1. Immunology and Inflammation
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TGFβ-dependent expression of PD-1 and PD-L1 controls CD8+ T cell anergy in transplant tolerance

  1. Marije Baas
  2. Alix Besançon
  3. Tania Goncalves
  4. Fabrice Valette
  5. Hideo Yagita
  6. Birgit Sawitzki
  7. Hans-Dieter Volk
  8. Emmanuelle Waeckel-Enée
  9. Benedita Rocha
  10. Lucienne Chatenoud
  11. Sylvaine You  Is a corresponding author
  1. University Paris Descartes, Sorbonne Paris Cité, France
  2. Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, France
  3. Institut Necker-Enfants Malades, France
  4. Juntendo University School of Medicine, Japan
  5. Charité University Medicine, Germany
  6. Pasteur Institute, France
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Cite this article as: eLife 2016;5:e08133 doi: 10.7554/eLife.08133

Abstract

CD8+ T cell anergy is a critical mechanism of peripheral tolerance, poorly investigated in response to immunotherapy. Here, using a pancreatic islet allograft model and CD3 antibody therapy, we showed, by single cell gene profiling, that intragraft CD8+ lymphocytes coexpressing granzyme B and perforin were selectively depleted through the Fas/FasL pathway. This step led to long-standing anergy of the remaining CD8+ T cells marked by the absence of cytotoxic/inflammatory gene expression also confirmed by transcriptome analysis. This sustained unresponsiveness required the presence of the alloantigens. Furthermore, tissue-resident CD8+ lymphocytes produced TGFβ and expressed the inhibitory receptors PD-1 and PD-L1. Blockade of TGFβ downregulated PD-1 and PD-L1 expression and precipitated graft rejection. Neutralizing PD-1, PD-L1 or TGFβRII signaling in T cells also abrogated CD3 antibody-induced tolerance. These studies unravel novel mechanisms underlying CD8+ T cell anergy and reveal a cell intrinsic regulatory link between the TGFβ and the PD-1/PD-L1 pathways.

https://doi.org/10.7554/eLife.08133.001

eLife digest

The immune system is always on guard for signs of infection or cells that have become diseased. When these signs are identified, a subset of white blood cells called CD8+ T cells leap into action, multiply in number and then act to eliminate the potential threat. While this response is essential to fighting off infections and other diseases like cancer, it can backfire in people with an organ transplant. Indeed, the CD8+ T cells can target and attack the cells of the transplanted organ causing the body to reject the organ.

One way to avoid transplant rejection would be to turn off CD8+ T cells that have learned to recognize cells from the transplant. In fact, studies in 2012 and 2013 showed that treating transplanted animals with an antibody that binds T cells protects a transplanted organ from attack. This treatment had to be given after the CD8+ T cells had recognized and began targeting the transplanted organ to be effective. But it was not clear exactly how this antibody treatment protected the transplant.

Now, Baas, Besançon et al. – including some of the same researchers involved in the earlier studies – show that the antibodies used in the treatment selectively target and eliminate the attacking CD8+ T cells. This leaves behind only inactive CD8+ T cells that don’t harm the transplant. To do this, Baas, Besançon et al. transplanted pancreatic cells from mice into other mice with a diabetes-like disorder. Next, the experiments compared gene expression in CD8+ T cells found within the transplanted tissue in mice that were treated with the antibody and those that were not treated. The expression of many genes for toxic molecules was stopped after treatment with the antibody leaving the CD8+ T cells in an inactive state.

In addition, the treated CD8+ T cells expressed more of a certain type of receptor (called PD-1 and PD-L1) that acts as inhibitory checkpoint for the immune system. So, Baas, Besançon et al. treated transplanted mice with both the T cell-eliminating antibody and antibodies that block these inhibitory receptors to see what would happen. The transplanted organs were quickly attacked and rejected. This shows that the inhibitory receptors play a crucial role in helping to shut down attacking CD8+ T cells in the initial antibody treatment and allowed long-term survival of the transplanted organs. Blocking another protein called TGFβ in antibody-treated mice also caused organ rejection. The findings help explain how these antibodies protect transplanted organs and may help scientists trying to develop new anti-transplant rejection drugs in the future.

https://doi.org/10.7554/eLife.08133.002

Introduction

Upon antigen recognition, CD8+ T lymphocytes proliferate vigorously and differentiate into effector cells characterized by their ability to produce cytokines and chemokines, to migrate to inflamed tissues and to use various cytolytic pathways to kill their targets (Williams and Bevan, 2007). CD8+ T cells play major role in transplant rejection. Through direct presentation, they get activated by donor antigen-presenting cells (APC), in particular dendritic cells, which migrate from the graft to secondary lymphoid organs (Gras et al., 2011; Ochando et al., 2006). This response is rapid, intense and induces acute graft rejection. Recipient APC also capture donor antigens from the transplant, present them to alloreactive CD8+ T cells through self MHC I molecules (cross-priming/indirect presentation) hereby contributing to acute and chronic graft rejection (Celli et al., 2011; Valujskikh et al., 2002). Cytotoxic CD8+ T lymphocytes migrate to the graft where they destroy target cells through granzyme B/perforin- and/or Fas/FasLigand-dependent cytolytic pathways.

