the bipotent stage may represent a distinct lineage of cells unique to fetal
development. In particular, although NKl. 1 % ~ 1 I7+ TNK precursor thymocytes are readily
detectable early in fetal ontogeny, ihey represent a small minority of CD1 17+ cells at later stages in
development and among adult thymocytes. For instance, NKl . 1' cells decrease proportionately
during fetal developrnent and only represent about 4% of adult C D ~ " TLPs (40, 126).
Furthemore, the identical population of TNK precursors found in the early fetal circulation
disappears after day 16 of gestation and is undetectable at birth (34, 100, 152). These observations
132
rnay be explained by the possibility that TNK cells represent a limited population of cells that
proportionately decreases during ontogeny as other more mature stages increase in number and
complexity .
However, other lines of evidence that support the uniqueness of bipotent TNK cells,
uncommon with the majority of T lineage precursors, are their level of CD 1 17 expression and their
relative proportion among CD 1 17+ precursors. Although TNK progenitors are predominantly
CD1 1 71°, the majority of pro-T cells clearly display high level expression of CD1 17. an unexpected
plienotype for a descendant of an NK 1.1 +/CD 1 17'0 precursor (Figure 35) ( 1 52). Furthemore.
TNK progenitors represent a minor proportion of CD1 17' thymocytes when compared to either
CD2S TLPs or ~ ~ 2 5 ' pro-T cells (Figure 35). Thus, the NKI. I + stage may not represent a
necessary pathway during the normal course of T lineage cornmitment. Rather. the differentiation
of pro-T ce1 1s rnay occur direct1 y from CD 1 1 7hi TLPs, w hi le CD 1 1 7'' TNK progeni tors rnay
predominantly give rise to pre-NK cells in vivo. However, there is evidence to suggest that not al1
CD1 1 7hi thymus-colonizing precursor cells are necessanly multipotent precursors (34,235). and
that early immature progenitors can be CD1 17'0 (46, 236).
Another possibility is that the NKI. 1' stage rnay represent an NK lineage commitment
event from a multipotent precursor which does not preclude T lineage potential. In this case, the
decline of TNK cells dunng thymic ontogeny rnay indicate that the thymic microenvironment grows
more efficient at inducing full T lineage cornmitment from multipotent progenitors later in
development ( 1 a), yet T lineage precursors rnay still pass through a transient TNK bipotent stage.
These NK1. I + precursors may represent an evolutionary remnant of a primordial T/NK-like cell,
from which T and NK cells evolved divergently.
On the other hand, as suggested previously by numerous investigators, the TNK stage
could represent a T lineage commitment event that does not preclude a default developmental
pathway for NK ce11 potential(34. 1 15, 1 19.237). The existence of such a close lineage
relationship would avoid cell waste in the event of an inadequate microenvironment capable of fully
133
supporting T ce11 differentiation (127). Insight into this possibility cornes from the finding that the
majority of TLP cells cultured in isolation (with exogenous cytokines to maintain viability)
efficiently and spontaneously upregulates NKl . 1 expression ex vivo within 48 hours (see Chapter
IV). This effect is specific to NKI . 1 (and CD 1 6/32), as spontaneous upregulation of CD25 to a
similar extent does not occur. hportantly, the efficient upregulation of NK 1.1 does not represent
an in vitro artifact, as it does not occur with fetal liver precursors, nor with fetal blood multipotent
cells. Thus, this spontaneous cell fate appears to be predetermined by prior exposure to fetal
thyrnic stroma. Moreover, it represents a true lineage commitment event, as the resulting NKI . l +
precursors possess both T and NK precursor activity, but not B lymphoid potential, while the
remaining NK1.1- fraction can generate al1 three lineages (see Chapter IV). This suggests that a
subset of TLPs has received a thymus-induced signal that results in their lineage cornmitment and
differentiation, while the rernaining subset has not yet received this signal, despite their residency
within the thymus. Therefore, either the duration of the NK1.1' stage is proportionately
pronounced in the absence of continued thymic influence, or this phenomenon represents a default
pathway of development that occurs upon removal from an optimal T lineage environment. Either
way. the interpretation remains that the majority of multipotent TLPs, en route to becoming T
lineage-committed pro-T cells, undergo lineage commitment to a stage which does not preclude
either of the T or NK lineages. Therefore, the TNK phenotype seems to represent a true cellular
commitment pathway induced by, but not dependent on, thymic stroma. This spontaneous
phenotype change in vitro also highlights a caveat in detemining precursor/product relationships;
that is, a phenotypically homogeneous population of cells may be functionally heterogeneous.
