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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Jul 24;103(31):11695–11700. doi: 10.1073/pnas.0601347103

Regulation of T cell activation and tolerance by PDL2

Yongliang Zhang *, Yeonseok Chung *, Caroline Bishop †, Betsy Daugherty ‡, Hilary Chute ‡, Paige Holst ‡, Carole Kurahara ‡, Fred Lott ‡, Ning Sun ‡, Andrew A Welcher ‡, Chen Dong *,§
PMCID: PMC1544232  PMID: 16864790

Abstract

T cell activation and tolerance are regulated by costimulatory molecules. Although PD-1 serves as a crucial negative regulator of T cells, the function of its ligands, PDL1 and PDL2, is still controversial. In this study, we created a PDL2-deficient mouse to characterize its function in T cell activation and tolerance. Antigen-presenting cells from PDL2−/− mice were found to be more potent in activation of T cells in vitro over the wild-type controls, which depended on PD-1. Upon immunization with chicken ovalbumin, PDL2−/− mice exhibited increased activation of CD4+ and CD8+ T cells in vivo when compared with WT animals. In addition, T cell tolerance to an oral antigen was abrogated by the lack of PDL2. Our results thus demonstrate that PDL2 negatively regulates T cells in immune responses and plays an essential role in immune tolerance.

Keywords: costimulation, cytokines, PD-1

During infection, pathogen-specific T cells are activated, undergo robust clonal expansion, and subsequently differentiate into effector cells. In contrast, peripheral tolerance mechanisms have been found to prevent autoreactive T cell function (1, 2). Oral tolerance is a form of peripheral tolerance, in which antigen-specific T cell tolerance is induced against oral antigens (3). Although oral tolerance has been tested for protection against autoimmune and allergic diseases, the cellular and molecular mechanisms underlying oral tolerance induction have remained unclear.

T cell activation and tolerance are critically regulated by costimulatory molecules, especially those in the B7 and CD28 superfamilies (4). PD-1, a novel member of the CD28 family, is expressed on activated T cells and B cells (5). PD-1 has been shown to be a negative regulator of T cell activation and is crucial for maintaining immune tolerance. PD-1 deficiency in mouse results in spontaneous autoimmune diseases (6, 7). Moreover, PD-1 deficiency (8) or blockade (9) accelerated autoimmune diabetes on NOD background. Blocking PD-1 also enhanced experimental autoimmune encephalomyelitis (EAE) disease (10).

Two ligands, B7-H1/PDL1 and PDL2/B7DC, have been found to bind to PD-1 (11–14). The function of PDL1 and PDL2 in T cell activation is still in debate. Contradictory results have suggested PDL2 serves as a negative and a positive regulator of T cell function. Latchman et al. (13) have shown that recombinant PDL2 protein inhibited the activation and cytokine production of CD4+ T cells via cell-cycle arrest, whereas Tseng et al. (14) published that B7DC-Ig costimulated the proliferation of naïve T cells at suboptimal anti-CD3 concentrations, and that it increased IFN-γ secretion. Others studied PDL2 function by using antibodies that block PDL2 binding to PD-1. Salama et al. (10) reported exacerbation of EAE disease when PDL2, but not PDL1, was blocked. In a model of airway hypersensitivity, Matsumoto et al. (15) found that anti-PDL2 antibody administered at the time of challenge increased eosinophilia. These data suggest that PDL2, through engaging PD-1, negatively regulates T cell priming. However, an antibody to PDL2 was found to enhance the ability of murine dendritic cells (DCs) to stimulate T cells (16, 17). This antibody in vivo allowed mice to reject poorly immunogenic or established tumors (18). Similarly, Liu et al. (19) expressed B7-DC on tumor cells and found that they were rejected more efficiently than WT tumors, and that this effect was independent of PD-1 (19). It is not easy to reconcile the above contrasting data on PDL2 function. It has been suggested that a second, positive receptor exists for PDL2. In support of this idea, Wang et al. (20) created mutants of PDL2 that no longer bound PD-1 but still possessed positive costimulatory functionality. Thus PDL2 may be a positive or negative costimulator depending on the context in which it functions and the receptor it preferentially engages.

In this study, we have created and analyzed a mouse model deficient in PDL2. We found that antigen-presenting cells (APCs) from PDL2−/− mice had enhanced ability to activate T cells compared with WT cells. Upon immunization, PDL2-deficient mice 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 is essential for regulation of T cell tolerance.

Results

Generation of PDL2 Knockout (KO) Mice.