In most tolerance promoting protocols, long-term graft survival was associated with CD8+ T cell dysfunction which mainly resulted from clonal deletion and/or anergy (Iwakoshi et al., 2000; Monk et al., 2003; Qian et al., 1997). Classically defined as the functional inactivation of T cells to cognate antigens, anergy was first described in vitro when T cells recognized antigens (signal 1) in absence of appropriate costimulation (signal 2), usually provided by CD28 (Schwartz, 2003). T cells were not able to produce IL-2, entered a hyporesponsive non proliferative state that prevented further responses upon antigen re-encounter. Over the last decade, better insight was gained into the signaling events leading to anergy, highlighting in particular the role of the transcription factors NF-AT (nuclear factor of activated T cells) and early growth response gene 2 and 3 (Egr-2, Egr-3) (Macian et al., 2002; Safford et al., 2005). However, characterization of the anergic phenotype and gene signature as well as the mechanisms that drive and sustain CD8 T cell anergy in vivo in the transplant setting are incompletely understood.

Recently, we reported that CD3 antibodies (CD3 Abs) induced tolerance in fully MHC-mismatched experimental models of pancreatic islet and cardiac transplantation (Goto et al., 2013; You et al., 2012). The timing of treatment was crucial; long-term graft acceptance was obtained only when CD3 Abs were administered once T cell priming had occurred, a few days post-transplant. Permanent survival of a second graft from the original donor but not from third-party donor demonstrated that antigen-specific tolerance had been induced. Purified spleen CD8+ T cells from CD3 Ab-treated recipients were unable to respond when stimulated with donor antigens while response to third-party antigens was conserved (You et al., 2012). In this model, CD3 Abs preferentially targeted and depleted activated effector T cells, notably within the graft.

In the present manuscript, we addressed the molecular mechanisms underlying CD8+ T cell tolerance induced by CD3 Ab therapy. We conducted detailed analysis of alloreactive CD8+ T cells combining single-cell gene profiling, transcriptome analysis and ex vivo/in vivo functional studies. We found that CD3 Abs selectively deleted Gzmb+Prf1+ CD8+ cytotoxic effectors within the transplant. CD8+ T cells escaping this deletion became anergic. The presence of the alloantigen was mandatory for the effect just as was TGFβ signaling to promote and sustain PD-1/PD-L1-mediated CD8+ T cell tolerance.

Results

CD3 Ab therapy selectively depletes Gzmb+Prf1+ CD8+ T cells and promotes anergy

We previously showed that CD3 Ab-induced transplant tolerance was associated with a drastic reduction of CD8+ T cell infiltrates and of peripheral donor-specific CD8+ T cell responses (You et al., 2012). Here we measured the anti-donor reactivity of graft infiltrating T cells using a 20 hr-IFNγ Elispot assay. Pancreatic islets from BALB/c mice were isolated and grafted under the kidney capsule of diabetic C57BL/6 recipients. Tolerogenic treatment with CD3 Ab F(ab’)2 fragments was applied for 5 days (50 µg/day) at day 7 after transplantation. Intragraft T cells recovered after CD3 Ab treatment, on days 14 or 100 post-transplant, did not respond to BALB/c donor antigens as opposed to graft infiltrating T cells of untreated recipients analyzed few days before rejection (day 14) (Figure 1—figure supplement 1).

To better dissect the impact of CD3 Ab therapy on alloreactive CD8+ T lymphocytes, we took advantage of a validated multiplex single cell PCR method established by the group of B. Rocha. This technique provides information on cell heterogeneity through the analysis of the simultaneous expression of selected inflammatory and/or cytotoxic genes by individual CD8+ T cells (Peixoto et al., 2007). We focused our analysis on Th1 and cytotoxic genes as it has been shown that the IFNγ, perforin and Fas/FasL pathways constituted predominant mechanisms of CD8+ T cell-mediated destruction of islet allografts (Diamond and Gill, 2000; Sleater et al., 2007). Individual CD8+ T cells were sorted from the islet allografts (72 cells) or spleen (48 cells) recovered from 3 individual recipients on day +14, that is right after the last injection of CD3 Abs, or on day +100 post-transplant, once tolerance was established. On day 14 post-transplant, in untreated recipients, graft infiltrating CD8+ T cells expressed the cytolytic molecules Gzmb, Prf1 and Fasl as well as Tbx21 and Klrg1 (Figure 1A). Thirty three percent of these cells co-expressed 3 or more of the 7 genes tested (Figure 1B). Interestingly, Gzmb was co-expressed with either Prf1 or Fasl which rarely overlapped, suggesting the presence of two distinct subsets of graft infiltrating CD8+ lymphocytes (Figure 1C). Tbx21, Eomes and Klrg1 were preferentially associated with Prf1 rather than Fasl (Figure 1C).

