Cutting Edge: Programed Death (PD) Ligand-1/PD-1 Interaction Is Required for CD8+ T Cell Tolerance to Tissue Antigens

Natalia Martin-Orozco, Yi-Hong Wang, Hideo Yagita and Chen Dong
J Immunol December 15, 2006, 177 (12) 8291-8295; DOI: https://doi.org/10.4049/jimmunol.177.12.8291

Abstract

Constitutive presentation of tissue Ags by dendritic cells results in tolerance of autoreactive CD8+ T cells; however, the underlying molecular mechanisms are not well understood. In this study we show that programmed death (PD)-1, an inhibitory receptor of the CD28 family, is required for tolerance induction of autoreactive CD8+ T cells. An antagonistic Ab against PD-1 provoked destructive autoimmune diabetes in RIP-mOVA mice expressing chicken OVA in the pancreatic islet cells, which received naive OVA-specific CD8+ OT-I cells. This effect was mediated by the PD ligand (PD-L) PD-L1 but not by PD-L2. An increased number of effector OT-I cells recovered from the pancreatic lymph nodes of anti-PD-L1-treated mice showed down-regulation of PD-1. Furthermore, the blockade of PD-1/PD-L1 interaction during the priming phase did not significantly affect OT-I cell division but enhanced its granzyme B, IFN-γ, and IL-2 production. Thus, during the presentation of tissue Ags to CD8+ T cells, PD-1/PD-L1 interaction crucially controls the effector differentiation of autoreactive T cells to maintain self-tolerance.

In addition to central tolerance, peripheral tolerance mechanisms exist to prevent autoimmune destruction by CD8+ T cells. Dendritic cells (DCs)3 uptake and process tissue Ags and migrate into peripheral lymph nodes to present Ags to naive CD8+ T cells (1). As a result, autoreactive CD8+ T cells undergo some initial cell divisions but are subsequently tolerized by deletion and/or anergy mechanisms (1, 2, 3). Active induction of CD8+ T cell tolerance to pancreatic islet Ags has been characterized in C57BL/6-Tg(Ins2-TFRC/OV)296Wehi/WehiJ mice (hereafter referred to as RIP-mOVA mice, where RIP is ran insulin promoter) that express membrane-bound chicken OVA (mOVA) in pancreatic islet β cells and the kidney proximal tube (4). When naive OT-I CD8+ T cells specific for the class I-restricted, OVA-derived SIINFEKL peptide were transferred into RIP-mOVA mice, OT-I cells proliferated in pancreatic lymph nodes (PLNs), but diabetes developed only with high numbers of OT-I cells or when OVA-specific CD4+ T cells were cotransferred (5). Small numbers of OT-I cells, when transferred, divided in PLNs but were largely deleted afterward.

Costimulation critically regulates the commitment to T cell activation or tolerance. The lack of positive costimulation, especially B7.1 and B7.2 engagement of CD28, has been attributed to the induction of T cell tolerance in the resting state (6). However, TLR ligands that up-regulate B7 expression on DCs did not prevent CD8+ T cell tolerance to pancreatic Ags in the RIP-mOVA model (7). Thus, additional mechanism(s) must exist to regulate CD8+ T cell tolerance.

Programmed death (PD)-1, an inhibitory receptor of the CD28-family, critically regulates T cell tolerance. PD-1 is expressed on activated CD4+ and CD8+ T cells and has two ligands, PD ligand (PD-L)-1 (called PD-L1 or B7-H1) and PD-L2 (or B7-DC) (8). PD-L1 is expressed in hemopoetic cells and can be up-regulated upon activation (9). PD-L1 is also found in several tissues including pancreatic islets, heart, endothelium, small intestine, and placenta (10). In contrast, PD-L2 expression is restricted to macrophages and DCs and can be up-regulated upon activation with IFN-γ, GM-CSF, and IL-4 (9). The expression pattern of PD1 ligands suggests overlapping and differential roles in immune regulation. Interestingly, we found that CD11c+CD8α+ DCs, which were previously shown to cross-present pancreatic Ags to CD8+ T cells in PLN (11), express PD-L1 but not PD-L2, whereas CD11c+CD11b+CD8α- cells express PD-L1 and PD-L2 (data not shown).