Although this emphasizes the importance of single-cell assays, it also cautions that the results
derived from these same assays w il1 also be heterogeneous. These findings reveal a new layer to
the nature and complexity of the signals responsible for lineage cornmitment, including the ligands,
receptors, and transcription factors involved.
Interestingly, some recent advances have been made already regarding the molecular
134
mechanisms for lymphocyte lineage commitment. For example, the Notc h receptor famil y has
recently k e n implicated in the control of both aB versus y6 as well as CD4 versus CD8 T cell
lineage commitment in the thymus (68,69,93,94). Furthemore, an inhibitory transcription factor.
Id3, was recently identified to have a role in T versus NK lineage commitment (237). Id3
represents a naturally occumng dominant-negative fom of the basic helix-loop-helix (bHLH)
family of transcription factors, known as Id (inhibitor of DNA binding) proteins. Expression of
selected bHLH family memben is required for B and T ce11 development and the Id proteins may
provide a natural molecular switch to block development of these lymphocyte lineages and perhaps
augment others. To this end, constitutive expression of Id3 in human precursor thymocytes
cultured in FTOC blocks T ce11 development and results in an apparent promotion of NK ce11
development. Thus, the Id family of proteins provide the first molecular evidence of enforced
lineage commitment from one lymphoid cell fate, predetermined by expression of a transcription
factor. to an altemate default pathway of lymphoid differentiation. induced by blockade of
transcription factor activity. This lends support io the notion that control of ce11 fate detemination
between the T and NK lymphocyte lineages involves an active recruitment to the T ce11 destiny,
without precluding a default NK pathway for differentiation of bipotent precursors. However, the
factors that control restriction to a bipotent cell fate and how the putative Id default pathway is
invoked remain a matter of speculation at present.
"The NK1.l antigen", 20 years later
During our initial investigation of TNK progenitors. we examined two NK 1 . I -expressing
strains. B6 and Sw. Surprisingly, although circulating progenitors from both strains were found to
express high levels of CDl6/32, those derived from B6 mice express low to negative staining for
NK 1. I (Figure 28). This suggested either that the precursors from the two strains were
fùnctionally distinct, that the developmental regulation of NKR-PIC (the ligand for NK1.1 (1 16.
135
120)) was disparate, or that NKl . 1 recognized a different ligand in each of the strains.
Nonetheless, both Sw and B6 fetal blood TNK cells are capable of giving rise to T and NK cells in
vitro. and the phenotypes of the two populations are othenvise identical (unpublished observations)
(152, 187).
As outlined in Chapter V, it now appears that the mAb to the NK 1.1 antigen, PK 136.
actually recognizes the products of at least two distinct genes (1 16, 120.226). This is not
surprising in retrospect, given that two of these genes, NKR-P1B and NKR-PIC. are approximately
96% identical in their extracellular C-type lectin domains (1 16, 120, 190,226). What is more
surprising is that these two "NKl. 1 antigens" appear to possess opposite roles in the regulation of
NK cell function (190.226). Thus, the NKR-PI family members appear to be sirnilar in both fom
and function to the Ly-49 gene family of NK ceIl receptors ( 162, 188, 190, 199.238.239). In
keeping with this, the demonstration that NKR-PIB associates with the SH2-containing tyrosine
phosphatase, SHP- 1 (205), provides a mechanism for inhibition through this receptor and
demonstrates that elements of its signalling pathway are likely shared with that of the inhibitory
Ly-49 molecules (226).
