Regulation of T cell activation and tolerance by PDL2Yongliang
Zhang*, Yeonseok Chung*, Caroline Bishop†, Betsy Daugherty‡, Hilary
Chute‡, Paige Holst‡,Carole Kurahara‡, Fred Lott‡, Ning Sun‡,
Andrew A. Welcher‡, and Chen Dong*§
*Department of Immunology, University of Texas M. D. Anderson
Cancer Center, Houston, TX 77030; †Department of
Immunology,University of Washington, Seattle, WA 98195; and ‡Amgen
Inc., One Amgen Center Drive, Thousand Oaks, CA 91320
Edited by Tasuku Honjo, Kyoto University, Kyoto, Japan, and
approved June 22, 2006 (received for review February 16, 2006)
T cell activation and tolerance are regulated by
costimulatorymolecules. Although PD-1 serves as a crucial negative
regulator ofT cells, the function of its ligands, PDL1 and PDL2, is
still contro-versial. In this study, we created a PDL2-deficient
mouse to char-acterize its function in T cell activation and
tolerance. Antigen-presenting cells from PDL2��� mice were found to
be more potentin activation of T cells in vitro over the wild-type
controls, whichdepended on PD-1. Upon immunization with chicken
ovalbumin,PDL2��� mice exhibited increased activation of CD4� and
CD8� Tcells in vivo when compared with WT animals. In addition, T
celltolerance to an oral antigen was abrogated by the lack of PDL2.
Ourresults thus demonstrate that PDL2 negatively regulates T cells
inimmune responses and plays an essential role in immune
tolerance.
costimulation � cytokines � PD-1
During infection, pathogen-specific T cells are activated,
un-dergo robust clonal expansion, and subsequently
differentiateinto effector cells. In contrast, peripheral tolerance
mechanismshave been found to prevent autoreactive T cell function
(1, 2). Oraltolerance is a form of peripheral tolerance, in which
antigen-specificT cell tolerance is induced against oral antigens
(3). Although oraltolerance has been tested for protection against
autoimmune andallergic diseases, the cellular and molecular
mechanisms underlyingoral tolerance induction have remained
unclear.
T cell activation and tolerance are critically regulated by
costimu-latory molecules, especially those in the B7 and CD28
superfamilies(4). PD-1, a novel member of the CD28 family, is
expressed onactivated T cells and B cells (5). PD-1 has been shown
to be anegative regulator of T cell activation and is crucial for
maintainingimmune tolerance. PD-1 deficiency in mouse results in
spontaneousautoimmune diseases (6, 7). Moreover, PD-1 deficiency
(8) orblockade (9) accelerated autoimmune diabetes on NOD
back-ground. Blocking PD-1 also enhanced experimental
autoimmuneencephalomyelitis (EAE) disease (10).
Two ligands, B7-H1�PDL1 and PDL2�B7DC, have been foundto bind to
PD-1 (11–14). The function of PDL1 and PDL2 in T cellactivation is
still in debate. Contradictory results have suggestedPDL2 serves as
a negative and a positive regulator of T cell function.Latchman et
al. (13) have shown that recombinant PDL2 proteininhibited the
activation and cytokine production of CD4� T cells viacell-cycle
arrest, whereas Tseng et al. (14) published that
B7DC-Igcostimulated the proliferation of naı̈ve T cells at
suboptimal anti-CD3 concentrations, and that it increased IFN-�
secretion. Othersstudied PDL2 function by using antibodies that
block PDL2 bindingto PD-1. Salama et al. (10) reported exacerbation
of EAE diseasewhen PDL2, but not PDL1, was blocked. In a model of
airwayhypersensitivity, Matsumoto et al. (15) found that anti-PDL2
anti-body administered at the time of challenge increased
eosinophilia.These data suggest that PDL2, through engaging PD-1,
negativelyregulates T cell priming. However, an antibody to PDL2
was foundto enhance the ability of murine dendritic cells (DCs) to
stimulateT cells (16, 17). This antibody in vivo allowed mice to
reject poorlyimmunogenic or established tumors (18). Similarly, Liu
et al. (19)expressed B7-DC on tumor cells and found that they were
rejectedmore efficiently than WT tumors, and that this effect was
inde-pendent of PD-1 (19). It is not easy to reconcile the
above
contrasting data on PDL2 function. It has been suggested that
asecond, positive receptor exists for PDL2. In support of this
idea,Wang et al. (20) created mutants of PDL2 that no longer
boundPD-1 but still possessed positive costimulatory functionality.