PD-1-deficient mice develop spontaneous autoimmune diseases (12, 13, 14), indicating an essential function of PD-1 in immune tolerance mechanisms. PD-1 blockade or deficiency resulted in accelerated type-I diabetes (T1D) in NOD/LtJ mice (15, 16), although it is not clear whether PD-1 functions in resistant animals to mediate protection against autoimmune diabetes. Investigations on the role of PD-1 ligands in tolerance, however, have led to contradictory results, with evidence supporting their roles as negative regulators in some scenarios and positive regulators in others (8). Recently it was reported that the PD-L1 protein expressed on islet cells mediates peripheral tolerance and prevents autoimmune attack in NOD/LtJ mice (17). Previously, an opposite conclusion was drawn on the transgenic expression of PD-L1 in the islet cells of C57BL/6J mice, where PD-L1 in the islet cells appears to promote spontaneous autoimmunity (18). In this study, we specifically analyze the function of PD-1 and its ligands in the regulation of CD8+ T cell tolerance to tissue Ags. We found that a blockade of PD-1 in the RIP-mOVA mice resulted in T1D mediated by adoptively transferred OT-I T cells. PD-L1 but not PD-L2 was required for CD8+ T cell tolerance induction. Blockade of PD-1/PD-L1 interaction during the priming phase did not significantly affect divisions of autoreactive T cells but greatly enhanced their effector differentiation.

Materials and Methods

Mice

C57BL/6J and OT-I transgenic mice (C57BL/6-Tg (TcraTcrb)1100 Mjb/J) were from The Jackson Laboratory, and the RIP-mOVA mice provided by W. Heath of the Walter and Eliza Hall Institute of Medical Research (Parkville, Australia) (4) were bred in M. D. Anderson Cancer Center animal facility.

Antibodies

Anti-PD-1 Abs clone J43 and clone RMP1-14 (19) were affinity purified using a protein G column. The anti-PD-L1 clone MIH5 was purchased from eBioscience (20). Anti-PD-L2 from clone TY25 was purified as previously described (9). Control IgGs were from Jackson ImmunoResearch Laboratories.

Flow cytometry

CD4, CD8, CD44, IL-2, IFN-γ, and Vα2 Abs were from BD Biosciences, and granzyme B (16G6) and PD-1 Abs and isotype controls were from eBioscience. An OT-I pentamer was purchased from ProImmune. All cell suspensions were incubated with anti-CD32/CD16 for 30 min at 4°C before staining with the Ab mixture. Cells were analyzed on a FACSCalibur cytometer (BD Biosciences).

Adoptive transfer and Ab treatment

CD8 cells from OT-I mice were purified using anti-CD8 Miltenyi beads and an autoMACS cell separator (Miltenyi Biotec). Cells (5 × 105 per 100 μl of PBS) were i.v injected into RIP-mOVA mice or C57BL/6J mice. Blocking Abs were i.p. administered at 100 μg per 100 μl of PBS on days 1, 4, and 7. Mice were monitored daily for urine glucose levels (Diastix; Bayer Pharmaceuticals) and high reads were confirmed by blood glucose measurements (Ascencia Elite; Bayer Pharmaceuticals). Diabetes was scored after three consecutive reads higher than 13.5 mM/L.

