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Keywords:

  • Costimulatory molecules;
  • Immunobiology;
  • Transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Programmed cell death-1 (PD-1, CD279) and its widely expressed, inducible ligand, PD-L1 (CD274), together dampen T cell activation, but whether they are essential for allograft tolerance is unknown. We show, using gene-deficient mice and blocking mAbs in wild-type mice, that costimulation blockade is ineffectual in PD-1–/– or PD-L1–/– allograft recipients, or in wild-type allograft recipients treated with anti-PD-1 or anti-PD-L1 mAb. Alloreactive PD-1–/– CD4 and CD8 T cells had enhanced proliferation and cytokine production compared to wild-type controls, and anergy could not be induced in PD-1-deficient CD4 T cells. We conclude that without inhibitory signals from PD-1 ligation, alloantigen-induced T cell proliferation and expansion cannot be regulated by costimulation blockade, and peripheral tolerance induction cannot occur.

Abbreviations:
DST:

donor-specific transfusion

PD-1:

programmed cell death-1

PD-L1:

PD-1 ligand

PI3K:

phosphoinositide 3-kinase

qPCR:

quantitative PCR

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Programmed cell death 1 (PD-1, CD279) is a type 1 transmembrane protein containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) 1, which is induced upon the activation of CD4 and CD8 T cells as well as B, NKT and myeloid cells 2. PD-1 has two ligands, PD-L1 (also known as B7-H1 or CD274), constitutively expressed by many tissues and up-regulated on activation 3, and PD-L2 (also known as B7-DC or CD273), expressed primarily by activated DC and macrophages 4. Upon ligation by either molecule, PD-1 recruits the SHP-1 and SHP-2 phosphatases to its ITSM, leading to dephosphorylation of effector molecules downstream of the TCR and the BCR, such as Syk and phosphoinositide 3-kinase (PI3K), as well as decreasing CD28-mediated activation of PI3K 5. Without PD-1, C57BL/6 mice develop a lupus-like glomerulonephritis and arthritis 6, BALB/c mice develop a fatal cardiomyopathy due to autoantibody to troponin 1 7, and NOD mice show an earlier onset and more severe diabetes than wild-type (WT) NOD mice 8. PD-L1 expression by resting T, B cells and DC, plus macrophages and various parenchymal cells, may contribute to peripheral T cell tolerance, as backcrossing of PD-L1–/– and NOD mice, or mAb blockade of PD-L1 in control NOD mice, accelerated development of diabetes 9, 10. In contrast to PD-1 and PD-L1, little is known of the in vivo functions of PD-L2, although mAb blockade of PD-L2 accelerated the onset and increased the severity of disease during murine experimental autoimmune encephalomyelitis 11.

We showed previously that ligation of PD-1 using a PD-L1.Ig fusion protein, but not PD-L2.Ig, dampened T cell activation and, when used in conjunction with a subtherapeutic regimen of cyclosporine or rapamycin, significantly enhanced cardiac and skin allograft survival and prevented the development of transplant arteriosclerosis and other features of chronic rejection 12. Similarly, injection of mAb to PD-1 13 or PD-L1 14 accelerated allograft rejection in immunosuppressed recipients. However, although most studies indicate that the PD-1/PD-L1 pathway negatively regulates T cell activation and proliferation, there is evidence for a positive costimulatory effect of PD-L1 on TCR activation of naive murine T cells 3, 15. Here, we report data showing that both PD-1 and PD-L1 are essential for the induction and maintenance of allograft tolerance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

