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: cdong@mdanderson.org.

© 2006 by The National Academy of Sciences of the USA

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vated in the presence of KO APCs proliferated much better thanT cells treated with WT APCs (Fig. 2A). To test whether this effectwas caused by lack of PD-1 signaling, a PD-1 blocking antibody was

added to the above culture and cell proliferation was measured.Naı̈ve T cells proliferated similarly, when stimulated with either WTor KO APCs in the presence of anti-PD-1 blocking antibody (Fig.

Fig. 2. PDL2 deficiency enhanced CD4� T cell activation and function in vitro. (A) Naı̈ve CD4� T cells were purified from B6 mice and activated with WT or KO APCsin the presence of various concentrations of plate-bound anti-CD3 antibody with or without 10 �g�ml of PD-1 blocking antibody (eBioscience). (B) Purified CD28���CD4� T cells were stimulated with indicated concentrations of anti-CD3 antibody in the presence of WT or KO APCs. [3H]thymidine incorporation was determined onday 3. (C) OT-II cells were activated in the presence of OVA323–339 peptide and WT or KO APCs for 4 days and then restimulated with 5 �g�ml of plate-bound anti-CD3Ab for 24 h. IFN-� and IL-4 secretion in the culture supernatant was determined by ELISA. Data are representative of three individual experiments.

Fig. 1. Generation of PDL2 KOmice. (A) Strategy of PDL2 gene tar-geting. Most of exon 2, encodingamino acid residues 27–113, was re-placed with a PGKneo cassette. Thelocation of the primers used forgenomic and mRNA analysis are asindicated. (B) Genomic PCR of WT(���), heterozygous (���), and ho-mozygous (���) PDL2 KO mice. The5� primer 3309-24 was multiplexedwith two 3� primers, 3081-24 and3309-25. A 234-bp band indicatesamplification from the WT allele,and a 150-bp band indicates amplifi-cation from the targeted allele. (C)RT-PCR analysis of PDL2 mRNA fromlung and liver of WT (���), het-erozygotes (���), and homozygous(���) PDL2 KO animals. Taqmananalysis and direct visualization ofamplified products indicates thatPDL2 mRNA is present in both WTand heterozygous animals but ab-sent in the PDL2-deficient animals.(D) Surface expression of PDL2, PDL1,B7.1, B7.2, and MHCII on WT andPDL2��� splenic APCs. CD11c� pop-ulation from LPS-activated totalsplenocytes was gated and analyzed.Data are representative of three in-dividual experiments.

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2A), indicating that PDL2 might mediate negative regulation of Tcells via engaging PD-1. In addition, WT APCs activated CD28���CD4�T cells poorly (Fig. 2B). These T cells, however, exhibitedgreatly enhanced proliferation when activated with KO APCs (Fig.2B). These data suggest that PDL2 may serve as a negativeregulator of CD4� T cells.

To substantiate the above results, we also used WT and KOAPCs to stimulate antigen-specific T cells. First, OT-II cells werestimulated with WT and KO APCs in the presence of OVA323–339peptide. Enhanced proliferation was observed in OT-II cells stim-ulated by KO APCs (data not shown). Furthermore, when restim-ulated with anti-CD3 after 4 days of activation, OT-II cells activated

in the presence of KO APCs exhibited enhanced IL-4 and IFN-�production compared with those activated via WT cells (Fig. 2C),indicating that effector generation was enhanced in the absence ofPDL2. Similarly, OT-I cells activated in the presence of KO APCsproduced significantly increased levels of IL-2 and exhibited en-hanced proliferation, compared with those activated with WTAPCs (Fig. 3A). In the presence of anti-PD-1 antibody, OT-I cellsexhibited similar IL-2 production and proliferation when activatedby WT or KO APCs (Fig. 3A). Furthermore, when restimulated,OT-I cells activated by KO APCs produced increased levels ofIFN-� and TNF-�, compared with those activated by WT APCs(Fig. 3B).