Proliferation, cytokine analysis, and CTL

Purified OT-I cells were adjusted to 20 × 106 cells/ml and labeled in RPMI 1640 containing 1% FBS and 10μΜ CFSE for 15 min at 37°C. Cells were then washed twice with RPMI 1640 and 10% FBS and one time with PBS before injection into RIP-mOVA mice or C57BL/6J mice. Blocking Abs were administered as described above. For intracellular cytokines, cell suspensions were culture for 5 h with 5 μg/ml SIINFEKL peptide and 1 μl/ml GolgiPlug, and further intracellular staining was performed with the Cytofix/Cytoperm kit (BD Biosciences). For IFN-γ ELISPOT, T cells from organs were cultured for 18 h with 3 μg/ml SIINFEKL peptide and, for the CTL assay, for 5 days with 1 μg/ml peptide. A CTL assay was done by the caspase-3 cleavage assay described in Ref. 21 using EL4 cells as targets.

Histology

Pancreata from RIP-mOVA mice were fixed in 10% formalin and embedded in paraffin. Six-micrometer sections were cut every 100 microns through the total tissue and H&E staining was performed. Fifty to 100 islets per mice were scored for the presence and type of insulitis.

Results and Discussion

PD-1 protects against CD8+ T cell-mediated autoimmunity

To study the role of PD-1 on peripheral tolerance of CD8+ T cells, we used the RIP-mOVA model (22). Purified CD8+ T cells from OT-I mice were transferred into RIP-mOVA mice, and a blocking Ab against PD-1 or a control IgG was administered three times in the recipient mice. Consistent with previous literature (22, 23), control mice did not develop diabetes for at least 30 days (Fig. 1⇓A). However, all mice that received anti-PD-1 developed diabetes; hyperglycemic mice were observed 6 days after transfer. Pancreata from mice treated with anti-PD-1 showed aggressive insulitis with infiltration of CD8+ cells in the islets and also their surrounding areas, whereas mice treated with control IgG showed only peri-insulitis or no infiltration (Fig. 1⇓, B and C). Thus, PD-1 is required for controlling the autoimmunity mediated by islet-specific CD8+ T cells.

FIGURE 1.
FIGURE 1.

PD-1 blockade leads to CD8+ T cell-mediated autoimmune diabetes. Purified OT-I cells were transferred into RIP-mOVA followed by treatment with anti-PD-1 (clone J43 or RMP1–14) or control hamster or rat IgG. A, Diabetes incidence. Arrowheads indicate time of Ab administration. The results are compiled from two independent experiments with three mice per group in each case. B, H&E-stained pancreas sections of RIP-mOVA recipient mice that were treated with anti-PD-1 or IgG. C, Quantification of infiltrated islets from pancreas of RIP-mOVA recipient mice. Each bar represents one individual mouse. Level 1 is insulitis in 50–60% of an islet and level 2 represents the islet completely infiltrated with cells invading neighboring tissues.

PD-L1 but not PD-L2 regulates tolerance of CD8+ T cells

Expression of PD-L1 but not PD-L2 in PLN CD11c+CD11b−CD8α+ DC suggests that PD-L1 mediates cross-tolerance of CD8+ T cells. We thus used antagonistic Abs against PD-L1 and PD-L2 in the same transfer system of OT-I cells into RIP-mOVA mice. We found that 100% of the mice treated with anti-PD-L1 developed diabetes (Fig. 2⇓). The insulitic lesions in mice treated with anti-PD-L1 were similar to the ones provoked by anti-PD-1, with predominant infiltrating CD8+ cells (data not shown). A blocking Ab against PD-L2 (9) did not have any effect; none of the RIP-mOVA mice that received OT-I cells and anti-PD-L2 developed diabetes (Fig. 2⇓). Thus, PD-1 engagement by PD-L1 is critical in protection against autoimmunity mediated by transferred OT-I cells.

FIGURE 2.
FIGURE 2.

PD-L1 mediates tolerance of autoreactive CD8+ T cells. OT-I cells were transferred into RIP-mOVA as in Fig. 1⇑ followed by treatment with anti-PD-L1 (♦), anti-PD-L2 (▴), or rat IgG (□). Arrows indicates time of Ab administration. The results are compiled from two independent experiments with two or three mice per group in each case.