However, in contrast to the well-known MHC class I ligands recognized by the Ly-49
receptors ( 162, 190, 199,238,240). the cognate protein ligands for the NKR-PI molecules remain
unknown ( 190, 193, 194). Nonetheless, the expression of two nearly identical receptors with
opposite regulatory function on the NK ceIl surface predicts a role for the NKR-Pl receptors in
mediating selfInon-self recognition in the immune system (226). ~viden'ce is already gathering that
these molecules may be involved in NK cell-mediated allorecognition, including the well-known
FI -anti-parent "hybrid resistance" phenornenon (223). The identification of a negative regulatory
rnember of the NKR-Pl family, NKR-PlB, now suggests a role for these molecules in the
"missing self' hypothesis ( 196, 197), in which the perception of self versus non-self appears to
rely on the presence or absence on the target ceIl surface of polymorphic MHC class 1 alleles (1 98).
Furthemore, these findings raise the possibility that the cognate ligands for the NKR-Pl molecules
136
are similar in form and function to MHC class 1 or related gene products. Indeed, recent evidence
has identified products of the HLA-E region as the cognate receptors for another related family of
KTRs/KARs in humans. the CD94/NKG2 complex (2 18,22 1,222,234). In a manner analogous to
the recent discovery of MHC class 1-related CD 1 ligands recognized by the ap TCR of mouse
NKI . l + T cells (133), it seems that the variety of receptors involved in NK cell-niediated
sel fhon-self recognition is still expanding .
More than twenty years after its original identification, "the NK 1 . 1 antigen" is still
incornpletely charactenzed. The identification of protein ligands for the NKR-Pl receptors should
elucidate the functions of these elusive molecules so long identified only as the target of an
aniibody on an enigmaiic ce11 type.
EXPERIMENTAL PROCEDURES
Timed-pregnant C57BV6, Swiss.NIH. and athymic nude (nuhu) mice were obtained from
the National Cancer Institute, Frederick Cancer Research and Development Center (Frederick.
MD). RAG-2-'- mice and (C57B1/6xSwis~.NIH)Fl were bred and maintained in our animal
facility.
Isolation of fetal cells
Intact uteruses were removed from timed-pregnant mice and washed 2 times with
Ca/Mg-free PBS to remove materna1 blood. Fetuses were removed from the uterus without
intempting the umbilical cord, and washed briefly with R O C medium (DMEM medium
supplemented with 12% FCS, 2 m M glutamine. 10 Ulm1 penicillin. 100 pglml streptornycin. 100
pglml gentamicin, 1 1 O pg/ml sodium pyruvate, 50 PM 2-mercaptoethanol, and 10 m M HEPES. pH
7.4) prior to isolation of fetal tissues. To obtain fetal blood, fetuses were separated from the
placenta. placed in medium plus 1UIml lithium heparin, and the jugular veins and cervical aneries
were severed using micro-dissection forceps and scissors. Fetuses were allowed to exsanguinate
for approximately 10 minutes. after which they were removed for further dissection. Fetal blood
(FB. in heparinized medium) was filtered once through nylon mesh. and low buoyant density cells
were enriched by discontinuous density gradient centrifugation using Lympholyte-Mammal or
Lympholyte-M (Cedar Lane. Homby. ON). Fetal thymus (FT), fetal liver (FL), and fetal spleen
(FS) tissues were subsequently harvested. washed twice in medium to remove any contaminating
fetal blood, and disrupted through 70 pm nylon mesh using a syringe plunger. Al1 fetal cells were
then washed. resuspended in medium. and viable cells were recovered using Lympholyte.
138
~ ~ 2 4 ' 0 1 ~ ~ 2 5 ' fetal cells were obtained by antibody- and cornplement-mediated lysis. Briefly.
50-500 pl of anti-CD24 (J 1 1 d.2) and anti-CD25 (7D4) culture supernatant and a 1/10 dilution of
Low-Tox rabbit complement (Cedar Lane) were added to single-ce11 suspension in 2-3 ml medium.
and cells were incubated at 37OC for 30 minutes. After incubation, viable cells were recovered using
Lympholyte and washed pnor to analysis.