PD-1 and PD-L1 promote allograft survival

Since there are no known data on allograft survival in PD-1–/– or PD-L1–/– mice, we first undertook heterotopic vascularized cardiac allografting across a full MHC mismatch using PD-1–/– and PD-L1–/– recipients. WT C57BL/6 (H-2b) recipients rejected BALB/c (H-2d) cardiac allografts by 6–8 days post transplant, whereas PD-1–/– recipients rejected their grafts by day 6 (Fig. 1a), and analogous findings were seen using PD-L1–/– recipients (Fig. 1b). The finding that neither lack of PD-1 nor PD-L1 in allograft recipients led to statistically significant acceleration of rejection likely reflects the extreme potency of the alloresponse, involving maximal activation of all arms of the immune system in a hitherto naive host. In contrast, clear-cut contributions of the PD-1/PD-L1 pathway to regulation of host alloresponses were apparent in studies of allograft tolerance. Whereas permanent (>120 days) allograft survival was achieved in WT recipients using CD154 mAb plus donor splenocyte transfusion (DST) or CD154 plus CTLA-4Ig, CD154/DST (Fig. 1c) or CD154/CTLA-4Ig (Fig. 1d) costimulation blockade was ineffectual in PD-1–/– recipients, each resulting in acute rejection within 1–2 wk of engraftment (p <0.01). Consistent with a key role of the PD-1/PD-L1 pathway in tolerance induction, CD154/DST therapy failed to induce long-term allograft survival in WT mice treated with anti-PD-1 mAb (Fig. 1e), or when administered to PD-L1–/– recipients (Fig. 1f). Collectively, these data show that the presence of the PD-1/PD-L1 pathway is essential for the in vivo induction of allograft tolerance by costimulation blockade.

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Figure 1. Costimulation-induced allograft tolerance requires PD-1 and PD-L1. (a) PD-1–/– recipients rejected cardiac allografts only marginally faster than WT mice (p >0.05). (b) PD-L1–/– recipients rejected cardiac allografts only marginally faster than WT mice (p >0.05). (c) Long-term cardiac allograft survival was induced in WT recipients by CD154 mAb + DST but was ineffective in PD-1–/– recipients (p <0.01). (d) Long-term cardiac allograft survival was induced in WT recipients by CD154 mAb + CTLA-4Ig but was ineffective in PD-1–/– recipients (p <0.01). (e) Beneficial effect of CD154 mAb + DST in WT allograft recipients was abrogated by administration of an anti-PD-1 mAb (p <0.01). (f) CD154 mAb + DST was also ineffective in PD-L1–/– allograft recipients (p <0.01). In each panel, cardiac allografts were transplanted from BALB/c to C57BL/6 mice, using six to eight allografts per group. (g) qPCR analysis of cytokine, chemokine and chemokine receptor expression in cardiac isografts (C57BL/6) and allografts (BALB/c to C57BL/6) harvested at 7 days post transplantation. Recipient WT and PD-1–/– mice were treated with CD154 mAb + DST; mean ± SD, using three grafts per group and *p <0.05 and **p <0.01 for comparisons between allograft groups.

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PD-1 suppresses intragraft gene expression

Cardiac transplant rejection is mediated by cytokine-secreting and activated mononuclear cells that are attracted to an allograft by multiple chemokines that bind to corresponding chemokine receptors 16. We have previously shown that intragraft expression of key chemoattractant pathways, especially involving CXCR3 and CCR5 in the case of fully MHC-disparate allografts, is markedly blunted by CD154/DST costimulation blockade 1719, as is intragraft expression of PD-1 and PD-L1 12. We now report that, compared to WT mice, use of costimulation blockade in PD-1–/– recipients had markedly reduced efficacy, resulting in significantly increased intragraft mRNA levels of IL-2 and IFN-γ cytokines, as well as components of the IP-10/CXCR3 and RANTES/CCR5 chemokine/chemokine receptor pathways (Fig. 1g). Hence, PD-1 expression by activated T cells attenuates cytokine and chemokine generation, and in the absence of this inhibition, costimulation blockade fails to suppress the generation of key immune reactants.

PD-1 regulates T cell alloantigen-induced activation and proliferation in vivo

To assess direct effects of PD-1 on T cell alloactivation and proliferation in vivo, we adoptively transferred CFSE-labeled parental C57BL/6 T cells into C57BL6/DBA F1 recipients. Analysis of cells harvested 3 days later showed only modest enhancement of proliferation of CD4 T cells from PD-1–/– and WT donors, consistent with the lack of a statistically significant difference in the tempo of allograft rejection using fully MHC-mismatched cardiac allografts in untreated WT and PD-1–/– recipients (Fig. 2a). However, whereas CD154 mAb therapy in vivo markedly decreased WT T cell proliferation, PD-1-deficient T cells showed persistence of alloactivation and proliferation (Fig. 2b). Similarly, expression of the activation markers, CD62Llow and CD25high, was significantly increased in the case of PD-1–/– CD4 and CD8 T cells versus WT cells (Fig. 2c). These data provide direct evidence of the inhibitory effects of PD-1 on alloactivation and proliferation in the context of costimulation blockade.