Fig. 4. Enhanced in vivo antigen-specific T cell activation in the absence of PDL2. WT and KO mice (three in each group) were immunized with OVA and CFA.On day 8, spleen cells from immunized mice were stimulated with indicated concentrations of SIINFEKL or OVA323–339 peptide in 96-well plates as triplicates.Proliferation was assayed after 3 days of treatment (A and C), and cytokine production was measured by ELISA (B and D). The results shown are representativeof two independent experiments.

Fig. 3. PDL2 deficiency enhanced CD8� T cell activation and function in vitro. Purified OT-I cells were stimulated with indicated concentrations of SIINFEKLpeptide in the presence of WT or KO APCs with or without 10 �g�ml of PD-1 blocking antibody. (A) IL-2 production and [3H]thymidine incorporation weredetermined. (B) Seven days after activation, effector OT-I cells were restimulated with 5 �g�ml of plate-bound anti-CD3 Ab for 24 h, and IFN-� and TNF-�production was determined by ELISA. Data are representative of three individual experiments.

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Together, these results indicate that in vitro PDL2 may negativelyregulate T cell activation that leads to effector function in aPD-1-dependent manner.

Enhanced Antigen-Specific T Cell Responses in PDL2��� Mice. Toexamine the effect of PDL2 deficiency on T cell activation in vivo,we immunized WT or KO mice with chicken ovalbumin (OVA)protein in complete Freund’s adjuvant (CFA). On day 8 postim-munization, splenic cells were isolated from these mice and stim-ulated with various concentrations of SIINFELK or OVA323–339peptide to examine antigen-specific T cell responses. Upon restimu-lation, splenocytes from immunized KO mice produced signifi-cantly increased levels of IL-2 and exhibited greatly enhancedproliferation than WT cells (Fig. 4 A and C). We further measuredthe effector cytokine production by both WT and KO cells uponrestimulation. KO cells produced significantly increased amounts ofIFN-� and TNF-� upon SIINFELK peptide stimulation (Fig. 4B).Upon OVA323–339 peptide restimulation, the KO cells also pro-duced significantly increased levels of both T helper 1 (IFN-�) andT helper 2 (IL-4, IL-5, IL-10, and IL-13) cytokines (Fig. 4D).Similarly, we observed enhanced proliferation and cytokine pro-duction of draining lymph node and spleen cells from myelinoligodendrocyte glycoprotein peptide-immunized KO mice uponantigenic restimulation, when compared with cells from immunizedWT mice (data not shown). These data altogether indicate that lossof PDL2 resulted in enhanced antigen-specific CD4� and CD8� Tcell immune responses in vivo.

Breakdown of Oral Tolerance in PDL2��� Mice. Absence of positivecostimulation has been thought to contribute to peripheral toler-ance (1, 2). Efficient activation of CD28��� CD4� T cells byPDL2��� APCs suggests a role of PDL2 in immune tolerance. Toaddress this, we applied an oral tolerance model on both PDL2���and PDL2��� mice. PDL2��� and PDL2��� mice were fedwith 2 mg of OVA or PBS once a day for 5 days, and thenimmunized with OVA in CFA. Splenocytes were subsequentlyisolated and stimulated with different doses of OVA protein tostudy the activation and function of antigen-specific T cells. Spleencells from PDL2��� mice subject to oral administration with OVAantigen exhibited greatly reduced production of IL-2, proliferation,and the secretion of IFN� and IL-4 upon restimulation, as com-pared with those only introduced with PBS (Fig. 5), indicating thatprofound T cell tolerance had been induced to oral antigen. On theother hand, OVA-specific T cells from OVA- and PBS-fedPDL2���mice produced similar amounts of IL-2 and proliferatedat the similar levels upon antigen stimulation (Fig. 5A). Further-more, IFN-� and IL-4 production by OVA-specific cells wascomparable between OVA-fed and PBS-fed PDL2��� mice (Fig.5B). These data demonstrate that PDL2 is essential for the induc-tion and�or maintenance of oral tolerance.

