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. Author manuscript; available in PMC: 2008 Oct 15.
Published in final edited form as: J Immunol. 2007 Oct 15;179(8):5204–5210. doi: 10.4049/jimmunol.179.8.5204

PDL1 Is Required for Peripheral Transplantation Tolerance and Protection from Chronic Allograft Rejection1

Katsunori Tanaka *, Monica J Albin *, Xueli Yuan *, Kazuhiro Yamaura *, Antje Habicht *, Takaya Murayama *, Martin Grimm †, Ana Maria Waaga †, Takuya Ueno *, Robert F Padera ‡, Hideo Yagita §, Miyuki Azuma , Tahiro Shin ∥, Bruce R Blazar #, David M Rothstein **, Mohamed H Sayegh *, Nader Najafian *,2
PMCID: PMC2291549  NIHMSID: NIHMS44068  PMID: 17911605

Abstract

The PD-1:PDL pathway plays an important role in regulating alloimmune responses but its role in transplantation tolerance is unknown. We investigated the role of PD-1:PDL costimulatory pathway in peripheral and a well established model of central transplantation tolerance. Early as well as delayed blockade of PDL1 but not PDL2 abrogated tolerance induced by CTLA4Ig in a fully MHC-mismatched cardiac allograft model. Accelerated rejection was associated with a significant increase in the frequency of IFN-γ-producing alloreactive T cells and expansion of effector CD8+ T cells in the periphery, and a decline in the percentage of Foxp3+ graft infiltrating cells. Similarly, studies using PDL1/L2-deficient recipients confirmed the results with Ab blockade. Interestingly, while PDL1-deficient donor allografts were accepted by wild-type recipients treated with CTLA4Ig, the grafts developed severe chronic rejection and vasculopathy when compared with wild-type grafts. Finally, in a model of central tolerance induced by mixed allogeneic chimerism, engraftment was not abrogated by PDL1/L2 blockade. These novel data demonstrate the critical role of PDL1 for induction and maintenance of peripheral transplantation tolerance by its ability to alter the balance between pathogenic and regulatory T cells. Expression of PDL1 in donor tissue is critical for prevention of in situ graft pathology and chronic rejection.

Tcell recognition of alloantigen is the central and primary event which ultimately leads to graft rejection, although several other factors may also contribute to the effector mechanisms of graft failure (1). T cells require two distinct signals for full activation. The first is provided by the engagement of the TCR with the MHC plus peptide complex on APCs, and that determines the Ag specificity of the response. The second positive costimulatory signal is provided by engagement of one or more T cell surface receptors with their specific ligands on APCs. The best characterized and perhaps most important costimulatory pathway is the CD28/CTLA4:B7-1/B7-2 pathway. Interaction of CD28, constitutively expressed on T cells, with B7-1 and B7-2, expressed on APCs, provides a second positive signal which results in full T cell activation including cytokine production, clonal expansion and prevention of anergy, and T cell survival (2). Ligation of CD28 by B7-1 or B7-2 is blocked by CTLA4Ig, a recombinant fusion protein which contains the extracellular domain of CTLA4 fused to an IgG H chain tail. We and others have shown that CD28-B7 blockade prevents acute allograft rejection and induces donor specific tolerance in several experimental animal models (3-7). Preclinical studies indicate the efficacy of B7 blockade by CTLA4Ig in primate renal (8, 9) and islet (10-13) transplantation models. A recent clinical trial in renal transplant recipients with Belatacept (LEA29Y), a mutant form of CTLA4Ig (14) suggest that it is as effective as cyclosporine as a means to prevent acute rejection, and may preserve the glomerular filtration rate and reduce CAN (15). Belatacept is currently undergoing clinical testing in phase III trials in kidney transplant recipients.

The programmed death 1 (PD-1)3 receptor and its ligands, PDL1 (B7-H1) and PDL2 (B7-DC) have been recently characterized (16, 17). PD-1 is induced on peripheral T cells, B cells and myeloid cells upon activation (18). PDL1 is widely expressed on resting cells and up-regulated on activated B, T, myeloid, dendritic cells and also on many non-hemopoietic tissues such as heart, placenta, pancreas, brain, muscle and endothelial cells, whereas PDL2 is expressed exclusively on dendritic cells and monocytes (19). Parenchymal expression of PDL1 may serve to regulate autoreactive T or B cell responses in peripheral tissues, and/or may serve to regulate inflammatory responses at these sites (20-22). Previous work by Ozkaynak et al. (23) showed that in the context of sub-maximal TCR or positive costimulatory signal blockade, PD-1 signaling can block allograft rejection and modulate T and B cell dependent pathologic immune responses in vivo. In an islet transplantation model, Gao et al. demonstrated that PDL1.Ig synergized with anti-CD154 mAb to induce long-term survival of islet allografts (24).