Figure 1 with 3 supplements see all
Coexpression of effector genes in graft-infiltrating CD8+ T cells after CD3 antibody therapy.

C57BL/6 mice were transplanted under the kidney capsule with BALB/c pancreatic islets and treated or not with CD3 Abs on day +7 post-transplant. Individual CD8+ T cells (n = 72) present within the graft were FACS sorted on day +14 or day +100 post-transplant and subjected to multiplex gene expression analysis. (A) Proportion of CD8+ T cells among the 72 cells tested that expressed Gzma, Gzmb, Prf1, Fasl, Tbx21, Eomes and Klrg1 mRNA in each group. (B) Polyfunctionality distribution of intragraft CD8+ T cells. (C) Coexpression of inflammatory and cytotoxic molecules by individual graft infiltrating CD8+ T cells. Each row represents one individual cell that is numbered. Each column represents a different gene. For better visualization of coexpression patterns, individual cells were ordered by the degree of gene coexpression. (D) FasL expression by intragraft CD8+ T cells 14 days after transplant and CD3 Ab therapy. (Figures 1A and 1B: χ2 test, *p<0.03, **p<0.01, ***p<0.0003, ****p<0.0001).

https://doi.org/10.7554/eLife.08133.003

In CD3 Ab-treated recipients, on day +14 after transplantation, expression of Prf1, Tbx21, Klrg1 and Gzmb by intragraft CD8+ T cells was clearly reduced as compared to untreated mice (Figure 1A). The frequency of cells coexpressing 3 or more genes was significantly decreased (from 33.3% to 15.3%) while the number of cells expressing only one gene doubled after CD3 Ab treatment (Figure 1B). A dramatic decrease in Gzmb+Prf1+ CD8+ T cells was observed (Figure 1C). Contrasting with these findings, Fasl expression was enhanced as compared to untreated controls (50% versus 30.6%). The increased FasL expression on intragraft CD8+ T cells was confirmed by flow cytometry (Figure 1D). Among this Fasl+ subset, 38% co-expressed Gzmb (versus 62% in untreated recipients) and 33.3% did not express any other effector genes.

On day 100 post-transplant, expression of Fasl, Prf1, Tbx21 and Eomes was detected in less than 5% of intragraft CD8+ T cells in CD3 Ab-treated tolerant mice (Figure 1A). Only 5.6% of CD8+ T cells coexpressed 3 or more genes and 43.1% of the cells did not express any of the 7 inflammatory/cytotoxic genes tested (Figure 1B,C). Gzmb was detected in 26% of the cells but was not associated with Fasl or Prf1 suggesting the absence of potent cytotoxic function (Figure 1A–C).

In the spleen, the single cell gene profile differed from that of intragraft T cells. A large proportion (56%) of CD8+ T cells from untreated mice coexpressed Gzmb, Tbx21 and Klrg1 but nor Prf1 or Fasl on day +14 post-transplant (Figure 1—figure supplement 2). Such coexpression was abrogated after CD3 Ab therapy as 52% and 37.5% of spleen CD8+ T cells expressed none or only one of the 7 selected genes, respectively (Figure 1—figure supplement 2B and 2C).

RT-qPCR on total islet allografts confirmed the decreased expression of Gzmb, Prf1, Tbx21 and Ifng (Figure 1—figure supplement 3A). Fasl expression was also reduced on day +14 post-transplant which contrasts with results from single cell PCR (Figure 1). We also analyzed expression of Fas mRNA which, interestingly, increased after administration of CD3 Abs on day +14 post-transplant and decreased hereafter. We confirmed by flow cytometry that CD3 Abs rapidly induced Fas expression on CD8+ T cells after a 24 hr in vitro stimulation and most of them co-expressed FasL at 48 hr (Figure 1—figure supplement 3B).

To investigate whether CD3 Ab-induced depletion of alloreactive CD8+ T cells was dependent on the Fas/FasL pathway, monoclonal antibodies neutralizing FasL were administered in C57BL/6 recipients at the time of CD3 Ab therapy. Long-term islet allograft survival was not observed in this condition (Figure 2A). FasL blockade abrogated CD3 Ab depleting effect: CD8+ T cells were detected within the islet allografts at levels comparable to that found in untreated recipients (Figure 2B). FasL expression was drastically reduced on infiltrating CD8+ T cells (Figure 2C).

FasL blockade reversed CD3 antibody-induced transplant tolerance.