B7H3 and B7S1 (also called B7x or B7-H4) are two other recentlyidentified B7 family members that may function to negativelyregulate T cell activation (23–27). To better understand the mech-anisms for oral tolerance induction, we also examined the roles ofB7H3 and B7S1. Oral tolerance could be equally induced in bothWT and B7H3-deficient mice (Fig. 6A), indicating that B7H3 didnot play an important role in oral tolerance. Moreover, anti-B7S1blocking antibody (23) did not affect oral tolerance (data notshown).

The above results revealed that PDL2 is selectively required forinduction of oral tolerance. Because antigen presentation, possiblyby DCs, in mesenteric lymph nodes (MLNs) is thought to beresponsible for oral tolerance induction (28), we examined theexpression of costimulatory molecules on DCs in MLNs. Expres-sion of PDL2 and PDL1 but not B7H3 or B7S1 was found onCD8��DCs from WT but not PDL2��� mice (Fig. 6B). Thisresult suggests that PDL2 on CD8�� DCs may be responsible fororal tolerance induction.

DiscussionThe decision of T cell tolerance or activation is determined bycostimulatory molecules. PD-1 has been shown to be a negativecostimulatory receptor. Here we analyze mice deficient in PDL2, aligand for PD-1, in T cell activation and tolerance. We show thatPDL2 negatively regulates T cell activation in vitro and in vivo andthat PDL2 is essential for induction of oral tolerance.

The roles of PDL2 in T cell regulation have been controversialin literature. Some studies indicate that PDL2 is an inhibitorycostimulatory molecule (13, 29), whereas others suggest that it is apositive costimulatory molecule and it exerts its function through areceptor other than PD-1 (14). Identification of a second receptorfor PD-1 and characterization of its expression may help resolve thisissue and define the context in which PDL2 may function as apositive or negative regulator of T cells.

Earlier, Shin et al. (30) published their studies on PDL2-deficientmice that had been backcrossed onto BALB�c background andfound that type I immune responses were inhibited in the deficientmice, indicating its specific costimulatory role in T helper 1 andcytotoxic T lymphocyte response. In this study, we analyzed ourPDL2 KO mouse on a different genetic background. RT-PCRanalysis indicated that a PDL2 null mutation was successfullyobtained. In contract to the report by Shin et al., we found thatPDL2 deficiency led to enhanced T cell activation in vitro and invivo. Our results are consistent with what was recently published byKeir et al. (31), who also analyzed a PDL2 KO mouse on BALB�cbackground. It is not easy to reconcile the difference between thesethree studies. Further studies to compare these mice on the same

Fig. 5. PDL2 deficiency abrogates oral tolerance. WT and PDL2���micewere fed five times with OVA or PBS. Seven days after the last feeding, all micewere immunized with OVA in CFA. Seven days later, mice were killed andsplenocytes were cultured with different concentrations of OVA. (A) IL-2production in culture supernatants was determined by ELISA after 24 h, and[3H]thymidine incorporation after 72 h. (B) IFN-� and IL-4 production in culturesupernatants was measured by ELISA. Data are representative of two individ-ual experiments. Each experimental group consisted of five mice.

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background in the same experiment, as well as determining thecontext of PDL2 function, are necessary. It is possible that PDL2may preferentially engage to different receptors on different back-grounds. In support of this idea, we found that blocking theincreased T cell priming in vitro by PDL2��� APC depended onPD-1. Thus, in our system, PDL2 may exert its negative regulationthrough the PD-1 receptor.