Blockade of PD-1/PD-L1 interaction increased autoreactive CD8+ effector T cell numbers

OT-I cells transferred into RIP-mOVA mice undergo deletion after brief proliferation (5). We thus analyzed OT-I cells from peripheral lymph nodes and spleens of recipient RIP-mOVA mice by staining with a MHC-I pentamer containing the SIINFEKEL peptide. Seven days after the transfer, the majority of tetramer-positive cells were localized in the PLNs of both anti-PD-L1- and control Ab-treated mice (Fig. 3⇓A). Moreover, OT-I cells recovered from both groups of mice expressed high levels of CD44, indicating that they were all Ag-experienced. However, mice treated with anti-PD-L1 contained 5–10 times more OT-I cells per lymph node when compared with the mice treated with control Ab (Fig. 3⇓A). Furthermore, the absolute number of OT-I cells was greatly increased in the anti-PD-L1-treated mice (Fig. 3⇓C). Thus, PD-1-PDL1 signaling appears to mediate immune tolerance by restricting the expansion or survival of autoreactive CD8+ T cells.

FIGURE 3.
FIGURE 3.

Anti-PD-L1 greatly increases the number of effector CD8+ T cells. Seven days after the transfer of OT-I cells into RIP-mOVA mice, lymph nodes (ILN, Inguinal lymph node; MLN, mesenteric lymph node) and spleens (Spln) from both anti-PD-L1-treated and rat IgG-treated mice were analyzed for the presence of OT-I cells with SIINFEKEL pentamers. A, Dot plots represent gated CD8+ cells from each tissue from both groups in their CD44 and OT-I pentamer staining. B, Histograms show PD-1 expression levels on OT-I+ cells: thicker black line is isotype, thinner black line is rat IgG, and filled gray histogram is anti-PD-L1. C, Total numbers of OT-I cells from each organ as determined by the proportion of OT-I+ cells from CD8+ cells multiplied by the percentage of live cells and with the number of live counted cells. This graph is a combination of results from three independent experiments. D, CTL activity showed as the percentage of target EL4 cells positive for the caspase 3 active form. E, IFN-γ ELISPOT. The results are a representative of three similar experiments.

Recently it has been reported that “exhausted” CD8+ T cells from mice suffering a chronic viral infection overexpressed PD-1 (24). We also found that tolerant CD4+ and CD8+ T cells generated in vitro by activating them in the absence of CD28 and ICOS costimulation exhibited enhanced PD-1 expression when compared with effector T cells (25). Interestingly on day 7 the remaining OT-I cells recovered from the PLNs of RIP-mOVA mice treated with control IgG expressed higher levels of PD-1 on their surface than those from anti-PD-L1-treated mice (Fig. 3⇑B). This finding suggests that OT-I cells from mice treated with anti-PD-L1 were not tolerant. Indeed, we found an increased percentage of OT-I cells from anti-PD-L1-treated mice that were more cytotoxic and produced more IFN-γ than those from control mice (Fig. 3⇑, D and E). Thus, PD-1/PD-L1 prevents the progression of autoreactive CD8+ T cells to long-lasting effector cells.

PD-1/PD-L1 interaction restricts effector differentiation of autoreactive CD8+ T cells