Flow cytometric analysis and cell sorting
Single-cell suspensions were stained for surface expression of various markers using
FiTC-. Cychrome-, APC-, PE-, or Red-61 3-conjugated mAbs obtained from Pharmingen (San
Diego, CA) or Gibco-BRL (Bethesda. MD), respectively. in staining buffer (Hank's balanced salt
solution (HBSS) with 1 % BSA and 0.05% NaN3). Cells were stained in 100 pl for 30 min on ice
and washed twice prior to analysis. Intracellular staining was performed as directed by the supplier
using the Cytofix/Cytoperm staining kit (Pharmingen). Stained cells were analyzed with a
FACScan or FacsCalibur flow cytometer using Lysis U or CellQuest software (Becton Dickinson,
Mountain View, CA); data was live-gated by fonvardhide light scatter and lack of propidium iodide
uptake (except PI was omitted for intracellular stains). AH contour plots display 10,000 events
contoured at 50% (log), except control dG-FTOC populations, which show al1 events: dot plots
show 5.000-20.000 events. Events contained in each quadrant are given as percentages in the upper
right corner. For cell sorting, single-cell suspensions were prepared andstained for FACS as
described above. except that no NaN3 was added to staining buffer. Cells were sorted using a
Coulter Elite cytorneter (Hialeah, FL); sorted cells were 98-99% pure, as determined by post-sort
analysis. Staining was not altered in the presence of blocking FcyRlyIII antibody (2.4G2).
Fetal thymic organ culture (FTOC) reconstitution
Lymphocyte-depleted thymic lobes were prepared by culturing day 15 fetal thymic lobes
frorn timed-pregnant Swiss mice in JTOC medium containing 1.00- 1.35 rnM 2-deoxyguanosine
(dG), as previously described ( 1 30, 13 1 ). Briefly , host dG-treated FïOCs were cd tured for 5-6
days, dG-containing medium was replaced with FTOC medium for one day, then lobes were rinsed
twice, resuspended in 10 pl medium, and placed in Terasaki plates at two lobes (one thymus) per
well. Sorted donor cells were washed twice with medium, resuspended at 10'-3x IO" cells in 20 pl
medium, and added to dGuo-treated alymphoid fetal thymic lobes in Terasaki plates. After adding
donor cells or medium alone, the plates were inverted ("hanging drop") and cultures were
incubated at 37°C in a humidified incubator containing 5% CO2 in air for 24-48 hrs. Lobes were
then transferred to standard FTOC for 10-12 days, except where indicated othenvise. Ce11
suspensions from reconstituted thymic lobes were analyzed by flow cytometry. Reconstituted
dG-FTOC lymphoid cells were >98% donor-derived as detemined by ffow cytometric analysis for
donor-specific MHC class 1 expression. Reconstitution of host dG-treated FTOC was consistently
successful only if 530 donor cells were used. Cell yields for each experiment using 1 x 10 3
precursors typically ranged from 1-5 x 1 o4 cellsAobe.
In vivo adoptive transfer
CD24KD25-depleted day 15 fetal thymocytes from Swiss mice were sorted for
NK 1 . 1 -/cD~@ (CD 1 1 7 7 and NK 1.1 + / ~ ~ 4 4 + (CD 1 17-) cells. Sorted cells ( 1 O' of each) were
washed twice and resuspended in 300 )il of culture medium, then injected into the tail vein of
sublethally-irradiated (750 cGy) adult RAG-2-'' mice. Mice were sacrificed by cervical dislocation
3 weeks later and tissues were harvested for analysis. Single-ce11 suspensions of spleen, thymus,
lymph node, and bone marrow were analyzed by flow cytornetry.
140
Cell lines
The OP9 bone marrow-derived stromal ceIl line was obtained from Dr. Tom Nakano
(Osaka University). The YTS-191 (a-CD4) and YTS-169 (a-CDS) hybridomas were obtained
from Dr. Brian Barber (University of Toronto). The 2.4G2 (a-CD161321 hybridoma was obtained
from Dr. Michael Julius (University of Toronto). The J1 ld.2 (a-CD24). 7D4 (a-CD25). and
PK 136 (a-NK 1 .1) hybridomas were obtained from the American Type Tissue Culture (ATCC)
collection. ELA, YAC-1. and P8 15 target cells were obtained from Dr. Brian Barber (University of
Toronto). Jurkat cells were obtained from Dr. Neil Bennstein (University of Toronto). The Sw-
derived MNK- I pre-NK cell line was derived in our own laboratory. Briefly, day 15 fetal
th y moc ytes were sorted for NK 1 . I + ce1 1s. infecied ovemight wi th a Bcl-2 retroviral producer ce1 1
line, then transfected by electroporation with linearized constnicts encoding a replication-deficient
SV40 genome and a human c-myc cDNA. Cells were then maintained in R O C medium plus
recombinant human L-2 ( 10 Ulml) for 6 weeks before adherent colonies formed. Phenotypic
analysis indicates that the resulting MNK- 1 cell line possesses a pre-NK ce11 phenotype.