To analyze the role of PDL2 in T cell activation and tolerance, we created a PDL2 gene KO mouse. A targeting strategy was designed to delete most of the second coding exon, comprising amino acid residues 27–113, of the mouse PDL2 gene (Fig. 1A). Confirmation of the successful ablation of the PDL2 gene was done by both genomic and mRNA analysis (Fig. 1 B and C). Importantly, the primers used for the Taqman/RT-PCR analyses (Fig. 1C) were derived from the third and fourth coding exons; the result indicates that no gene product is detectable and thus a null mutation was created. Homozygous KO animals were born at the expected frequency and were fertile. Routine necropsy of 3-month-old animals revealed no obvious changes in organ weights, hematology, clinical chemistries, or obvious signs of inflammation or gross changes in histology (data not shown).

Fig. 1.

Fig. 1.

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Generation of PDL2 KO mice. (A) Strategy of PDL2 gene targeting. Most of exon 2, encoding amino acid residues 27–113, was replaced with a PGKneo cassette. The location of the primers used for genomic and mRNA analysis are as indicated. (B) Genomic PCR of WT (+/+), heterozygous (+/−), and homozygous (−/−) PDL2 KO mice. The 5′ primer 3309-24 was multiplexed with two 3′ primers, 3081-24 and 3309-25. A 234-bp band indicates amplification from the WT allele, and a 150-bp band indicates amplification from the targeted allele. (C) RT-PCR analysis of PDL2 mRNA from lung and liver of WT (+/+), heterozygotes (+/−), and homozygous (−/−) PDL2 KO animals. Taqman analysis and direct visualization of amplified products indicates that PDL2 mRNA is present in both WT and heterozygous animals but absent in the PDL2-deficient animals. (D) Surface expression of PDL2, PDL1, B7.1, B7.2, and MHCII on WT and PDL2−/− splenic APCs. CD11c+ population from LPS-activated total splenocytes was gated and analyzed. Data are representative of three individual experiments.

The expression of PDL2 and other cell-surface molecules by splenic and bone marrow-derived DCs from both WT and KO mice were analyzed by flow cytometry. After overnight LPS stimulation, 45.5% WT splenic DCs expressed PDL2 (Fig. 1D). In contrast, the PDL2 KO splenic DCs did not express PDL2. PDL2 was found highly expressed by mature WT, but not by KO, bone marrow-derived DCs (data not shown). The expression of other costimulatory molecules and MHC II on both WT and KO DCs was comparable (Fig. 1D). These data indicate a specific ablation of PDL2 expression in the KO animals.

PDL2−/− APCs Exhibit Enhanced T Cell Activation in Vitro.

Expression of PDL2 on the cell surface of DCs and macrophages (21) suggests its roles in APC function. To address this, we purified splenic APCs from both WT and KO mice as described (22) and used them to stimulate naïve CD4+ T cells from B6 mice in the presence of various concentrations of anti-CD3 antibody. CD4+ T cells activated in the presence of KO APCs proliferated much better than T cells treated with WT APCs (Fig. 2A). To test whether this effect was caused by lack of PD-1 signaling, a PD-1 blocking antibody was added to the above culture and cell proliferation was measured. Naïve T cells proliferated similarly, when stimulated with either WT or KO APCs in the presence of anti-PD-1 blocking antibody (Fig. 2A), indicating that PDL2 might mediate negative regulation of T cells via engaging PD-1. In addition, WT APCs activated CD28−/−CD4+T cells poorly (Fig. 2B). These T cells, however, exhibited greatly enhanced proliferation when activated with KO APCs (Fig. 2B). These data suggest that PDL2 may serve as a negative regulator of CD4+ T cells.

Fig. 2.

Fig. 2.

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PDL2 deficiency enhanced CD4+ T cell activation and function in vitro. (A) Naïve CD4+ T cells were purified from B6 mice and activated with WT or KO APCs in the presence of various concentrations of plate-bound anti-CD3 antibody with or without 10 μg/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 on day 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 μg/ml of plate-bound anti-CD3 Ab for 24 h. IFN-γ and IL-4 secretion in the culture supernatant was determined by ELISA. Data are representative of three individual experiments.

To substantiate the above results, we also used WT and KO APCs to stimulate antigen-specific T cells. First, OT-II cells were stimulated with WT and KO APCs in the presence of OVA323–339 peptide. Enhanced proliferation was observed in OT-II cells stimulated by KO APCs (data not shown). Furthermore, when restimulated with anti-CD3 after 4 days of activation, OT-II cells activated in the presence of KO APCs exhibited enhanced IL-4 and IFN-γ production compared with those activated via WT cells (Fig. 2C), indicating that effector generation was enhanced in the absence of PDL2. Similarly, OT-I cells activated in the presence of KO APCs produced significantly increased levels of IL-2 and exhibited enhanced proliferation, compared with those activated with WT APCs (Fig. 3A). In the presence of anti-PD-1 antibody, OT-I cells exhibited similar IL-2 production and proliferation when activated by WT or KO APCs (Fig. 3A). Furthermore, when restimulated, OT-I cells activated by KO APCs produced increased levels of IFN-γ and TNF-α, compared with those activated by WT APCs (Fig. 3B).