(A) Graft survival of BALB/c islets was measured in C57BL/6 mice treated at day 7 with CD3 antibodies (50 μg, 5 days) alone (n = 10) or combined with neutralizing antibodies against FasLigand injected at the dose of 500 μg i.p. on day 6, 7 and 8 post-transplant (n = 4) (*p<0.02 between anti-CD3 and anti-CD3+anti-FasL Ab-treated mice). (B) Additional untreated (n = 6), CD3 Ab (n = 6) or CD3+FasL Ab (n = 2)-treated mice were sacrificed on day +14 post-transplant and proportion of CD8+ T cells was analyzed in the spleen, renal draining lymph nodes (dLN) and the islet allografts (**p<0.003, ***p<0.0006). (C) Expression of FasL by graft infiltrating CD8+ T cells isolated on day +14 from untreated (n = 6), CD3 Abs (n = 6) or CD3+FasL Abs (n = 2)-treated recipients (**p<0.002).

https://doi.org/10.7554/eLife.08133.007

Graft infiltrating CD8+ T cells present an inhibitory phenotype after CD3 Ab therapy

We next characterized intragraft CD8+ T cells not eliminated by CD3 Ab treatment. On day 14 post-transplant, most of them were CD69+ CD44highCD62LlowCD45RBlow (Figure 3A and Figure 3—figure supplement 1). They expressed low levels of CD122 (the β subunit of the IL-2 and IL-15 receptors) and CD25 as opposed to intragraft CD8+ T cells from untreated mice (Figure 3A). Expression of the proliferation marker Ki67 was also strongly reduced after CD3 Ab therapy. In contrast, the inhibitory receptors PD-1, PD-L1 and LAG3 were upregulated (58.5%, 60.9% and 58.6% versus 25%, 26.1% and 27% without treatment, respectively) and were mostly co-expressed by the same cells (Figure 3A,B). This phenotype was maintained over long-term since a large proportion of intragraft CD8+ T cells still expressed these inhibitory receptors on day +100 post-transplant. A similar trend was observed in the spleen and draining lymph nodes where CD8+ T cells showed an increase in PD-1, PD-L1 and LAG-3 expression after CD3 Ab therapy (Figure 3—figure supplement 2).

Figure 3 with 2 supplements see all
Phenotypic and functional characteristics of tolerant CD8+ T cells.

Pancreatic islet allografts were recovered from C57BL/6 mice after CD3 Ab treatment administered at day +7 post-transplant. (A) Expression of CD44, CD62, CD69, CD45RB, CD122, Ki67, CD25, PD-1, PD-L1 and LAG-3 (6–16/group) was evaluated on CD8+ T cells on day +7, day +14 and day +100 post-transplant (*p<0.05, **p<0.01, ***p<0.001). (B) Co-expression of PD-1/PDL-1, PD-1/LAG-3 and PD-L1/LAG-3 on graft-infiltrating CD8+ T cells recovered from CD3 Ab-treated mice on day +14 post-transplant. (C) Expression of PD-L1, CD25 and T-bet by graft infiltrating CD8+ T cells isolated on day14 from untreated, CD3 Abs or CD3FasL Abs-treated recipients (n = 2–6/group) (**p<0.005).

https://doi.org/10.7554/eLife.08133.008

Interestingly, CD3 antibody-induced upregulation of PD-L1 on intragraft CD8+ T cells was inhibited following in vivo FasL blockade (Figure 3C). In addition, expression of CD25 and T-bet were significantly increased in recipients treated with the combination of CD3 and FasL Abs as compared to CD3 Abs alone, reaching levels similar to those observed in untreated mice (Figure 3C).

Continuous presence of alloantigen and triggering of the PD-1/PD-L1 pathway are mandatory for the induction and maintenance of CD3 Ab-mediated CD8+ T cell tolerance

To investigate the role of the PD-1/PDL-1 pathway in CD3 Ab-induced transplant tolerance, we treated C57BL/6 mice at day 7 with CD3 Abs together with monoclonal Abs to PD-1 or PD-L1. In both cases, recipients rejected their graft with a median survival of 33.7 ± 1.5 days or 28.6 ± 5.1 days, respectively (Figure 4A,B). After administration of PD-1 Abs, most graft infiltrating CD8+ T cells were CD44highCD62LlowTbet+ and strongly proliferated (Figure 4—figure supplement 1A). Anti-PD-L1 Abs were also injected in tolerant recipients, on day 100 after transplantation and CD3 Ab therapy. Blockade of PD-L1 at the time of established tolerance precipitated graft rejection (Figure 4C).

Figure 4 with 1 supplement see all
Transplant tolerance and CD8+ T cell anergy rely on the PD-1/PDL-1 pathway.

Graft survival of BALB/c islets was measured in C57BL/6 mice treated at day 7 with a combination of anti-CD3 F(ab’)2 and neutralizing antibodies against PD-1 (panel A, n = 5) or PD-L1 (panel B, n = 5) injected at the dose of 500 μg i.p. every other day for a total of 5 injections. (****p<0.0001 between anti-CD3 and anti-CD3+anti-PD-1/anti-PD-L1 Ab-treated mice). (C) C57BL/6 mice showing long-term islet graft acceptance after CD3 Ab therapy were treated on day +100 post-transplant with anti-PD-L1 antibodies or isotype control IgG2a (n = 5). Graft rejection occurred 2–3 weeks later (*p<0.03). (D) CD8+ T lymphocytes were purified from the spleen of CD3 Ab-treated tolerant C57BL/6 mice and were transferred into C57BL/6 Rag-/- mice (3x106/recipient). Recipient mice were grafted with pancreatic islets from BALB/c on day 0 and graft survival was monitored. On day +100 post-transplant, anti-PD-L1 antibodies or isotype control IgG2a were injected (n = 5) (**p<0.007). (E) Tolerant CD8+ T cells were detected in the spleen, draining lymph nodes and islet allograft of C57BL/6 Rag-/- recipients. IFNγ production and CD44 expression were compared to the ones of CD8+ T cells recovered after treatment with anti-PD-L1 antibodies or of CD8+ T cells issued from untreated C57BL-6 mice and rejecting the islet graft.