OP9 stromal cell line coculture
Sorted donor cells were prepared as described above. In parallel with FTOC
reconstitutions, donor cells ( I -3x 1 o3 of each) were cocultured for 7- 1 1 days on confluent
monolayers of OP9 cells ( 134, 135) in R O C medium containing IL-3, IL-6, IL-7, and SCF (50
@ml of each cytokine), then stimulated with LPS ( 1 0 @ml) and IL-7 (and IL-2, where indicated)
for 4-6 days prior to harvesting for flow cytometry. Cells and culture supernatant were then
harvested for fiow cytometry and ELISA analysis, respectively. ELISA analysis (136). with a
sensitivity of 220 nglml of sIgM. revealed the presence of sIgM from the supernatant of FL and
FïLP but not from the FTNK cocultures. Cell yields from OP9 coculture experiments with 1 - 10
x 10' FlZP or FTNK cells showed a clear difference in total number of lymphocytes recovered
after 7 day cultures, Le. pnor to LPS activation; moreover, ceIl yields were typically 2100 fold
higher in FTLPlOP9 than in FïNWOP9 cocultures after four days of LPS activation. This
increase is due to presence of LPS responsive B-lineage cells in the FTLP/OP9 cocultures and the
tkeir absence in the FTNKfOP9 cocultures.
In vitro cell culture
Sorted NK 1.1-/CD 1 17+ cells (on ice) were washed twice and cultured in FïOC medium in
96-well round bottom plates at lo4 - 5 x 105 cellslwell at 37°C in a hurnidified incubator containing
5% CO,. Where indicated, the cytokines IL-3, IL-6, IL-7, and SCF were added (50 ng1m.L each - cytokine) to wells. Cocultures with fibroblasts or OP9 cells were perforrned by adding sorted
precursors to wells containing confluent layers of stroma1 cells. RNA polymerase 11 transcriptional
inhibition was achieved by adding a-amanitin ( IO pg/mL) (Boehringer Mannheim, Indianapolis,
IN) to wells immediately prior to incubation ai 37OC. PMA (10 ng/mL) (Sigma, St. Louis. MO)
and ionomycin ( 1 ng/rnL) (Sigma) were added immediately prior to incubation at 37OC.
CFSE vital dye labelling
5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes,
Eugene. OR) was a kind gift of Dr. Parnela Ohashi (Toronto, ON). Cells were labelled with CFSE,
as previously described ( 1 86). Briefly. cells were washed in HBSS to remove protein, and
resuspended in HBSS plus CFSE (final concentration, 0.5 pM; stock CFSE, 0.5 mM in DMSO).
Cells were labelled for 10 minutes at 37°C. then washed three times in complete medium pior to
culture. Control cultures for staining calibration and compensation were camied out in paralle1 by
incubating cells at 4OC. After incubation, cells were prepared and analyzed by flow cytometry.
142
Relative CFSE fluorescence was monitored in the FLl parameter on a FacsCalibur flow cytometer.
"~r-release cell-mediated cytotoxieity assay
Single-ce11 suspensions from freshly-isolated day 15 fetal thymocytes from timed-pregnant
C57BU6 mice and adult RAG-~-'- rnice were sorted for a ~ ~ 3 7 ~ ~ 9 0 ' (Thy-1 ) phenotype with or
without NK 1. I expression. Sorted cells were assayed for cytolytic activity using a standard
' Cr-release assay ( 1 14). Soned NK 1.1 '/CD~O+/CD~- or NK 1.1 -/cD~o+/cD~- ce1 1s were
washed twice, and aliquoted at different effector to target ratios in 100 pl of culture medium
(DMEM medium supplemented with 1 2% FCS, 2 mM glutamine, 10 U/ml penicil lin, 100 p g / d
streptomycin, 100 @ml gentamicin, 1 10 pgml sodium pyruvate, 50 $VI 2-mercaptoethanol. and 10
m M HEPES, pH 7.4). Target YAC- I or E U cells were labelled with for 1 hour and used ai
3x 1 o3 cells in 100 pl per well (U-bottom, 96 well plates). Cells were mixed at the indicated
effectortarget (E:T) ratios. then plates were centrifuged for 30 seconds and placed in culture for 4
hours at 37OC. 100 pl of culture supernatant were collected and rneasured in a gamma-counter.
