Development
Dec 24, 2015
Where do T cells come from?
1. Multipotent Lymphoid Progenitors Migrate from the Bone Marrow to the Thymus
2. In the Thymus, the Lymphoid Progenitors Differentiate to pre-T Cells and are Educated to Differentiate Self from Non-self
3. Positively Selected T Cells Emigrate from the Thymus to Mediate and Effect the Cognate Immune Response
Reciprocal Regulation
Pax-5 induces the expressionOf B lineage genes (CD19, BLNK)And represses the expression of Notch
But really location controls the T cell versus B cell Lineage Choice
So what controls homing of CLPs to the thymus?
And what controls the expression of these homing receptors?
DN1 Cells in the Thymus Are Not Committed to become T Lymphocytes
• TCR loci are in germline configuration
• Cell can differentiate to become a B lymphocyte, Natural Killer cell, or Dendritic cell (Michie et al, JI, 2000; Ikawa et al, JEM, 1999;Sanchez et al, JEM, 1994)
The DN2 Subset of Thymocytes are More Committed, but Not Quite Exclusive
• TCR loci are in germline configuration
• DN2 thymocytes may give rise to dendritic cell(Moore et al, 1995; Wu et al 1996; Ardavin et al, Nature 1993; Shortman et al, Imm Rev. 1998)
• DN2 thymocytes no longer differentiate to the NK cell or B lymphocyte lineages
DN3 Cells are Committed to the T Lineage
• Downregulation of CD44 expression
• Upregulation of RAG genes
• V-D-J recombination of TCR chain locus
• Expression of pre-TCR chain
The pre-TCR chain (DN3)
• Invariant Type I TM protein
• Contains only one Ig Domain
• Physically associated with TCR chain
• Signaling appears to be ligand-independent– TCR chain in this complex
lacking extracellular domain is sufficient to allow progression to DP stage
From Fundamental Immunology, 4th ed. (Paul)
After Assembly of a Functional pre-TCR
• Shut down of TCR rearrangement; TCR allelic exclusion
• Onset of proliferation/expansion
• Differentiation to DN4, CD8ISP, and then DP
Three Models of vs. Lineage Determination
• Earliest (fetal) waves of T cells are mostly
• Later production is skewed toward lineage
• Conflicting Data Supporting Each Model
• Mature T cells have rearranged locus; ’s have rearranged locus
• Even DN4 cells (which have selected a chain) can become lymphocytes in transfer experiments
From Fundamental Immunology, 4th ed. (Paul)
V(D)J Recombination in developing T cells
• chain occurs first (DN3 stage) – VDJ• chain occurs second – VJ, no D region• Allelic exclusion• Combinatory diversity of the adaptive immune system
• This is a big problem
Positive Selection: Host MHC determines CTL immune responsiveness (Bevan, Nature, 1977)
d(with peptides) b(with peptides) B10(H-2d) B10(H-2b)H-2d/b BM -> H-2b/d +++ +++H-2d/b BM -> H-2d +++ +H-2d/b BM -> H-2b + +++
Balb/c H-2d/b BM -> H-2b/d Balb/c H-2d/b BM -> H-2d Balb/c H-2d/b BM -> H-2b
immunize with B10H-2d/b splenocytesrestim in vitro look for MHC restricted cytotoxicity
Evidence for MHC Restriction of CD4+ and CD8+ T Lymphocytes is Apparent in MHC KO Mice
CD4
CD8
(Grusby et al, PNAS, 1993)
Negative SelectionHY TCR Tg is negatively selected by a male specific
antigen
HY
TC
R fe
mal
eH
Y T
CR
Mal
e
(Kisielow et al, Nature, 1988)
Disruption of the ERK Pathway Interferes With Positive but not Negative Selection (an effect on
differentiation and proliferation?