Fig. 3.

Fig. 3.

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

Together, these results indicate that in vitro PDL2 may negatively regulate T cell activation that leads to effector function in a PD-1-dependent manner.

Enhanced Antigen-Specific T Cell Responses in PDL2−/− Mice.

To examine 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 postimmunization, splenic cells were isolated from these mice and stimulated with various concentrations of SIINFELK or OVA323–339 peptide to examine antigen-specific T cell responses. Upon restimulation, splenocytes from immunized KO mice produced significantly increased levels of IL-2 and exhibited greatly enhanced proliferation than WT cells (Fig. 4A and C). We further measured the effector cytokine production by both WT and KO cells upon restimulation. KO cells produced significantly increased amounts of IFN-γ and TNF-α upon SIINFELK peptide stimulation (Fig. 4B). Upon OVA323–339 peptide restimulation, the KO cells also produced significantly increased levels of both T helper 1 (IFN-γ) and T helper 2 (IL-4, IL-5, IL-10, and IL-13) cytokines (Fig. 4D). Similarly, we observed enhanced proliferation and cytokine production of draining lymph node and spleen cells from myelin oligodendrocyte glycoprotein peptide-immunized KO mice upon antigenic restimulation, when compared with cells from immunized WT mice (data not shown). These data altogether indicate that loss of PDL2 resulted in enhanced antigen-specific CD4+ and CD8+ T cell immune responses in vivo.

Fig. 4.

Fig. 4.

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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 representative of two independent experiments.

Breakdown of Oral Tolerance in PDL2−/− Mice.

Absence of positive costimulation has been thought to contribute to peripheral tolerance (1, 2). Efficient activation of CD28−/− CD4+ T cells by PDL2−/− APCs suggests a role of PDL2 in immune tolerance. To address this, we applied an oral tolerance model on both PDL2+/+ and PDL2−/− mice. PDL2+/+ and PDL2−/− mice were fed with 2 mg of OVA or PBS once a day for 5 days, and then immunized with OVA in CFA. Splenocytes were subsequently isolated and stimulated with different doses of OVA protein to study the activation and function of antigen-specific T cells. Spleen cells from PDL2+/+ mice subject to oral administration with OVA antigen exhibited greatly reduced production of IL-2, proliferation, and the secretion of IFNγ and IL-4 upon restimulation, as compared with those only introduced with PBS (Fig. 5), indicating that profound T cell tolerance had been induced to oral antigen. On the other hand, OVA-specific T cells from OVA- and PBS-fed PDL2−/−mice produced similar amounts of IL-2 and proliferated at the similar levels upon antigen stimulation (Fig. 5A). Furthermore, IFN-γ and IL-4 production by OVA-specific cells was comparable between OVA-fed and PBS-fed PDL2−/− mice (Fig. 5B). These data demonstrate that PDL2 is essential for the induction and/or maintenance of oral tolerance.

Fig. 5.

Fig. 5.

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

B7H3 and B7S1 (also called B7x or B7-H4) are two other recently identified B7 family members that may function to negatively regulate T cell activation (23–27). To better understand the mechanisms for oral tolerance induction, we also examined the roles of B7H3 and B7S1. Oral tolerance could be equally induced in both WT and B7H3-deficient mice (Fig. 6A), indicating that B7H3 did not play an important role in oral tolerance. Moreover, anti-B7S1 blocking antibody (23) did not affect oral tolerance (data not shown).

Fig. 6.

Fig. 6.

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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 immunized with OVA in CFA. Seven days later, mice were killed and splenocytes were cultured with different concentrations of OVA. (A) IL-2 production in culture supernatants was determined by ELISA after 24 h and [3H]thymidine incorporation after 72 h. Each experimental group consisted of five mice. (B) Selective expression of PDLs on DCs in MLNs. Surface expression of PDL2, PDL1, B7h, B7S1, and B7H3 from WT and PDL2 KO mice was analyzed on CD11c+ population. Data are representative of five individual experiments.