Supernatant from tiuget cells cultured alone or target cells plus 1 % SDS gave the spontaneous or
maximal release counts, respectively. Spontaneous release was < 10% of maximal release. Counts
obtained from culture supematanis at different E:T ratios (experimental release) were used to
determine percent specific iysis, as previously described ( 1 14).
5'~r-release antibody-induced redireeted lysis (AIRL) assay
Spleen cell suspensions from B6, S w, and (B6xSw)F 1 mice were depleted C D ~ + , C D ~ + ,
C D ~ + , and ~ ~ 2 4 + cells by antibody/complement-mediated lysis, then sorted for DXS+ cells using
Midi-MACS (Miltenyi Biotec Inc., Auburn, CA). Sorted cells were grown for one week in R O C
medium containing 100 U/ml of recombinant human IL-2. Effector cells were pre-labelled with 10
1 43
pg/ml antibody (anti-NK 1.1. PK 136; anti-CD 16/32.2.462) for 15 min in complete medium. then
washed once prior to use. Target P815 cells were labelled with " ~ r for 1 h and used at 2-3 X 10'
cells in 100 fl per well (V-bottom, 96 well plates). Cells were mixed at different effectortarget
ratios (ET), and plates were centrifuged for 1 min at 200xg and placed in culture~for 4 hrs at 37°C.
After lysis, 100 pl of each culnire supernatant was collected and radioactivity was measured in a
gamma-counter. Supernatant from target cells alone or target cells plus 1% SDS gave the
spontaneous or maximal release counts, respectively. Percent specific lysis (%SL) values were
detemined as previously described ( 1 14). Percent specific lysis values for anti-NK 1 . 1 -mediated
AIRL were indexed relative to those for anti-CD 1 6/32 AIRL (anti-FcyRmm) and spontaneous
lysis (no antibody) controls for each effector subset and ratio tested, according to the formula: Cï%
NK 1 . 1 cytotoxicity = [%SL(NK 1 . 1 ) - %SL(no mAb)] / [%SL(CD 16/32) - %SL(no mAb)] x
100%. Data shown represent the mean (*SEM) of 5 independent experiments performed at a 30: 1
E:T ratio (sirnilar results were obtained with 10: 1 and 3: 1 E:T ratios).
RT-PCR analysis
Single ceIl suspensions were prepared as described above. Total RNA was obtained from
ceil pellets using the Trizol RNA isolation protocol (Gibco-BRL). RNA was resuspended in 25 pi
DEPC-treated (O. 1 %) dH,O and residual genomic DNA was digested using RNAse-free DNAse - (Boehringer Mannheim, Indianapolis, IN). RNA was re-extracted using the Trizol protocol and
resuspended in 25 pl dieth y l py rocarbonate (DEPC)-treated dH,O. cDN A was prepared from 1 pg - of each RNA using random hexamer primers and the cDNA Cycle kit (Invitrogen, San Diego, CA).
Subsequent PCR analysis was performed using titrations of cDNA in a 1/5 dilution series in
dH,O. dH,O and RT reactions done in the absence of avian myeloblastosis virus (AMV) reverse - - transcriptase were included as negative controls. PCR was perfomed on an automated GeneAmp
9600 thennocycler (Perkin Elmer, Nonvalk, Ci') using 20-30 seconds denaturation at 94OC. 30-45
seconds annealing at 50-55"C, and 30-60 seconds extension at 72°C for 32-35 cycles. with a hot
start at 94°C for 2 minutes and a final extension at 72°C for 6 minutes, using annealing
temperatures specific for primer pairs as detemiined using the OJ,IGO program (NB1 Software).