The PD-1 pathway has been shown to play an important role in regulating the alloimmune response in experimental models of skin and heart transplantation (25, 26), and graft vs host disease (27). PDL1 has also been demonstrated to play an important role in fetomaternal tolerance (28). We have demonstrated that blockade of PD-1:PDL1 pathway resulted in accelerated rejection of fully MHC-mismatched cardiac allografts in CD28 and B7-1/B7-2 double deficient recipients (25), suggesting that the interplay between positive stimulatory and negative regulatory costimulatory signals may also be an important determinant of the outcome of an alloimmune response. More recently, Fife et al. reported that PD-1:PDL1 signaling is required for protection from autoimmune diabetes by two different therapeutic strategies in the NOD mouse model (29). These results prompted us to investigate the role and mechanisms of PD-1:PDL pathway in acquired transplantation tolerance.

Materials and Methods

Mice

C57BL/6 (B6, H-2b) and BALB/c (H-2d) mice were purchased from The Jackson Laboratory (Bar Habor, ME). PDL1-/- (28) and PDL2-/- (obtained from Tahiro Shin PhD) mice on the B6 background were maintained as a breeding colony in our animal facility. All mice were used at 8-12 wks of age and housed in accordance with institutional and National Institutes of Health guidelines.

Fusion proteins and mAbs, and in vivo treatment protocols

The anti-mouse PDL1 mAb was generously provided by Dr. M. Azuma (30). The anti-mouse PDL2 mAb (TY25) was supplied by Dr. H. Yagida. We have previously demonstrated the blocking properties of the mAbs against PDL1, and PDL2 (25, 26, 31). All mAbs were manufactured and purified by Bioexpress Cell Culture. CTLA4Ig (Abatacept) is a human IgG1 fusion protein (Bristol Myers Squibb).

PDL1 and PDL2 mAbs were given i.p. according to the following protocol: 0.5 mg of mAb on the day of transplantation and 0.25 mg on days 2, 4, 6, 8, and 10 after transplantation. CTLA4Ig was given i.p.: 0.5 mg of mAb on the day of transplantation and 0.25 mg on days 2, 4, and 6 after transplantation.

Heterotopic heart transplantation

Vascularized heart grafts were transplanted using microsurgical techniques as described by Corry et al. (32). Rejection was defined as complete cessation of cardiac contractility as determined by direct visualization. Graft survival is shown as the median survival time (MST) in days.

Histology

Cardiac graft samples from transplanted mice harvested post transplant were fixed in 10% formalin, embedded in paraffin, coronally sectioned, and stained with H&E and Verhoeff’s elastin for evaluation of the degree of rejection according to the International Society for Heart and Lung Transplantation guidelines (33), and for cellular infiltration and vasculopathy by light microscopy (34, 35). The severity of vasculopathy was graded according to the percentage of luminal occlusion by intimal thickening using a scoring system previously described (34, 35). Only vessels that were cut orthogonally and displayed a clear internal elastic lamina were scored. An examiner blinded to the groups read all the samples. Immunohistochemical staining for FOXP3 was performed as previously published (36). The mean percentage of positive stained cells by immunohistochemical analysis for FOXP3 was semiquantitatively classified based on cell counting in six individual magnified fields (×400 magnification).

ELISPOT assay

Splenocytes from transplanted recipient mice were harvested 2-3 wk post transplant and were used as responders. Splenocytes obtained from naive wild-type BALB/c mice were used as stimulators. The ELISPOT assay was adapted to measure the frequency of alloreactive T cells producing IFN-γ (Th1) and IL-4/IL-5 (Th2) secreting cells, as previously described (25). The resulting spots were counted on a computer-assisted enzyme-linked immunospot image analyzer (Cellular Technology), and frequencies were expressed as the number of cytokine-producing spots per 0.5 × 106 splenocytes.