https://doi.org/10.7554/eLife.08133.011

To further dissect the role of this pathway in CD8+ T cell tolerance, we adoptively transferred purified spleen CD8+ T cells from CD3 Ab-treated tolerant mice into immunodeficient RAG-/- recipients transplanted on the same day with BALB/c pancreatic islets. Islet grafts survived in these recipients. Transferred CD8+ T cells were detected in significant proportion in the spleen, draining lymph nodes and within recipients’ transplants (Figure 4D–E); they stained CD44high but did not produce high amounts of IFNγ in response to PMA/ionomycin stimulation (Figure 4E and Figure 4—figure supplement 1B). Administration of PD-L1 Abs on day +100 post-transplant provoked graft rejection (Figure 4C) and restored the capacity of CD8+ T cells to secrete IFNγ (Figure 4D–E and Figure 4—figure supplement 1B).

To assess the role of donor alloantigens in sustaining CD8+ T cell unresponsiveness, RAG-/- C57BL/6 recipients were grafted under the kidney capsule with BALB/c pancreatic islets either on the same day or more than two months after transfer of purified CD8+ T cells recovered from tolerant mice, to exclude any impact of homeostatic proliferation on T cell functional properties. In this later condition, all grafts were rejected contrasting with the long-term survival obtained when cell transfer and islet grafts were both performed on day 0. (Figure 5). A 4-week interval between infusion of tolerant CD8+ T cells and islet allografts also led to graft rejection (Figure 5). Control syngeneic islet grafts were tolerated.

CD8+ T cell anergy depends on the presence of the antigens.

CD8+ T lymphocytes were purified from CD3 Ab-treated tolerant C57BL/6 mice on day +100 post-transplant and were adoptively transferred into C57BL/6 Rag-/- mice (3x106/recipient, day 0). C57BL/6 Rag-/- recipients were transplanted with pancreatic islets from BALB/c donors either on day 0 (D0, n = 4) or 4 weeks (4wks, n = 4) or 2 months (2mo, n = 4) after cell transfer (**p<0.007). Syngeneic islets from C57BL/6 donors were grafted 4 weeks after cell transfer and were used as controls.

https://doi.org/10.7554/eLife.08133.013

Efficient TGFβ/TGFβR signaling promotes PD-1 and PD-L1 expression on graft infiltrating CD8+ T cells and is required for CD3 Ab-induced transplant tolerance

We next analyzed the role of TGFβ in our model. RT-qPCR results on islet grafts recovered from CD3 Ab-treated recipients showed a significant increase of Tgfb1 mRNA, as compared to untreated controls (Figure 6A). In addition, the intragraft Tgfb1/Ifng ratio was increased over long-term. To further identify the source of TGFβ, we sorted individual T lymphocytes, recovered on day 14 post-transplant, either from the spleen or from the islet grafts of 3 recipient mice treated with CD3 Abs. Expression of Tgfb1 mRNA was determined by single cell qPCR on 60 CD8+ and 48 CD4+ T cells. Results showed that Tgfb1 mRNA was expressed in 52% and 65% of CD4+ and CD8+ T cells, respectively (Figure 6B). In untreated recipients, only 9% of CD4+ and 6% of CD8+ intragraft T cells expressed Tgfb1. Tgfb1 mRNA was expressed in very few T cells recovered from the spleen of the same untreated or CD3 Ab-treated mice. Administration of a neutralizing antibody to TGFβ partially, yet significantly, abrogated CD3 Ab-induced tolerance (Figure 6C). To assess whether TGFβ signaling within T cells was involved in the effect observed, we used as recipients transgenic DnTGFβRII C57BL/6 mice that express a dominant negative form of the human TGFβ receptor II under the control of the mouse CD4 promoter (Gorelik and Flavell, 2000). In these mice, the dnTGFBRII-CD4 transgenic construct lacks the CD8 silencer. Thus, expression of the transgene blocks TGFβ signaling specifically in both CD4+ and CD8+ T cells. Treatment of these transgenic recipients with CD3 Ab on day +7 post-transplant did not induce long-term survival of islet allografts (median survival of 36.8 ± 6.1 days) (Figure 6D).

CD3 antibody-induced transplant tolerance depends on in situ TGFβ production and signaling in T cells.