Ali PCR reactions were perfonned using the same cDNA batches as shown for pactin. and al1 PCR
products correspond to the expected molecular sizes. Gene-specific primers used for PCR are as
follows (5'->3'): P-actin (5 ' ) . GAT GAC GAT ATC GCT GCG CTG; P-actin (3'). GTA CGA
CCA GAG GCA TAC AGG; NKR-Pi A/B/C (genes 2.34.40; 5')' AAG G T ( m ) CAC ATT GCC
AGA CAT; NKR-P 1 A (gene 2; 3'). GTA GAC ATG GCT CAG TGA 'ITG; NKR-P 1 B (gene 34:
Y), GGA CAG GGG AGA GAT GGA GAT; NKR-PIC (gene 40; 3')' GAG TCA ACG AAT
GGA AAG GAA; Ly-49A (5 ' ) , TTC TGC 'ITC CTT CIT C'TG GTA: Ly-49.4 (3'), TGT G7T
CAA GGC AAG m AGA; L+C ( 5 7 , AGA CCA GAA AAA CGC CAA CTT: L Y - 4 9 ~ (37.
TTC ACT G ï T CCA TCT GTC CTG; perforin ( 5 7 , ATG T C CCC AGT CGT GAG AGG;
perforin (3'), AAG GTG GAG TGG AGG T'TT TïG; CD95L (Fas-ligand; 5'). AAG AGA ACA
GGA GAA ATG GTG; CD95L (39, AGA TTT GTG TTG TGG TCC TTC; Ikaros ( 5 7 , CGT
GGA TAA CTï GCT GCT GCT; lkaros (3'), CAA ACG CCA AAC AAC CAA CAT; GATA-3
(Sv), CTC CTT TTT GCT CTC C'IT TTC TAT; GATA-3 (3'). ACA TTT TGC rlrlrCr CTG CCT
TCA; TCF-1 ( 5 ' ) , GGA GCA CAC TTC GCA GAG ACT TTT; TCF- 1 (3'), TTG GAG ACT
TAG GGG CTG GAA; TCR Cp (5 ' ) . GAG GAT CTG AGA AAT GTG ACT; TCR Cp (37, TTT
CTT TTG ACC ATA GCC ATC; pre-Ta ( 5 9 , CAG AGC CTC CTC CCC CAA CAG; pre-Ta
(3'). GCT CAG AGG GGT GGG TAA GAT; C D ~ E (Y), ACT TGC CAG GAC GAT GCC GAG
A; CD3e (3')' TGC GGA TGG GCT CAT AGT CTG G; RAG-l(5'). TGC AGA CAT TCT AGC
ACT CTG G; RAG- 1 (3'). ACA TCT GCC 'ITC ACG TCG AT; RAG-2 (5') ' CAC ATC CAC
AAG CAG GAA GTA CAC; RAG-2 (3'), TCC CTC GAC TAT ACA CCA CGT CAA; Lck (5 ' ) ,
CAT TCC CTï CAA CTï CGT GG; Lck (3'). TAA TGG CGG ACT AGA TCG TG; IL-7Ra
(57, AGC AAG GGG TGA AAG CAA CTG; IL-7Ra (3')' AGG AAA GGA GTG GAG GGG
CAT; IL-1 SRa (57, AGG CTG ACA CCA TCC CAA ACA; IL- 1SRa (37, TCT TCA TCC TCC
145
ï T G CTG CTG (127. 148, 177,241.242). Products were separated by agarose gel electrophoresis
on a 1.6% gel. and visualized by ethidium bromide staining; reverse photo images are shown.
RT-PCR for cDNA cloning
RNA and cDNA were prepared as described above. PCR was perfonned using Expand
DNA polymerase (Boehringer Mannheim, Indianapolis, IN) with 10 seconds denaturation at 94°C.