The above results indicate a critical role of PD-1/PD-L1 in the control of autoimmune diabetes mediated by CD8+ T cells. Because PD-L1 is broadly expressed, it is unclear whether its interaction with PD-1 regulates CD8+ T cell tolerance during the initial priming or beyond. We thus tested the effect of anti-PD-L1 treatment on OT-I cells when they first encountered Ag-presenting DC in PLN. Cell division of CFSE-labeled OT-I cells in PLNs and spleens was first evaluated at 60 h after their transfer into RIP-mOVA or C57BL/6J mice. Divisions of OT-I cells occurred only in RIP-mOVA mice and primarily in PLNs (Fig. 4⇓A). Anti-PD-L1 only moderately increased (by ∼7%) OT-I cell division in PLN, which was reflected in cells recirculating into the spleen (Fig. 4⇓A). This suggests that PD-1 signaling does not primarily control the activation or proliferation of OT-I cells in vivo. In contrast, a profound effect on IL-2 and especially IFN-γ production was observed after blocking PD-L1, where the number of cytokine-producing cells was doubled in the dividing OT-I cells (Fig. 4⇓B). Interestingly, cytokine-expressing cells were among those exhibiting the highest numbers of cell division, which suggests a role of PD-1 in sustaining T cell activation programs. In addition, the blockade of PD-1 also resulted in greatly enhanced granzyme B expression in dividing OT-I cells (Fig. 4⇓C). In contrast, we did not observe an enhanced proliferation or effector differentiation of OT-I cells when an anti-PD-L2 blocking Ab was administered in a similar manner (data not shown). These results indicate that PD-L1/PD-1 interaction regulates the tolerance of autoreactive CD8+ T cells during the priming phase by controlling their differentiation into effector cells. Subsequently, “nonexhausted” effector T cell likely exhibits prolonged survival because larger numbers of OT-I cells can be recovered from RIP-m OVA mice treated with anti-PD-L1 Abs at day 7 (Fig. 3⇑, A and C).

FIGURE 4.
FIGURE 4.

PD-1 controls effector differentiation of autoreactive CD8+ T cells. Purified OT-I cells were labeled with CFSE and transferred into RIP-mOVA or C57BL/6J mice followed by treatment with anti-PD-L1 (A and B), anti-PD-1 (C), or control IgG. Sixty hours after the transfer, proliferation in PLN (A) and cytokine (B) and granzyme B expression (C) of OT-I cells were assessed. The numbers in the dot plots represent the percentage of dividing cells. ALN, Axillary lymph node; ILN, inguinal lymph node; MLN, mesenteric lymph node; and SLN, spleen.

In summary, we show here that PD-1/PDL1 interaction is required for maintaining peripheral tolerance of autoreactive CD8+ T cells on a genetic background (C57BL/6J) resistant to T1D. Furthermore, although our results at this stage do not rule out PD-1 function beyond priming in lymph nodes, they have indicated a novel function of PD-1/PD-L1 in peripheral tolerance by restricting effector differentiation of autoreactive CD8+ T cells. This observation is consistent with our recent in vitro data showing that a PD-1 blockade restored the effector function of CD8+ T cells activated in the absence of CD28 and ICOS costimulation (25). Our results thus agree on the roles of PD-1 and PD-L1 as negative costimulatory molecules in T1D as described by others (15, 16, 17). Interestingly, PD-L2 was recently found to be important in CD4+ T cell tolerance to oral Ags (26). Impairments of an immune tolerance mechanism regulated by PD-1 could thus lead to susceptibility to autoimmunity, while inhibiting PD-1 may be explored in the boosting of immunity to infectious agents and to cancer.

Acknowledgments

We thank Dr. William Heath for RIP-mOVA mice, Ingrid Udris, Ying Wang, and Yang Yang for technical assistance and the entire Dong laboratory for their help and discussion.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work is supported in part by grants from the National Institutes of Health (to C.D.). C.D. is a Cancer Research Institute Investigator and a Trust Fellow of the M. D. Anderson Cancer Center.

  • ↵2 Address correspondence and reprint requests to Dr. Chen Dong, Department of Immunology, M. D. Anderson Cancer Center, 7455 Fannin, Unit 906, Houston, TX 77030. E-mail address: cdong{at}mdanderson.org

  • ↵3 Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; mOVA, membrane-bound chicken OVA; PD, programmed death; PD-L, PD ligand; PLN, pancreatic LN; RIP, rat insulin promoter.

  • Received September 11, 2006.
  • Accepted October 17, 2006.

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