(A) Expression of Tgfb1 mRNA and evaluation of the Tgfb1/Ifng ratio in pancreatic islet allografts recovered at day 14 from untreated mice or day 14, 21 and 100 after transplantation from CD3 Ab-treated recipients. Data are shown as mean ± SEM of 5–9 individual samples (*p<0.05, **p<0.01). (B) Single cell PCR: individual CD4+ (n = 48) and CD8+ (n = 60) T cells were sorted on day +14 post-transplant from the spleen or islet allografts recovered from C57BL/6 mice treated or not with CD3 antibodies. Expression of Tgfb1 mRNA was measured in each cell. Results show the proportion of CD4+ and CD8+ T cells positive for Tgfb expression (*p<0.05). (C) Graft survival of BALB/c islets in wild-type C57BL/6 mice treated at day +7 with anti-CD3 F(ab’)2 and neutralizing TGFβ antibodies (1 mg/injection, twice a week for 3 weeks) (n = 4 to 8 per group, *p<0.05). (D) Abrogation of tolerance in DnTGFβRII C57BL/6 mice transplanted with BALB/c pancreatic islets and treated with CD3 antibodies on day7 post-transplant (n = 5 per group, ***p = 0.0002).

https://doi.org/10.7554/eLife.08133.014

We asked whether TGFβ could influence the expression of PD-1 or PD-L1 on CD8+ T cells. T cells were stimulated in vitro for 24 hr with CD3 Abs in presence of neutralizing Abs to TGFβ or recombinant TGFβ. Co-expression of PD-1 and PD-L1 was induced after TCR stimulation (Figure 7—figure supplement 1). Blockade of TGFβ downregulated expression of PD-1 and even more drastically that of PD-L1. In contrast, addition of recombinant TGFβ to the culture further promoted PD-1 but not PD-L1 expression. To assess the relevance of this pathway in vivo, islet allograft recipients were treated with CD3 Abs either alone or combined with TGFβ Abs. Paralleling the in vitro data, in vivo neutralization of TGFβ downregulated PD-1 and PD-L1 co-expression on graft infiltrating CD8+ T cells (Figure 7).

Figure 7 with 1 supplement see all
Induction of PD-1 and PD-L1 expression on intragraft CD8+ T cells is regulated by TGFβ.

C57BL/6 mice were transplanted with BALB/c pancreatic islets and treated at day 7 with anti-CD3 F(ab’)2 with or without neutralizing TGFβ antibodies. Mice were sacrificed on day 14 post-transplant and PD-1 and PD-L1 expressions were analyzed on graft-infiltrating CD8+ T cells. (A) Co-expression of PD-1 and PD-L1 on CD8+ T cells. (B) Histograms representing PD-1 and PD-L1 expression. (C) Median fluorescence intensity of PD-1 and PD-L1 (*p<0.02, **p<0.005).

https://doi.org/10.7554/eLife.08133.015

Gene signature of intragraft CD8+ T cells

Using the Agilent platform, we compared intragraft CD8+ T cells from CD3 Ab-treated tolerant mice to those from untreated mice showing acute rejection. Functional grouping analysis showed that gene categories related to effective T cell responses were downregulated in CD3 Ab treated mice as compared to controls (Figure 8A). Accordingly, pathways induced by IL-2 or TCR signaling were also downregulated (Figure 8B and Figure 8—figure supplement 1). Analysis of immune genes differentially expressed between the two groups revealed a decreased expression of genes involved in cell migration (Cxcl9, Ccr10, Nrp1, Cd44), proliferation (Fos, Jun, Ccnd1), co-stimulation (Cd80, Icos, Cd200, Ctla4), inflammatory cytokine signaling (Tnf, Il2ra, Il1r2, Il18r1, Tnfrsf21) and environmental sensing (hypoxia-inducible factor Hif1a) (Figure 8C).

Figure 8 with 1 supplement see all
Transcriptomic analysis of tolerant intragraft CD8+ T cells after CD3 Ab therapy.

Five hundred graft-infiltrating CD8+ T lymphocytes were sorted from untreated or CD3 Ab-treated C57BL/6 mice, on day +14 and day +100, respectively (n = 8 per group). Agilent Whole Mouse Genome Microarrays were performed after amplification of RNAs. Functional grouping analysis used annotations derived from Gene Ontology (fold-change >2, p<0.05). (A) Bar chart showing the frequency of representative categories downregulated (left panel) or upregulated (right panel) in tolerant CD8+ T cells as compared to CD8+ T cells from untreated recipient mice. Below, statistically enriched categories were indicated by their adjusted p-value (only the top 10 categories, Fisher’s exact test with Benjamini-Hochberg correction for multiple testing). (B) Bar chart showing the frequency of target pathways induced by IL-2 ou TCR signaling. (C) Selection of immune genes that were downregulated (grey bars) or upregulated (red bars) in tolerant CD8+ T cells as compared to CD8+ T cells from untreated recipient mice (fold-change >2, p<0.05).

https://doi.org/10.7554/eLife.08133.017

In tolerant CD8+ T cells, categories related to metabolic processes (nucleotides, energy, carbohydrates, lipids…) as well as cell cycle arrest were enriched. In addition, these cells showed upregulation of genes inhibiting or regulating T cell functions: inhibitors of cell proliferation (Hopx, Cdkn2c, Map2k6, Mapk9), of cytokine signaling (Socs2, Socs6), of differentiation (Eid2, Id2), of co-stimulation (Btla) (Figure 8C and Figure 8—source data 1). Eomes, encoding for the IFNγ transcription factor eomesodermin, was also upregulated in tolerant CD8 T cells.