ThusPDL2 may be a positive or negative costimulator depending on
thecontext in which it functions and the receptor it
preferentiallyengages.
In this study, we have created and analyzed a mouse
modeldeficient in PDL2. We found that antigen-presenting cells
(APCs)from PDL2��� mice had enhanced ability to activate T
cellscompared with WT cells. Upon immunization, PDL2-deficientmice
exhibited enhanced T cell activation in vivo. Furthermore,PDL2 is
required for induction of T cell tolerance to oral
antigen.Therefore, PDL2 is a negative regulator of T cell
activation and isessential for regulation of T cell tolerance.
ResultsGeneration of PDL2 Knockout (KO) Mice. To analyze the
role of PDL2in T cell activation and tolerance, we created a PDL2
gene KOmouse. A targeting strategy was designed to delete most of
thesecond coding exon, comprising amino acid residues 27–113, of
themouse PDL2 gene (Fig. 1A). Confirmation of the
successfulablation of the PDL2 gene was done by both genomic and
mRNAanalysis (Fig. 1 B and C). Importantly, the primers used for
theTaqman�RT-PCR analyses (Fig. 1C) were derived from the thirdand
fourth coding exons; the result indicates that no gene productis
detectable and thus a null mutation was created. HomozygousKO
animals were born at the expected frequency and were
fertile.Routine necropsy of 3-month-old animals revealed no
obviouschanges in organ weights, hematology, clinical chemistries,
orobvious signs of inflammation or gross changes in histology
(datanot shown).
The expression of PDL2 and other cell-surface molecules
bysplenic and bone marrow-derived DCs from both WT and KO micewere
analyzed by flow cytometry. After overnight LPS stimulation,45.5%
WT splenic DCs expressed PDL2 (Fig. 1D). In contrast, thePDL2 KO
splenic DCs did not express PDL2. PDL2 was foundhighly expressed by
mature WT, but not by KO, bone marrow-derived DCs (data not shown).
The expression of other costimu-latory molecules and MHC II on both
WT and KO DCs wascomparable (Fig. 1D). These data indicate a
specific ablation ofPDL2 expression in the KO animals.
PDL2��� APCs Exhibit Enhanced T Cell Activation in Vitro.
Expressionof PDL2 on the cell surface of DCs and macrophages (21)
suggestsits roles in APC function. To address this, we purified
splenic APCsfrom both WT and KO mice as described (22) and used
them tostimulate naı̈ve CD4� T cells from B6 mice in the presence
ofvarious concentrations of anti-CD3 antibody. CD4� T cells
acti-
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS
office.
Abbreviations: EAE, experimental autoimmune encephalomyelitis;
DC, dendritic cell; APC,antigen-presenting cell; KO, knockout; OVA,
ovalbumin; CFA, complete Freund’s adjuvant;MLN, mesenteric lymph
node.
§To whom correspondence should be addressed. E-mail:
[email protected].
© 2006 by The National Academy of Sciences of the USA
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these cells and the fate of T cells stimulated by these DC may
unveilnovel mechanisms of T cell tolerance.
Materials and MethodsGeneration of PDL2 KO Mice. A PDL2
gene-target construct thatreplaces most of the second exon
(encoding amino acid residues27–113) of the PDL2 gene with a PGKneo
cassette was transfectedinto GS-1 ES cells (129 SVJ, Genome
Systems, St. Louis, MO).Homologous recombinants were identified and
introduced intoC57BL�6NHsd blastocysts (Harlan, Indianapolis, IN),
followed byimplantation into the recipient CD-1 mouse strain
(Charles RiverLaboratories, Wilmington, MA). After successful
germ-line trans-mission, mice heterozygous for the PDL2 targeting
event wereinterbred to obtain homozygous PDL2 KO mice. Targeting
wasconfirmed by both genomic and mRNA PCR analysis.