Flow cytometry

Splenocytes were obtained from recipient mice posttransplantation and stained with fluorochrome-labeled mAbs against CD8, CD62 ligand (CD62L), CD44 (BD Biosciences). Percentages of effector CD8 T cells expressing the CD44highCD62Llow phenotype are calculated as described (25).

To evaluate the percentage of chimeric donor cells in recipients, cells were stained with fluorochrome-conjugated Abs (anti-CD8, anti-CD4 and anti-H2Kb and isotype controls) purchased from BD Biosciences. Flow cytometry analysis was performed using a FACSCaliber system (BD Biosciences, San Jose, CA) and analyzed using CellQuest software.

Mixed allogeneic chimerism model

Bone marrow was isolated from B6 wild-type donor mice. RBCs were lysed using ACK lysis buffer and a single cell suspension of the bone marrow derived cells (BMC) in sterile PBS was prepared. BALB/c mice were conditioned with 250rad of whole body irradiation one day before transfer of 40 million B6 donor BMC on day 0, followed by MR1 at 0.2 mg on days 1-5, 8, 11, 14 and rapamycin 1.5 mg/kg on days 1-13 (37). To investigate the effect of PD-1:PDL pathway on induction of chimerism and tolerance, we administered blocking Abs against PDL1 or PDL2 i.p. at a dose of 0.5 mg on day 0 and 0.25 mg on day 2, 4, 6, 8 and 10. Donor skin grafts are transplanted 60 days after bone marrow transfer. The percentage of H-2Kb chimeric donor cells in recipient peripheral blood is measured by flow cytometry 8 wk after chimerism induction in various treatment groups.

Statistics

Kaplan-Meier survival graphs were constructed and a log rank comparison of the groups was used to calculate p values. Student’s t test was used for comparison of means between experimental groups examined by ELISPOT assay. Differences were considered to be significant at values p < 0.05.

Results

PDL1 but not PDL2 blockade abrogates the induction and maintenance of tolerance by CTLA4Ig therapy

We have previously demonstrated that blockade of PD-1:PDL1 pathway resulted in accelerated rejection of fully MHC-mismatched cardiac allografts in CD28 and B7-1/B7-2 double deficient recipients (25). These results prompted us to investigate the role and mechanisms of PD-1:PDL pathway in induction and maintenance of acquired transplantation tolerance. To this end we first used a previously described fully MHC-mismatched cardiac transplant model using BALB/c (H-2d) hearts as donors and C57BL/6 (H-2b) mice as recipients where tolerance was induced by CD28-B7 T costimulatory blockade using CTLA4Ig (38). CTLA4Ig treatment induced long-term graft survival in all recipients (MST > 100 days, n = 8; p < 0.0001 vs. MST 7 days; n = 18 for IgG isotype-treated controls) (Fig. 1A). Administration of anti-PDL1 mAb starting on the day of transplantation resulted in graft rejection in the CTLA4Ig-treated recipients (MST = 31.5 days; n = 12; p < 0.0005) (Fig. 1A). In contrast, administration of anti-PDL2 mAb did not result in any significant change in graft survival in the CTLA4Ig-treated recipients (MST > 100 days; n = 5; p = ns) (Fig. 1A). Furthermore, administration of the anti-PDL1-mAb beginning on day 60 posttransplantation also precipitated rejection in CTLA4Ig treated recipients (MST = 83, n = 6, p = 0.02) (Fig. 1B).

FIGURE 1.

FIGURE 1

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A, Early PDL1 but not PDL2 blockade abrogates tolerance induction by CTLA4Ig. CTLA4Ig treatment induced long-term graft survival in all recipients (MST > 100 days, n = 8; p < 0.0001vs MST 7 days; n = 18 for IgG isotype-treated controls). Administration of anti-PDL1 mAb starting on the day of transplantation resulted in graft rejection in the CTLA4Ig-treated recipients (MST = 31.5 days; n = 12; p < 0.0005). In contrast, administration of anti-PDL2 mAb did not result in any significant change in graft survival in the CTLA4Ig-treated recipients (MST > 100 days; n = 5; p = ns). B, Delayed PDL1 blockade abrogates tolerance induction by CTLA4Ig. Although long-term allograft survival was achieved by CTLA4Ig treatment, administration of the anti-PDL1-mAb beginning on day 60 posttransplantation also precipitated rejection in CTLA4Ig treated recipients (MST = 83, n = 6, p = 0.02).