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Figure 2. The PD-1/PD-L1 pathway regulates T cell proliferation in vivo. Comparison of proliferation of WT and PD-1–/– donor T cells recovered from each F1 recipient's lymph nodes and spleen 3 days after transfer of 30 million CFSE-labeled cells shows (a) similar proliferation by both CD4 and CD8 T cells in untreated recipients, but (b) CD154 mAb therapy had considerably less inhibitory effects on CD4 and CD8 T cell alloactivation and proliferation in the case of PD-1–/– donors versus WT mice. (c) Lack of PD-1 led to greater immune activation despite CD154 mAb therapy, compared to WT controls, as assessed by flow cytometric analysis of CD25high and CD62Llow cell populations. Data are representative of three independent experiments; right-hand histograms (mean ± SD) in (b) and (c) show statistical analysis of the results in CD154 mAb-treated mice, with *p <0.01 and ** p < 0.005 for PD-1–/– cells versus the corresponding responses using WT cells.

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PD-1 regulates T cell activation and proliferation in vitro

To assess direct effects of PD-1 on TCR-driven activation in vitro, purified T cells were labeled with CFSE and stimulated with plate-bound anti-CD3 mAb ± anti-CD28 mAb for 72 h. PD-1-deficient CD4 and CD8 T cells were activated significantly more than corresponding WT T cells following anti-CD3 mAb or anti-CD3/CD28 mAb stimulation (Fig. 3a) and proliferated significantly more in the case of anti-CD3 mAb stimulation, whereas the addition of CD28 costimulation led to comparable proliferation for WT and PD-1–/– T cells (Fig. 3b); these differences were also seen by assessment of absolute cell numbers (Fig. 3c). Hence, as with the in vivo data, PD-1 controls the extent of TCR-driven T cell activation in vitro, whereas under condition of maximal activation and costimulation, the inhibitory effects of PD-1 are overridden.

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Figure 3. PD-1/PD-L1 pathway regulates T cell proliferation in vitro. Activation and proliferation in vitro of T cells purified from WT or PD-1–/– C57BL/6 mice using plate-bound anti-CD3 mAb ± anti-CD28 mAb. (a) Flow cytometric analysis of CD25 expression showed that both CD4 and CD8 PD-1–/– T cells underwent increased activation in response to CD3 or CD3/CD28 stimulation as compared to WT controls; results of quantitative analysis of CD25 expression are shown on the right. (b) Flow cytometric analysis of CFSE dilution profiles showed enhanced proliferation of CD4 and CD8 T cells from PD-1–/– mice versus WT controls upon CD3 stimulation. (c) Assessment of absolute cell numbers acquired, and normalization to allow for equal acquisition, showed that CD3 stimulation significantly enhanced the proliferation of CD4 and CD28 T cells from PD-1–/– mice compared to WT controls (**p <0.01). Data in all panels are representative of three independent experiments.

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PD-1 contributes to anergy induction in CD4 T cells

Restimulation of T cells that underwent initial TCR activation plus costimulation blockade typically leads to anergy, characterized by markedly decreased T cell proliferation and IL-2 production. Given exuberant T cell activation by PD-1–/– T cells and the inability to suppress rejection in costimulation blockade-treated allograft recipients, we assessed the effects of PD-1 expression on anergy induction. WT or PD-1–/– CD4 T cells were activated for 72 h with anti-CD3 + anti-CD28 mAb or CTLA-4Ig, rested for 24 h in fresh media and then restimulated overnight with anti-CD3 mAb or anti-CD3 mAb + IL-2 (Fig. 4a). Compared with WT CD4 T cells, CD4 T cells from PD-1–/– mice had markedly enhanced proliferation upon restimulation, despite costimulation blockade during the primary stimulation (Fig. 4b), as well as significantly increased production of Th1 cytokines (IL-2 and IFN-γ), and decreased production of Th2 cytokines (IL-4 and IL-10) (Fig. 4c). Hence, PD-1 contributes to CD4 T cell anergy induction by curtailing T cell activation, proliferation and cytokine production.