30 seconds annealing at 55°C. and 2 minute extensions at 72°C for 35 cycles, with a hot start at
94°C for 2 minutes and a final extension at 72OC for 6 minutes. Gene-specific primers used for
PCR cloning are as follows (5'->3'): NKR-PI A (5 ' ) . GCA CAA TGG ACA CAG CAA:
NKR-P 1 A (3'), GTA GAC ATG GCT CAG TGA T'TG; NKR-P I B (Sv), CAA TGG ATT CAA
CAA CAC TGG TC: NKR-PI B (3'), GGA CAG GGG AGA GAT GGA GAT; NKR-P 1 C ( 5 ' ) ,
TGA AAT GGA CAC AGC AAG TAT C; NKR-PIC (3')' GAG TCA ACG AAT GGA AAG
GAA. PCR products were cloned using the Eukaryotic TOPO TA cloning kit (Invitrogen. San
Diego. CA) and sequenced prior io use in transfections.
Genomic PCR
Single-cell suspensions were prepared from d 14- 15 fetal thymus, d 15- 16 fetal blood and
spleen. and adult RAG-~-'- thymocytes. For thyrnocytes, total (unsorted) cells were used for PCR
analysis. For remaining sarnples, ~ ~ 2 4 " cells were prepared as describe above and sorted for a
CD I 1 ~+/cD~-/DxS phenotype, gated on either NK 1 .1+ or C D ~ O + cells. as indicated. Genomic
DNA was prepared using an EasyDNA kit (Invitrogen). 100 ng of each DNA sarnple was
amplified using an automated thennocycler (Perkin Elmer). Primers used for the PCR analysis
have been previously described (100). Products were separated by agarose gel electrophoresis,
transferred by Southern blotting ont0 nitrocellulose filters (Nytran), and visualized using an -800
146
bp (Jp) genomic probe. Al1 PCR products correspond to expected molecular sizes.
Plasmid DNA transfections
Jurkat cells were electroporated using a BTX (San Diego, CA) ECM600, using the
following conditions: 300 V, 186 n, 1600 @, in 4 mm gap cuvettes. Cells were resuspended in
250 pl of RPMI with 20% FCS prior to electroporation, then cotransfected with 20 pg of NKR-Pl
plasmid DNA, as indicated, and with 6 pg of the GFP plasmid DNA. Transfected cells were then
cultured for an additional 6 hours prior to analysis of expression by flow cytometry.
Immunoprecipitation and Western blotting
50- 1 ûûx 1 o6 MNK- I cells were left unstimulated or stimulated with pervanadate for 20 min.
as described previously (2 16), and lysed for 15 min in 0.5 ml ice cold lysis buffer (0.5% TNTE
[OS% Triton X- 100, 150 m M NaCl, 20 m M Tris-CI (pH 7.4), I m M EDTA] plus 50 mM NaF, 10
m M sodium pyrophosphate. 1 mM sodium orthovanadate, and protease inhibitors [pepstatin,
leupeptin, aprotinin, trypsin inhibitor]). Lysates were centrifuged at 14,000xg for 1 O minutes to
remove ce11 debris, then 5 pg anti-NK 1 . 1 (PK 136) or isotype control rnouse IgG2ali (anti-TNP,
G 1 55- 1 78) antibody were added to pre-cleared lysates and incubated with gentle agitation for 1 h.
50 pl of protein G sepharose beads ( 1 :5 [wlv] in O. 1 % Triton lysis buffer) were added to lysates
and incubated further for 1 h with gentle agitation. Beads were washed 6 times with 1 ml wash
buffer (O. 1 % TNTE plus 1 m M sodium orthovanadate). Beads and lysate supernatant were boiled
for 2 minutes in protein loading buffer containing 2-mercaptoethanol, and proteins were resolved on
10% SDS-PAGE gels and eiectroblotied ont0 membranes. Membranes were blocked with 3%
BSA in TBST (50 mM Tris-CI [pH 8.0],500 m M NaCI, O. 1 % Tween 20) for 1 hour. and analyzed
by Western blotting using polyclonal rabbit anti-SHP- 1 antibody (Upstate Biotechnology
147
Incorporated, Lake Placid, NY), affinity-punfied rabbit anti-SHP-2 antibody (Santa Cruz
Biotechnology, Santa Cruz. CA), or rabbit anti-SHP antibody, a kind gift of Dr. D.J. Dumont
(Toronto) at 1 : 2 0 dilutions in 1 % BSA-TBST. Ali blots were visualized using 1 : 10,000 anti-
rabbit-HRP in 1 % BSA-TBST and an ECLPlus kit (Arnersham, Arlington Heights. IL).
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