Discussion

In the present study, using a pancreatic islet allograft model and CD3 Ab therapy, we provide novel insights into the immune mechanisms driving and sustaining CD8+ T cell anergy thus leading to long-term graft survival. We demonstrate that CD3 Abs selectively deplete graft-infiltrating Gzmb+Prf1+ cytotoxic CD8+ T cells by a FasL-mediated pathway. This depletion was accompanied by the onset of anergy in remaining CD8T cells which was dependent on the presence of the alloantigens and on an in situ crosstalk between the PD-1/PD-L1 and TGFβ/TGFβRII pathways.

We previously reported that a short-term course with CD3 Abs induced long-term graft survival and antigen-specific tolerance provided the treatment was applied in a defined therapeutic window, that is at the time of effector T cells priming to the alloantigens (Goto et al., 2013; You et al., 2012). Mechanistic studies revealed that regulatory and effector T cells were differentially affected by the treatment. Foxp3 Tregs were relatively spared from CD3 Ab-induced depletion and could transfer antigen-specific tolerance, suggesting that they play an important role in sustaining graft survival (Baas et al., 2013; Goto et al., 2013; You et al., 2012). In contrast to Tregs, CD3 Abs preferentially induced apoptosis of activated effector T cells (You et al., 2012). Long-term graft survival correlated with an absence of donor-specific CD8+ T cell responses at the periphery (You et al., 2012). Here, T cell unresponsiveness was confirmed at the tissue level as graft infiltrating T cells from CD3 Ab-treated recipient mice did not mount efficient IFNγ responses to donor antigens.

By single cell gene profiling using a method which proved optimal to discriminate functional patterns of CD8 T cells facing an immune stimulation (Peixoto et al., 2007; Ribeiro-dos-Santos et al., 2012), we identified two distinct subsets of graft infiltrating CD8+ T cells, co-expressing either Gzmb/Fasl or Gzmb/Prf1 mRNAs. Interestingly, Gzmb+Prf1 cytotoxic CD8 T cells were selectively depleted after CD3 Ab therapy. The frequency of individual CD8 T cells expressing Fasl was enhanced at the end of CD3 Ab treatment but it was not associated with Gzmb coexpression in contrast to what observed in untreated recipients. Flow cytometry analysis confirmed the increase of FasL+CD8+ T cells. Previous studies showed that signaling through the TCR/CD3 complex promotes activation-induced cell death (AICD) in activated effector T cells through a Fas/FasL interaction, but independently of perforin and granzymes (Brunner et al., 1995; Sobek et al., 2002). T cells expressed both Fas and FasL after CD3 Ab treatment. Our results suggest, together with previous reports using T cell hybridomas or clones, that apoptosis has occurred by triggering of Fas by FasL-expressing neighboring T cells (Ayroldi et al., 1997; Brunner et al., 1995). Rejection of islet allograft following FasL blockade further confirmed the Fas-dependence of CD3 antibody-induced depletion of alloreactive T cells. Therefore, our data concur to show that CD3 Abs induced apoptosis of Gzmb+Prf1 CD8+ T cells by a Fas/FasL-mediated pathway.

Apoptosis of alloreactive effectors was followed by the establishment of anergy in the remaining graft-infiltrating CD8+ T cells. Single cell gene profiling showed that expression of Gzmb, Prf1, Tbx21, Eomes and Klrg1 mRNAs decreased after CD3 Ab therapy as well as the simultaneous expression of 3 and more of the 7 genes tested. This downregulated gene pattern was even more obvious on day +100 post-transplant as intragraft CD8+ T cells were characterized by a complete absence of co-expression of cytotoxic and inflammatory genes (i.e. 80% of the cells expressed no or only one of the selected genes), a hallmark of profound intrinsic unresponsiveness. Gzmb was detected in 25% of the cells but was not associated with Prf1 or Fasl showing that these CD8+ T cells were deprived of effective killing ability.

Our findings are reminiscent of the pioneer experiments of Rocha and Von Boehmer who demonstrated the existence of anergy in vivo using TCR transgenic models. They described that female CD8 T cells specific for the male antigen HY rapidly expanded when transferred into male nude recipients and most of them died by AICD. The remaining transgenic CD8+ T cells were intrinsically unresponsive to TCR stimuli (Rocha and von Boehmer, 1991). Importantly, when parked into a second female nude recipient for 2 months and subsequently transferred into a third male nude mice, the T cells regained their ability to respond to the HY antigen showing that antigen persistence was required to maintain CD8 T cell anergy (Rocha et al., 1993). In our model, the continuous presence of the alloantigen was essential to sustain CD8+ T cell unresponsiveness in vivo as islet allografts transplanted 2 months after infusion of CD8+ T cells from tolerant mice into RAG-/- recipients were rapidly rejected. Our data suggest that, within islet allografts, chronic antigen encounter induces a suboptimal activation that reinforces anergic signals in CD8+ T cells and therefore sustains CD8 T cell tolerance.