Histological evaluation of rejected grafts harvested from PDL1 mAb treated recipients two weeks after blockade showed scattered inflammatory cell infiltrates, vasculitis with inflammation and variable degrees of vascular luminal obliteration (Fig. 2). In comparison, grafts from CTLA4Ig-treated animals were protected from acute and chronic rejection (Fig. 2).

FIGURE 2.

FIGURE 2

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Pathological analysis of cardiac tissue after CTLA4Ig treatment 2-3 wk after PDL1 blockade. Histological evaluation of grafts harvested from PDL1 mAb-treated recipients 2 wk after blockade showed scattered inflammatory cell infiltrates, vasculitis with inflammation and variable degrees of vascular luminal obliteration. In comparison, grafts from CTLA4Ig-treated animals were protected from acute and chronic rejection.

PDL1 blockade alters the balance between pathogenic and regulatory T cells

We then determined the frequency of alloantigen-specific alloreactive Th1 (IFN-γ) and Th2 (IL-4 and IL-5) cells in recipients treated with CTLA4Ig and anti-PDL1 mAb 2-3 wk posttransplant, using a previously published ELISPOT assay (25, 39). In the CTLA4Ig treatment group, the frequency of alloreactive IFN-γ-producing splenocytes was significantly decreased as compared with the untreated control recipients (19.8 ± 6 vs 469.6 ± 9.8, p < 0.0001) (Fig. 3A). Anti-PDL1-mAb administration during the induction phase resulted in significant increase of the frequency of alloreactive IFN-γ-producing splenocytes as compared with CTLA4Ig alone (66.1 ± 12.8 vs 19.8 ± 6, p = 0.003) (Fig. 3A).

FIGURE 3.

FIGURE 3

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A-C, ELISPOT analysis of Th1 (IFN- γ (A) and Th2 (B and C) cytokine profile of splenocytes derived from transplant recipients treated with CTLA4Ig 2-3 wk after PDL1 blockade. In the CTLA4Ig treatment group, the frequency of alloreactive IFN-γ-producing splenocytes was significantly decreased as compared with the untreated control recipients (A). Anti-PDL1-mAb administration during the induction phase resulted in significant increase of the frequency of alloreactive IFN-γ-producing splenocytes (A). CTLA4Ig treatment produced a slight but nonsignificant decrease in the frequency of IL-4-producing cells as compared with controls, whereas administration of anti-PDL1-mAb significantly increased the IL-4-producing T cell frequency (B). Interestingly, CTLA4Ig treatment significantly increased in the frequency of IL-5-producing cells as compared with controls (C), and anti-PDL1 mAb further increased the IL-5-producing T cell frequency as compared with the CTLA4Ig-treatment group (C). Data are representative of three independent experiments and indicate the mean of triplicate results in each experiment. D and E, Frequency of alloreactive CD8 effector T cells in splenocytes derived from transplant recipients treated with CTLA4Ig 2-3 wk after PDL1 blockade. The frequency of effector CD8+ T cells (expressing a CD62LlowCD44high phenotype) was significantly decreased in the CTLA4Ig treatment group as compared with controls (3.6 ± 1.0% vs 23.7 ± 0.4%, p < 0.001). Anti-PDL1 mAb significantly increased the frequency of effector CD8+ T cells, as compared with the CTLA4Ig treatment group (12.1 ± 2.5% vs 3.6 ± 1.0%, p < 0.001). A representative experiment is shown (D).

CTLA4Ig treatment produced a slight but nonsignificant decrease in the frequency of IL-4-producing cells as compared with controls, whereas administration of anti-PDL1-mAb significantly increased the IL-4-producing T cell frequency vs CTLA4Ig alone (289.9 ± 24.9 vs 149.4 ± 5.3, p < 0.0001) (Fig. 3B). Interestingly, CTLA4Ig treatment significantly increased the frequency of IL-5-producing cells as compared with controls (12.3 ± 2.9 vs 4.6 ± 1.0, p = 0.025) (Fig. 3C), and anti-PDL1 mAb further increased the IL-5-producing T cell frequency as compared with the CTLA4Ig treatment group (22.3 ± 2.1 vs 12.3 ± 2.9, p = 0.014) (Fig. 3C). Taken together, these data show that PDL1 blockade significantly expands both Th1 and Th2 alloreactive T cells in CTLA4Ig-treated recipients. However, the magnitude of the Th1 effect is far greater than Th2 effects.