CD28 is the major costimulatory molecule for CD4� activationand prevention of tolerance. Efficient priming of CD4� T cellactivation by PDL2��� APCs in the absence of CD28 led us tostudy the role of PDL2 in T cell tolerance. Oral tolerance is a classicform of peripheral tolerance in which T cells are toleralized againstoral antigens (32). We found that multiple doses of oral antigencould induce antigen-specific T cell tolerance in WT but not inPDL2��� mice. In addition, the other two B7 family members,B7S1 and B7H3, had no significant role in our oral tolerance model.Therefore, PDL2 is selectively required in oral tolerance. Theseresults well correlate to the expression of these molecules in MLNs,which have a crucial role in the induction of mucosal immunity andtolerance (33). We found that PDL2, but not B7S1 and B7H3, ishighly expressed by DCs in MLN, supporting a selective role ofPDL2 in oral tolerance. Interestingly, PDL2 is expressed only onCD8��CD11c� DCs. Mouse CD8�� and CD8�� DC populationspossess different antigen-presenting features (34). It has beenpreviously shown that CD8��CD11b� DCs but not CD8�� DCspresent intestinal antigens to CD8� T cells and play a critical rolefor induction of cross-tolerance to dietary proteins (35). Our resultssuggest that CD8�� DCs in MLNs may also be responsible for theinduction of CD4� T cell tolerance in a PDL2-dependent manner.Recently, inducible costimulator was also shown to play a crucialrole in the development of mucosal tolerance (36). However, itsligand, B7h, was not expressed on MLN DCs (Fig. 6B). Additionalcells or mechanisms may thus exist to regulate mucosal tolerance.

Interestingly, although PDL1 and PDL2 are frequently expressedon the same APCs, for example splenic DCs and mesenteric CD8a�DC, the presence of PDL1 does not appear to compensate the

deficiency of PDL2 in regulating T cell activation and function.Latchman et al. (37) showed that CD4� T cells activated byPDL1��� DCs produced increased amounts of IFN-�. Keir et al.(31) also showed recently that CD4� T cells activated by eitherPDL1��� or PD-L2��� APCs produced increased amounts ofIL-2 and IFN-�. Thus, both PDL1 and PDL2 are required foroptimal PD-1 signaling. Possibly, PDL1, also expressed on CD8��DCs in MLN, also regulates oral tolerance. On the other hand, theymay have overlapping function in early T cell activation as T cellsproduced even more IL-2 and IFN-� when both PDL1 and PDL2were absent (31). PDL1 and PDL2 thus appear to be similar to theother binary costimulatory systems, such as CD80�CD86 (4) andCD28�inducible costimulator (38). In addition to their overlappingfunction, PDL1 and PDL2 were reported to play distinct roles inimmune responses to Leishmaniasis mexicana (39). Compared withthe WT mice, PDL1��� mice showed resistance to L. mexicanainfection with a reduced IL-4 producing cell development, whereasPDL2��� mice exhibited increased susceptibility to the infectionwith enhanced T-dependent and T-independent humoral immuneresponses. Two PD-1 ligands also play distinct roles in regulatingautoreactive T cells in EAE, which is influenced by the geneticbackground of the mice. For instance, in MOG35–55-induced EAEin C57BL�6 mice, blockade of PD-L2, but not PD-L1, in bothpriming and effector phases significantly enhanced disease severity(10, 40). In BALB�c mice, blocking PD-L1, but not PD-L2,significantly increased disease incidence (40). In 129Sv mice, bothPD-L1 and PD-L2 deficiency resulted in severe clinical EAE withearly onset and rapid progression (ref. 37 and unpublished data).These differential functions of PDL1 and PDL2 are possiblyregulated by their distinct expression and regulatory mechanisms,although it is possible that they may be selectively used or recruitedto the immunological synapse.

In summary, we showed that PDL2 negatively regulates T cellactivation in vitro and in vivo. In addition, we found a uniquefunction of PDL2 in oral tolerance. PDL2� DC may be a DCpopulation important for T cell toleralization. Further studies on

Fig. 6. B7H3 is not essential for oral tolerance. B7-H3���and B7-H3��� mice were fed five times with OVA or PBS.Seven days after the last feeding, all mice were immunizedwith OVA in CFA. Seven days later, mice were killed andsplenocytes were cultured with different concentrations ofOVA. (A) IL-2 production in culture supernatants was deter-mined by ELISA after 24 h and [3H]thymidine incorporationafter 72 h. Each experimental group consisted of five mice.(B) Selective expression of PDLs on DCs in MLNs. Surfaceexpression of PDL2, PDL1, B7h, B7S1, and B7H3 from WT andPDL2 KO mice was analyzed on CD11c� population. Data arerepresentative of five individual experiments.

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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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