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Figure 4. PD-1 contributes to the development of CD4 T cell anergy. (a) Flow cytometric analysis of BrdU incorporation by T cells following two rounds of in vitro stimulation. In contrast to WT cells, restimulated PD-1-deficient CD4 T cells did not show any decrease in BrdU uptake if primary stimulation occurred in conjunction with costimulation blockade. (b) Statistical analysis of BrdU incorporation (mean ± SD) after two rounds of stimulation showed that the proliferation of WT CD4 T cells was significantly decreased compared to that of PD-1–/– CD4 T cells (**p <0.01). (c) Analysis of mRNA (qPCR) and protein (ELISA) expression by corresponding cells from WT and PD-1–/– CD4 T cells; lack of PD-1 led to increased Th1 and decreased Th2 cytokines (mean ± SD, *p <0.05 and **p <0.01 for WT versus PD-1–/– cells treated with CD3/CTLA-4Ig during primary stimulation).

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PD-1 and regulatory T cell numbers and function

An inability to limit T cell responses might also reflect alterations in the number or function of naturally occurring CD4+CD25+ regulatory T (Treg) cells. However, the numbers of Foxp3+ Treg cells in lymph nodes and spleens of WT and PD-1–/– mice were comparable (Fig. 5a), and Treg cells from the latter mice showed intact suppressive activity in standard in vitro assays (Fig. 5b). Hence, the inability to tolerize PD-1 allograft recipients does not appear related to changes in Treg cell numbers or suppressive functions. Comparable findings of a lack of a contribution of PD-L1 to Treg cell suppressive activity was also shown, using cells isolated from PD-L1–/– mice (data not shown).

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Figure 5. PD-1 does not affect Treg cell production or suppressive function. (a) Flow cytometric analysis of the proportions of Foxp3+CD4+CD25+ Treg cells in lymph nodes and spleens of WT and PD-1–/– mice were similar. (b) Treg cells from PD-1–/– also had similar suppressive functions to Treg cells isolated from WT mice; data are representative of three separate experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Of the three known inhibitory pathways involving receptors induced on T cell activation (CD28/CTLA-4/B7, PD-1/PD-L1/PD-L2, BTLA/HVEM), the most attention has been focused on the CD28/CTLA-4/B7 pathway. This reflects the clinical development of CTLA-4Ig to block the positive signal mediated by CD28 ligation by CD80 and CD86. Less attention has been paid to targeting the CTLA-4/B7 interaction, though induction of indoleamine 2,3-dioxygenase in DC, following ligation of surface B7 by CTLA-4, may be of relevance – in some models at least – to the development of tolerance 20. Similarly, no agonistic anti-CTLA-4 mAb are yet described, although transgenic NOD mice expressing a single-chain, membrane-bound anti-CTLA-4 antibody on B cells show protection against development of autoimmune diabetes 21. By contrast, agonist mAb to PD-1 and fusion proteins, such as PD-L1.Ig, have proven effective in disease models by increasing inhibitory signals within activated T cells 12, 22. The current studies suggest that PD-1 signaling serves to curtail T cell alloactivation, proliferation and cytokine production, and promotes anergy following costimulation blockade. Ongoing efforts to develop clinical allograft tolerance can likely exploit such knowledge and may benefit from agonist activity post transplantation.

Since the requirements and conditions required for maintenance of anergy differ somewhat between CD4 and CD8 T cells, and given the CD4-predominant nature of allograft rejection in this well-established model 23, we focused on induction of CD4 T cell anergy. The PD-1/PD-L1 pathway has received attention as a key mechanism of “immune exhaustion” in chronic infections and malignancies 2426. Thus, during chronic viral infections, the functions of virus-specific T cells can decline as a result of IFN-γ-induced up-regulation of PD-L1 on host CD8+ T cells 24, and a similar mechanism was noted for declining cytolytic activity by tumor-specific CD8+ T cells 27. However, these effects are distinct from the contribution of PD-1 to the development of CD4 T cell anergy shown in the current study.