Next, to assess the effect of PDL1 blockade on generation of alloreactive effector CD8+ T cells, we measured the percentage of effector CD8+ cells (expressing a CD62LlowCD44high phenotype) generated 2-3 wk after transplantation, in untreated control recipients and in recipients treated with CTLA4Ig or CTLA4Ig with anti-PDL1-mAb (25). As seen in Fig. 3, D and E, the frequency of effector CD8+ T cells was significantly decreased in the CTLA4Ig treatment group as compared with controls (3.6 ± 1.0% vs 23.7 ± 0.4%, p < 0.001). Anti-PDL1 mAb significantly increased the frequency of effector CD8+ T cells, as compared with the CTLA4Ig treatment group (12.1 ± 2.5% vs 3.6 ± 1.0%, p < 0.001), albeit to levels lower than the no treatment controls (Fig. 3, D and E). These data indicate that PDL1 blockade enhanced generation of effector T cells in CTLA4Ig-treated recipients.

We then determined the percentage of Foxp3+CD4+CD25+ T cells in the spleens of CTLA4Ig-treated animals with or without concomitant anti-PDL1 mAb administration, as compared with untreated rejecting controls. There was no difference in the percentage of these cells between the groups (10 ±1.5 vs 9 ± 1.7% when gating on CD4+ T cells). However, Foxp3 staining in the heart grafts demonstrated significant increase in the percentage of infiltrating cells expressing Foxp3 in CTLA4Ig-treated recipients (Fig. 4). Importantly, PDL1 blockade resulted in significant decrease in the percentage of cells expressing Foxp3 as compared with CTLA4Ig treatment alone (11.6 ± 4.6 vs 25 ± 2.2%, n = 6, p = 0.02) (Fig. 4). Therefore, abrogation of tolerance by PDL1 blockade is associated with an imbalance favoring significant expansion of alloreactive T cells in the periphery (Fig. 3) concomitant with decrease infiltration of Tregs in the graft (Fig. 4).

FIGURE 4.

FIGURE 4

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Immunohistochemical analysis of FOXP3 in cardiac tissues after CTLA4Ig and PDL1 blockade. Foxp3 staining in the heart grafts demonstrated significant increase in the percentage of infiltrating cells expressing Foxp3 in CTLA4Ig-treated recipients. Importantly, PDL1 blockade resulted in significant decrease in the percentage of cells expressing Foxp3 as compared with CTLA4Ig treatment alone (25 ± 2.2% vs 11.6 ± 4.6, n = 6, p = 0.02).

Role of donor vs recipient PDL1 in tolerance and chronic rejection

PDL1 has been shown to be expressed on APCs as well as parenchymal tissue including the heart (40, 41). To further dissect its role in tolerance, we first used PDL1/L2 deficient mice as recipients in an acquired tolerance model by CTLA4Ig. Although CTLA4Ig-treated PDL2 deficient mice (similar to wild-type recipients) demonstrated significant prolongation of allograft survival as compared with nontreated controls (MST 121 days, n = 6 vs MST 9, n = 5, p = 0.0009), PDL1 deficient vs wild-type recipients demonstrated significantly shortened graft survival after CTLA4Ig therapy (MST = 30, n = 5 vs >100 days, n = 4, p = 0.0007) (Fig.5A). Similar to the Ab therapy data (Fig. 2), histological assessment showed cellular rejection within the myocardium and vasculitis with inflammation, and vascular luminal obliteration in BALB/c wild-type allografts transplanted into CTLA4Ig-treated PDL1 deficient recipients (not shown). In contrast, PDL1 deficient heart allografts were accepted by wild-type BALB/c recipients treated with CTLA4Ig (MST > 100, n = 4) (Fig. 5B). However, pathological examination of the grafts (>100 days) showed severe chronic rejection and vasculopathy in PDL1 deficient hearts as compared with wild-type grafts from the CTLA4Ig-treated mice which showed protection from chronic rejection and vasculopathy (Fig. 5C). These data indicate that expression of PDL1 on recipient cells, likely host APCs, is critical for induction and maintenance of graft acceptance, while expression of PDL1 in the graft protects from local pathology and chronic rejection.

FIGURE 5.