FACS Staining. To analyze PDL2 expression, total splenocytes
werestained with biotin-labeled anti-PDL2 (eBioscience, San
Diego,CA), followed by incubation with streptavidin-APC and
FITC-conjugated anti-CD11c (BD Pharmingen, San Diego, CA).
FITC-conjugated anti-CD11c together with one of the following
phyco-erythrin (PE)-conjugated antibodies: anti-PDL1, anti-B7.1,
anti-B7.2, or anti-I-A�I-E (BD Pharmingen) was used to stain
othercostimulatory molecules. Total cells from MLNs were stained
withPE-conjugated anti-PD-L1 or biotin-labeled anti-PDL2, B7S1,
B7h,and B7H3 followed by incubation with streptavidin-APC
andFITC-conjugated anti-CD11c and PerCPCy 5.5-conjugated anti-CD8�
(BD Pharmingen). Cells were analyzed in a FACSCaliber(Becton
Dickinson, Mountain View, CA).
In Vitro T Cell Assays. Naı̈ve CD4� T cells from C57BL�6
andCD28���mice were purified as described (41). CD4� T cellsfrom
OT-II TcR transgenic mice and CD8� T cells from OT-ITcR transgenic
mice were purified by AutoMACS sorting.Splenic PDL2��� and PDL2���
APCs were prepared bycomplement-mediated lysis of Thy1� T cells. T
cells wereincubated with these APCs in the presence of different
concen-trations of plate-bound anti-CD3 antibody or specific
antigenicpeptide. IL-2 production was measured 24 h after T cell
activa-tion. Cell proliferation was determined 72 h after
incubationwith [3H]thymidine in the last 8 h. To analyze the effect
of PDL2
on effector T cell function, purified OT-I and OT-II cells
werestimulated with 10 ng�ml of SIINFEKL peptide or 5 �g�ml
ofOVA323–339 peptide, respectively, in the presence of 30
units�mlof IL-2 and WT or KO APCs for 4 days. Cells were then
washedand treated with 5 �g�ml of plate-bound anti-CD3 Ab for 24
h.The culture supernatants from the above experiments werecollected
for cytokine measurement. IFN-� and TNF-� produc-tion by OT-I cells
and IFN-� and IL-4 secretion by OT-II cellswere measured by ELISA
(Pharmingen, San Diego, CA).
Chicken OVA Immunization. PDL2��� and PDL2��� mice wereimmunized
as described (22) with chicken OVA protein (Sigma-Aldrich, St.
Louis, MO) emulsified in CFA at the base of the tail.On day 8, the
immunized mice were killed, and three mice fromeach group were
analyzed individually for their immune responses.Splenocytes were
restimulated with SIINFEKL or OVA323–339peptides to measure IL-2
expression, T cell proliferation, andeffector cytokine
production.
Induction and Assessment of Oral Tolerance. PDL2���,
PDL2���,B7-H3���, and B7-H3��� mice were daily administrated
intra-gastrically with 2 mg of chicken OVA protein (grade V, Sigma,
St.Louis, MO) dissolved in PBS for a total of five times. Control
micewere given PBS alone. One week after the last treatment, all
micewere immunized s.c. with 50 �g of OVA protein emulsified in
CFA.Seven days later, spleens were obtained from the mice,
andsplenocytes were restimulated with OVA protein to measure
IL-2production, T cell proliferation, and effector cytokine
production.All animal studies were approved by the appropriate
InstitutionalAnimal Care and Utilization Committee.
We thank Chris Paszty for comments; Laura Martin for
microinjections;the Amgen Laboratory Animal Resources group for
animal husbandry;Ms. Ying Wang for her assistance; and the entire
Dong laboratory fortheir help and discussion. This work was
supported in part by grants fromthe National Institutes of Health
(to C.D.). Y.Z. is an Odyssey Scholarof the M. D. Anderson Cancer
Center. C.B. was a recipient of a HowardHughes Medical Institute
predoctoral fellowship. C.D. received anInvestigator award from the
Cancer Research Institute, an ArthritisInvestigator award from the
Arthritis Foundation, and a Trust Fellow-ship from the M. D.
Anderson Cancer Center.
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