Our data are also in direct contrast to two recent reports that PD-1 plays a key role in mediating Treg cell suppression 28, 29, though the latter studies were not definitive. When PD-1 expression by Treg cells was first reported 30, it was noted that their suppressive function was only modestly affected by PD-1/PD-L1 blockade, with no effect at high Treg-to-effector T (Teff) cell ratios, and comparable data was subsequently reported for mouse Foxp3+ Treg cells 31. Despite this, the PD-1/PD-L1 pathway was recently invoked as a key mechanism of action of Treg cells in vitro and in the context of a murine model of acute GVHD 28. However, the relevant data involved the addition of an anti-PD-L1 mAb to in vitro Treg cell suppression assays or in vivo administration following adoptive transfer of splenocytes. Although use of anti-PD-L1 mAb in each setting enhanced Teff cell proliferation, this effect could not rigorously be shown to result from direct effects on Treg cells, since the effector T cells also expressed PD-L1. The second study concluding that the PD-1/PD-L1 is a central mechanism for Treg cell suppression, involving the use of the same PD-1–/– mice as employed in the current study, tested the effect of estrogen on T cell functions in vitro and found that Treg cells from PD-1–/– mice completely lacked any suppressive activity 29. The latter report is in marked contrast to our own data and also to knowledge that PD-1–/– mice experience autoimmunity only late in life (after 1 year of age) 6, 7, whereas mice lacking Treg cell suppressive function, such as Foxp3-mutant Scurfy mice, or mice lacking CTLA-4, IL-2 or TGF-β, die from autoimmunity within a few weeks of birth 32.

In summary, the current studies using mice lacking PD-1 or PD-L1, or WT mice treated with corresponding mAb, show that the PD-1/PD-L1 pathway is essential for costimulation blockade-induced allograft tolerance, and that this requirement reflects roles for the pathway in controlling Teff cell proliferation as well as induction of T cell anergy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Animals and transplantation

We used commercial (The Jackson Laboratory) WT BALB/c (H-2d), C57BL/6 (H-2b) and C57BL6/DBA F1 (H-2b/d) mice, as well as PD-1–/–6 and PD-L1–/– (Millennium Pharmaceuticals, Inc.) mice backcrossed on a C57BL/6 background for more than eight generations before use. Studies were performed with a protocol approved by the institutional animal care and use committee of the Children's Hospital of Philadelphia. Survival of heterotopic abdominal cardiac allografts was assessed using six allografts per group 17. Tolerance was induced with CD154 mAb (Bio-Express, 200 µg, i.v.) + DST (5 × 106 splenocytes, i.v.) or CTLA-4Ig (Bio-Express, 200 µg, i.p) 33. WT recipients were treated i.p. with anti-PD-1 mAb, anti-PD-L1 mAb or control IgG at days 0, 2, 4 and 6 (100 µg/day).

T cell proliferation and anergy

Alloreactive T cell responses in vivo were generated by i.v. injection of pooled 40 × 106 CFSE-labeled WT or PD-1–/– (H-2b) spleen and lymph node cells into C57BL/6 × DBA F1 (H-2b/d) recipients 14. In vitro responses used T cells purified by negative selection (Miltenyi Biotec), labeled with CFSE (5 mM) and cultured with 1 µg/mL of anti-CD3e mAb ± anti-CD28 mAb. For anergy studies, splenocytes were cultured for 72 h with soluble anti-CD3 mAb (1 µg/mL) + either anti-CD28 mAb (1 µg/mL) or CTLA-4Ig (5 µg/mL) so as to induce anergy. CD4 T cells were purified with magnetic beads, rested for 24 h and restimulated overnight with plate-bound anti-CD3 mAb (2 µg/mL) or anti-CD3 mAb + IL-2 (10 U/mL). Cells were used for quantitative PCR (qPCR), and supernatants for IL-2, IFN-γ, IL-4 and IL-10 ELISA (eBioscience). BrdU (1 µg/million cells) was added 6 h before staining with PE-conjugated anti-BrdU antibody.

Treg cell assay

CD4+CD25+ (Treg), CD4+CD25 (effector) and APC (CD90-negative) populations isolated with magnetic beads, and effector cells (1 × 105/well) labeled with CFSE + APC (1 × 105/well) were added to 96-well plates; Treg cells were added to each well in varying ratios to effector cells and cultured with 1 µg/mL of soluble anti-CD3 mAb for 72 h, followed by flow cytometric analysis.

qPCR

Primer and probe sequences specific for murine IL-2, IL-10, IFN-γ, MCP-1, IP-10, CCR2, and CXCR3 genes (TaqMan PDAR, Applied Biosystems) were used for qPCR amplification of total cDNA (50 ng), with normalization to ribosomal RNA.

Statistics

Cardiac allograft survival data were used to generate Kaplan–Meier survival curves, and comparisons between groups were performed by log-rank analysis. ELISA data (mean ± SE) were evaluated by one-way ANOVA; p <0.05 indicates a significant result.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This work was supported by an NIH grant (R01-AI54720) to W.W.H.

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