FIGURE 5

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Role of recipient versus donor PDL1 expression after treatment with CTLA4Ig. A, PDL1-deficient recipients demonstrated significantly shortened graft survival after CTLA4Ig therapy (MST = 30, n = 5, p = 0.0007) as compared with wild-type B6 recipients. B, PDL1-deficient heart allograft were accepted by wild-type BALB/c recipients treated with CTLA4Ig (MST > 100, n = 4). C, Pathological examination of the grafts (>100 days) showed severe chronic rejection and vasculopathy in PDL1-deficient hearts after CTLA4Ig treatment as compared with wild-type grafts which showed protection from chronic rejection and vasculopathy.

Role of PD-1:PDL pathway in induction of mixed allogeneic chimerism

Bone marrow alloengraftment under a nonmyeloablative-conditioning regime using costimulatory blockade strategies has been effective in inducing robust tolerance to solid organs in transplantation. We wanted to study the role of PD-1:PDL1/2 pathway in a model of central tolerance induced by mixed allogeneic chimerism involving a nonmyeloablative regimen of donor bone marrow transplantation, sirolimus and anti-CD40L mAb (see methods) (37). In this model, fully MHC-mismatched B6 skins transplanted 60 days post bone marrow transplantation are accepted by conditioned BALB/c transplant recipients (MST > 150 days, n = 4). In the context of sirolimus and anti-CD40L mAb, the coadministration of mAb against PDL1/L2 at the time of bone marrow transplantation did not substantially alter the percentage of H-2Kb chimeric donor cells in recipient peripheral blood 8 wk after chimerism induction regardless of treatments administered (controls: 47.23 ± 6.98, PDL1 blockade: 51.73 ± 7.8, and PDL2 blockade: 52.03 ± 2.46%) (Fig. 6). Consistent with these data there was no effect of coadministering anti-PDL1/L2 mAbs on the long-term survival of skin grafts (Fig. 7A, MST > 150 days, n = 4). These findings are in stark contrast to using CD40-CD154 blockade by MR1 to induce long-term allograft survival (peripheral tolerance) in B6 recipients of BALB/c hearts (MST > 100 days, n = 6). Interestingly, in this case anti-PDL1 mAb results in acute allograft rejection in MR1-treated (MST 28, n = 8, p = 0.0003) recipients (Fig. 7B). Therefore, PDL1 is not required for induction of central tolerance by mixed allogeneic chimerism, using this particular model of conditioning regimen and strain combination.

FIGURE 6.

FIGURE 6

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Effect of PDL1/L2 blockade on mixed allogeneic chimerism in a model of central tolerance. The percentage of H-2Kb chimeric donor cells in recipient peripheral blood 8 wk after chimerism induction was not affected regardless of treatments administered (controls: 47.23 ± 6.98, PDL1 blockade: 51.73 ± 7.8, and PDL2 blockade: 52.03 ± 2.46%). One representative example is shown in the top panel. Data are representative of four independent experiments and indicate the mean of duplicate results in each experiment.

FIGURE 7.

FIGURE 7

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Differential effect of PDL1/L2 blockade on allograft tolerance induced by mixed allogeneic chimerism based on a nonmyeloablative regimen using CD40-CD154 blockade versus tolerance induced by CD40-CD154 blockade alone. A, Fully MHC-mismatched skin allografts are accepted by conditioned transplant recipients. Tolerance is abrogated neither by PDL1 nor PDL2 blockade. B, Tolerance to fully MHC-mismatched cardiac allografts induced by CD40-CD154 blockade is only abrogated by PDL1, but not PDL2 blockade.

Discussion

In this paper, we set out to explore the role of PD-1:PDL1/PDL2 pathway in acquired transplantation tolerance. First, using blocking mAbs against PDL1 ligands we clearly demonstrate that recipient but not donor PDL1 is necessary for induction and maintenance of acquired transplantation tolerance after CTLA4Ig therapy. The data in PDL1 deficient mice used as recipients complement our findings with PDL1 blockade using Ab, and provide a definitive proof that PDL1 delivers a negative signal to regulate allogenic T cell responses in acquired tolerance by CTLA4Ig. Furthermore, we demonstrate a key role of PDL1 expression in recipients rather than donor tissue for the tolerogenic effects of CTLA4Ig.

Another interesting finding in this study is that PDL2 blockade does not appear to play an important role in regulating the alloimmune response after CD28/CTLA4:B7 T cell costimulatory blockade, regardless of whether studies were performed using Ab blockade or PDL2 deficient recipients. These results are consistent with previously published data in the fully allogeneic cardiac transplant model (25).