The above results revealed that PDL2 is selectively required for induction of oral tolerance. Because antigen presentation, possibly by DCs, in mesenteric lymph nodes (MLNs) is thought to be responsible for oral tolerance induction (28), we examined the expression of costimulatory molecules on DCs in MLNs. Expression of PDL2 and PDL1 but not B7H3 or B7S1 was found on CD8α−DCs from WT but not PDL2−/− mice (Fig. 6B). This result suggests that PDL2 on CD8α− DCs may be responsible for oral tolerance induction.

Discussion

The decision of T cell tolerance or activation is determined by costimulatory molecules. PD-1 has been shown to be a negative costimulatory receptor. Here we analyze mice deficient in PDL2, a ligand for PD-1, in T cell activation and tolerance. We show that PDL2 negatively regulates T cell activation in vitro and in vivo and that PDL2 is essential for induction of oral tolerance.

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

Earlier, Shin et al. (30) published their studies on PDL2-deficient mice that had been backcrossed onto BALB/c background and found that type I immune responses were inhibited in the deficient mice, indicating its specific costimulatory role in T helper 1 and cytotoxic T lymphocyte response. In this study, we analyzed our PDL2 KO mouse on a different genetic background. RT-PCR analysis indicated that a PDL2 null mutation was successfully obtained. In contract to the report by Shin et al., we found that PDL2 deficiency led to enhanced T cell activation in vitro and in vivo. Our results are consistent with what was recently published by Keir et al. (31), who also analyzed a PDL2 KO mouse on BALB/c background. It is not easy to reconcile the difference between these three studies. Further studies to compare these mice on the same background in the same experiment, as well as determining the context of PDL2 function, are necessary. It is possible that PDL2 may preferentially engage to different receptors on different backgrounds. In support of this idea, we found that blocking the increased T cell priming in vitro by PDL2−/− APC depended on PD-1. Thus, in our system, PDL2 may exert its negative regulation through the PD-1 receptor.

CD28 is the major costimulatory molecule for CD4+ activation and prevention of tolerance. Efficient priming of CD4+ T cell activation by PDL2−/− APCs in the absence of CD28 led us to study the role of PDL2 in T cell tolerance. Oral tolerance is a classic form of peripheral tolerance in which T cells are toleralized against oral antigens (32). We found that multiple doses of oral antigen could induce antigen-specific T cell tolerance in WT but not in PDL2−/− 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. These results well correlate to the expression of these molecules in MLNs, which have a crucial role in the induction of mucosal immunity and tolerance (33). We found that PDL2, but not B7S1 and B7H3, is highly expressed by DCs in MLN, supporting a selective role of PDL2 in oral tolerance. Interestingly, PDL2 is expressed only on CD8α−CD11c+ DCs. Mouse CD8α+ and CD8α− DC populations possess different antigen-presenting features (34). It has been previously shown that CD8α−CD11b+ DCs but not CD8α+ DCs present intestinal antigens to CD8+ T cells and play a critical role for induction of cross-tolerance to dietary proteins (35). Our results suggest that CD8α− DCs in MLNs may also be responsible for the induction of CD4+ T cell tolerance in a PDL2-dependent manner. Recently, inducible costimulator was also shown to play a crucial role in the development of mucosal tolerance (36). However, its ligand, B7h, was not expressed on MLN DCs (Fig. 6B). Additional cells or mechanisms may thus exist to regulate mucosal tolerance.

Interestingly, although PDL1 and PDL2 are frequently expressed on 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 by PDL1−/− DCs produced increased amounts of IFN-γ. Keir et al. (31) also showed recently that CD4+ T cells activated by either PDL1−/− or PD-L2−/− APCs produced increased amounts of IL-2 and IFN-γ. Thus, both PDL1 and PDL2 are required for optimal PD-1 signaling. Possibly, PDL1, also expressed on CD8α− DCs in MLN, also regulates oral tolerance. On the other hand, they may have overlapping function in early T cell activation as T cells produced even more IL-2 and IFN-γ when both PDL1 and PDL2 were absent (31). PDL1 and PDL2 thus appear to be similar to the other binary costimulatory systems, such as CD80/CD86 (4) and CD28/inducible costimulator (38). In addition to their overlapping function, PDL1 and PDL2 were reported to play distinct roles in immune responses to Leishmaniasis mexicana (39). Compared with the WT mice, PDL1−/− mice showed resistance to L. mexicana infection with a reduced IL-4 producing cell development, whereas PDL2−/− mice exhibited increased susceptibility to the infection with enhanced T-dependent and T-independent humoral immune responses. Two PD-1 ligands also play distinct roles in regulating autoreactive T cells in EAE, which is influenced by the genetic background of the mice. For instance, in MOG35–55-induced EAE in C57BL/6 mice, blockade of PD-L2, but not PD-L1, in both priming 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, both PD-L1 and PD-L2 deficiency resulted in severe clinical EAE with early onset and rapid progression (ref. 37 and unpublished data). These differential functions of PDL1 and PDL2 are possibly regulated by their distinct expression and regulatory mechanisms, although it is possible that they may be selectively used or recruited to the immunological synapse.