• Also:– DN Mek1
• Alberola-Ila et al, 1995• Alberola-Ila et al, 1996
– DN p21ras
• Swan et al, 1995• Alberola-Ila et al, 1996
– DN Raf• O’Shea et al, 1996
– RasGRP• Dower et al, 2000
– Indirect (ERK Induction from CD3 chain)
• Werlen et al, 2000• Delgado et al, 2000
ERK1 ERK1
Pages et al 1999 Science 286:1376
Cnb1deficient and Control Thymocytes are Equally Sensitive to TCR Crosslinking and
Peptide Complexed with MHC
MCC-Mediated Cell Death dose response
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100Cnbf/
Lck-Cre+ Cnbf/
concentration MCC peptide [2 X 10 X M]
%P
I-A
V-D
P t
hym
ocy
tes
rela
tive
to
co
ntr
ol
Neilson et al, 2004
Via
bili
ty
Which downstream pathways contribute to thymocyte selection?
(positive selection)(positive selection)
Active Rac1 changes positive to negative selection
Female Female/active Rac1
(Gomez et al, Immunity, 2001)
Data = Explanation?• Synapse during negative selection (Richie et al, Immunity, 2002)
• Synapse formation in peripheral T cells requires Vav/actin dynamics (Holsinger et al, Curr. Bio, 1998; Wulfing et al, PNAS, 2000)
• Positive selection likely does not form a synapse (Peter
Ebert unpublished)
• Therefore, lacking Vav/Rac etc. changes negative to positive selection via an effect on synapse formation and subsequent signaling
Which downstream pathways contribute to thymocyte selection?
(positive selection)(positive selection) (Modulator
Both)
Negative Selection is Impaired in DP Thymocytes Deficient in the Pro-Apoptotic Bcl-2 Family Member Bim
Bouillet et al 2002 Nature 618:108
Modulation of lck activity can alter CD4/8 lineage commitment (based on the fact that the cytoplasmic tail of CD4 binds to
more lck than CD8)
Dn-lck Act-lck
Other studies indicate that the duration of signal controls CD4 versus CD8 with CD4 requiring longer(Yasutomo et al, Nature, 2000)
Regulatory T cells may develop in the Thymus
HA specific TCR Transgenic mouseMouse expressing HA peptide
TCR Tg CD25
What a DP Thymocyte Needs to Progress to the SP Stage
• Everything it Needed to Become DP
• Functional TCR chain rearrangement
• CD4 and MHC II (To be a CD4+ cell)
• CD8, MHC I and TAP (To be a CD8+ cell)
• ERK signaling
• Calcineurin signaling
T Lymphocyte Maturation after leaving the Thymus
Some evidence that cells that haverecently left the thymus may have special “status” in the periphery in regards to tolerance induction or the ability to homeostatically proliferate
Antigen presentation and MHC expression control thymocyte selection
•DC•Medullary Epithelial cells•Cortical Epithelial cells•Endothelial cells
DO CERTAIN CELL TYPE CONTROL DIFFERENT T CELL DEVELOMENTAL FATES?-Positive selection-Negative selection-Receptor editing
Aire-/- mice develop autoimmunity because Aire Mediates
Ectopic Gene Expression in Medullary Stroma
Ohashi 2002 Science, 298:1348
The Transcription Factor Autoimmune Regulator (Aire) Mediates Ectopic Gene
Expression in the Thymic Medullary Stroma
Ohashi 2002 Science, 298:1348
Take Home Messages• Notch steers CLP’s to T lymphocyte Fate• TCR chains undergo V(D)J recombination to generate
diversity; they also exhibit allelic exclusion– TCR chain is selected with an invariant pT chain at the DN3
stage– TCR chain is selected with pre-existing TCR chain at the DP
stage
• DP Thymocytes Undergo Positive and Negative Selection to Generate a Population of Mature T Lymphocytes that can Recognize Self MHC with Intermediate Affinity
• Recent Evidence Indicates that Positive and Negative Selection Are Mediated by Distinct Pathways
• APC in the thymus may present self antigen that effect negative selection
T Lymphocyte Development and Function is Controlled at Various Stages of Development
by Signals through the T Cell Receptor