Our results appear to be unique to peripheral tolerance strategies in that PDL1/L2 blockade did not abrogate induction of mixed allogeneic chimerism in a model in which central transplantation tolerance has been shown to be mediated by clonal deletion. These findings are consistent with the notion that the major mechanism of central tolerance is the intrathymic deletion of alloreactive T cells (42).

Our mechanistic data show that blocking or genetically deleting the PDL1 costimulatory molecule in recipients promotes cardiac allograft rejection by expansion of alloreactive Th1 cells and CD8+ effector cells. Thus, PD-1:PDL interaction may contribute to peripheral transplantation tolerance by limiting the expansion of alloreactive T cells (26), possibly by cell cycle arrest (43), by increasing apoptosis of T cells (26, 44, 45), or by active regulation of the alloimmune response by a subpopulation of CD4+CD25+ regulatory T cells (26).

Consistent with a previous study showing that Foxp3 expression was specifically up-regulated within allografts of mice displaying donor-specific tolerance after CD154/donor-specific transfusion therapy (36), we showed that Foxp3+ T cells existed in the grafts of recipients treated with CTLA4Ig by immunohistological staining, and their frequency decreased after PDL1 blockade. Interestingly, PD-1 mRNA is highly expressed in CD4+CD25+ regulatory T cells and anergic T cells, suggesting several means by which PD-1 may be involved in regulating T cell tolerance (46, 47). Another study demonstrated that PD-1:PDL1 interaction is essential for induction of regulatory cells by intratracheal delivery of alloantigen (48). In addition, alloantigen presentation by vascular endothelium to CD4+ T lymphocytes induced CD4+CD25+ Foxp3+ regulatory T cells which could inhibit T cell proliferation in an alloantigen-specific fashion (49). In this study, the conversion of CD4+CD25- T cells was dependent on PDL1 costimulation. Collectively, our data support a shift in the balance in favor of effector T cells after PDL1 blockade by prohibiting negative signaling to these cells. Whether there is an additional direct effect on function or induction of Tregs after PDL1 blockade will require further investigation.

One important and novel finding in this manuscript is the observation that donor deficiency of PDL1 but not PDL2 accelerated chronic allograft vasculopathy but did not abrogate allograft acceptance after treatment with CTLA4Ig. PDL1 is constitutively expressed in T cells, B cells, macrophages and dendritic cells (DCs), and further up-regulated following activation of these cells (30). The expression of PDL1 is also detected on nonlymphoid cells, for example, endothelial cells in the heart, β cells in the pancreas, glial cells in inflamed brain and muscle cells (31, 50 - 53). In addition PDL1 expression has been detected on parenchymal cells including vascular endothelial cells (41). Koga et al. previously demonstrated increased severity of graft arterial disease in anti-PDL1 mAb treated recipients (40). These results are also consistent with recent observations by Kier et al. in the autoimmune diabetes model in NOD mice showing that islet cell expression of PDL1 regulated the autoimmune response against islet autoantigens in vivo (21). Our data suggest that PDL1 expression in donor tissue, possibly on vascular endothelium of the donor organ, can down-regulate recipient alloreactive T cell responses by interaction with the PD-1 receptor expressed on alloreactive T cells, thus limiting the local inflammation leading to chronic rejection and vasculopathy.

Our studies have important implications for understanding physiologic mechanisms that promote transplantation tolerance and shed light on the complex interactions between positive and inhibitory/negative costimulatory pathways. Our observations are clinically relevant for two important reasons. First, targeting PD-1 or its ligands to deliver an inhibitory signal may provide an adjunct strategy to promote transplantation tolerance (54). Second, since CTLA4Ig is being developed clinically in autoimmune diseases and transplantation (55), its important to understand the signals that may abrogate its beneficial therapeutic effects and avoid inhibiting the expression or blockade of these signals in humans.

Footnotes

1

This work was supported by National Institutes of Health Grants R01-AI51559, R01-AI34495, 2 R37 HL56067, R01-HL63452, P01-AI041521, and P01-AI56299. N.N. is a recipient of the American Heart Association Scientist Development Grant and National Institutes of Health 1K08AI064335.

3
Abbreviations used in this paper:
PD-1
programmed death 1
MST
median survival time.

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