In summary, we showed that PDL2 negatively regulates T cell activation in vitro and in vivo. In addition, we found a unique function of PDL2 in oral tolerance. PDL2+ DC may be a DC population important for T cell toleralization. Further studies on these cells and the fate of T cells stimulated by these DC may unveil novel mechanisms of T cell tolerance.

Materials and Methods

Generation of PDL2 KO Mice.

A PDL2 gene-target construct that replaces most of the second exon (encoding amino acid residues 27–113) of the PDL2 gene with a PGKneo cassette was transfected into GS-1 ES cells (129 SVJ, Genome Systems, St. Louis, MO). Homologous recombinants were identified and introduced into C57BL/6NHsd blastocysts (Harlan, Indianapolis, IN), followed by implantation into the recipient CD-1 mouse strain (Charles River Laboratories, Wilmington, MA). After successful germ-line transmission, mice heterozygous for the PDL2 targeting event were interbred to obtain homozygous PDL2 KO mice. Targeting was confirmed by both genomic and mRNA PCR analysis.

FACS Staining.

To analyze PDL2 expression, total splenocytes were stained 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 phycoerythrin (PE)-conjugated antibodies: anti-PDL1, anti-B7.1, anti-B7.2, or anti-I-A/I-E (BD Pharmingen) was used to stain other costimulatory molecules. Total cells from MLNs were stained with PE-conjugated anti-PD-L1 or biotin-labeled anti-PDL2, B7S1, B7h, and B7H3 followed by incubation with streptavidin-APC and FITC-conjugated anti-CD11c and PerCPCy 5.5-conjugated anti-CD8α (BD Pharmingen). Cells were analyzed in a FACSCaliber (Becton Dickinson, Mountain View, CA).

In Vitro T Cell Assays.

Naïve CD4+ T cells from C57BL/6 and CD28−/−mice were purified as described (41). CD4+ T cells from OT-II TcR transgenic mice and CD8+ T cells from OT-I TcR transgenic mice were purified by AutoMACS sorting. Splenic PDL2+/+ and PDL2−/− APCs were prepared by complement-mediated lysis of Thy1+ T cells. T cells were incubated with these APCs in the presence of different concentrations of plate-bound anti-CD3 antibody or specific antigenic peptide. IL-2 production was measured 24 h after T cell activation. Cell proliferation was determined 72 h after incubation with [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 were stimulated with 10 ng/ml of SIINFEKL peptide or 5 μg/ml of OVA323–339 peptide, respectively, in the presence of 30 units/ml of IL-2 and WT or KO APCs for 4 days. Cells were then washed and treated with 5 μg/ml of plate-bound anti-CD3 Ab for 24 h. The culture supernatants from the above experiments were collected for cytokine measurement. IFN-γ and TNF-α production by OT-I cells and IFN-γ and IL-4 secretion by OT-II cells were measured by ELISA (Pharmingen, San Diego, CA).

Chicken OVA Immunization.

PDL2+/+ and PDL2−/− mice were immunized 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 from each group were analyzed individually for their immune responses. Splenocytes were restimulated with SIINFEKL or OVA323–339 peptides to measure IL-2 expression, T cell proliferation, and effector cytokine production.

Induction and Assessment of Oral Tolerance.

PDL2−/−, PDL2+/+, B7-H3−/−, and B7-H3+/+ mice were daily administrated intragastrically with 2 mg of chicken OVA protein (grade V, Sigma, St. Louis, MO) dissolved in PBS for a total of five times. Control mice were given PBS alone. One week after the last treatment, all mice were immunized s.c. with 50 μg of OVA protein emulsified in CFA. Seven days later, spleens were obtained from the mice, and splenocytes were restimulated with OVA protein to measure IL-2 production, T cell proliferation, and effector cytokine production. All animal studies were approved by the appropriate Institutional Animal Care and Utilization Committee.

Acknowledgments

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 for their help and discussion. This work was supported in part by grants from the National Institutes of Health (to C.D.). Y.Z. is an Odyssey Scholar of the M. D. Anderson Cancer Center. C.B. was a recipient of a Howard Hughes Medical Institute predoctoral fellowship. C.D. received an Investigator award from the Cancer Research Institute, an Arthritis Investigator award from the Arthritis Foundation, and a Trust Fellowship from the M. D. Anderson Cancer Center.

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.

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

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