Transcriptome analysis of graft infiltrating CD8+ T cells recovered on day +100 post-transplant allowed us to identify a gene signature for tolerant T cells. Genes encoding for negative regulators of proliferation or differentiation (Eid2, Cdkn2c, Hopx, Id2) were overexpressed. Hopx (homeodomain-only protein), identified as a master regulator of the anergic state of induced Tregs, inhibits the expression of the AP-1 complex (Hawiger et al., 2010). Accordingly, Fos and Jun that compose the AP-1 complex were downregulated in tolerant CD8+ T cells as well as their downstream target cyclin D1 (Ccnd1) (Kang et al., 1992; Sundstedt et al., 1996). Additionally, the decreased expression of Cxcl9, encoding an IFNγ inducible chemokine contributing to the recruitment of allograft reactive T cells (Medoff et al., 2006), as well as that of Tnf, Il2ra, Lgals3, Il1r2, Il18r1 and Tnfrsf9 revealed the inhibition of Th1 inflammatory responses. Tolerant CD8+ T cells also downregulated the hypoxia-inducible factor Hif1a that has been shown to positively regulate T cell differentiation and effector functions by promoting aerobic glycolysis (Doedens et al., 2013; Finlay et al., 2012). Finally, we found an overexpression of Eomes in tolerant CD8+ T cells. This was unexpected as eomesodermin (Eomes) plays well-described roles in cytotoxic CD8+ T cell differentiation and memory formation (Intlekofer et al., 2005; Pearce et al., 2003). However, investigations in models of chronic infections in human and mouse (HIV, LCMV) have demonstrated that expression of Eomes by virus-specific CD8 T cells was associated with high expression of inhibitory receptors and impaired functions (Buggert et al., 2014; Doering et al., 2012; Paley et al., 2012). In our transplant model, as detailed below, the PD-1/PD-L1 pathway is mandatory for CD8 T cell tolerance. Thus, increased expression of Eomes in conjunction with PD-1/PD-L1 may characterize anergized CD8+ T cells induced after CD3 Ab therapy. This finding highlights the issue of a molecular link between Eomes and PD-1/PD-L1. Lastly, one important feature common to our model and the infectious setting is the continuous presence of the cognate antigens. Therefore, we may hypothesize that chronic antigenic stimulation delivers signals inducing a preferential and sustained expression of Eomes.

Our study highlighted a key contribution of the PD-1/PD-L1 and the TGFβ/TGFβRII pathways and we identified a new role for TGFβ in modulating PD-1 and PD-L1 expression on CD8+ T cells. After CD3 Ab therapy, graft-infiltrating CD8+ T cells exhibited a CD44highCD62LlowCD69+CD45RBlow antigen-experienced phenotype and coexpressed the inhibitory receptors PD-1, PDL-1 and LAG-3. The PD-1/PD-L1 pathway played a predominant role in the induction and the maintenance phase of CD3 Ab-mediated tolerance as neutralization of either receptor completely abrogated the therapeutic effect. More precisely, we demonstrated that the PD-1/PD-L1 signaling was mandatory for CD8+ T cell unresponsiveness. Indeed, after treatment with CD3 antibodies, intragraft CD8+ T cells showed a defective ability to proliferate, to produce IFNγ and to mount donor-specific responses. In vivo administration of anti-PD-1 or anti-PD-L1 antibodies restored the effector functions of allogeneic CD8+ T cells, as shown by the up-regulation of the proliferation marker Ki67 and the IFNγ transcription factor T-bet as well as the regained ability to secrete IFNγ, and resulted in islet allograft rejection. Our findings are in accordance with the well-described role of the PD-1/PD-L1 pathway as a master regulator of immune responses notably through its ability to limit effector T cell interaction with dendritic cells and to abort their activation (Fife et al., 2009; Francisco et al., 2010). In various settings, PD-1 has been identified as a marker of dysfunctional CD8+ T cells, and it contributed to the establishment of tolerance (Barber et al., 2006; Haspot et al., 2008; Ito et al., 2005; Lucas et al., 2011; Riella et al., 2012).

We found that CD8+ T cells coexpressed PD-1 and PD-L1, which may contribute to the formation of stable interactions with other CD8+ T cells as well as other cells. PD-L1 is widely expressed on hematopoietic and nonhematopoietic cells and its expression increases with activation (Keir et al., 2008). In addition, aside from PD-1, PD-L1 can interact with B7-1 and a recent report demonstrated that this interaction contributed to the control of allogeneic T cell responses (Keir et al., 2008; Yang et al., 2011). We can thus hypothesize that PD-L1 engagement on graft-infiltrating CD8+ T cells may deliver inhibitory signals and that these bidirectional interactions may promote and sustain T cell anergy and graft survival.

Another novel finding points to TGFβ as a regulator of PD-1 and PD-L1 expression and as a key mediator of CD3 Ab-induced allotolerance. We have previously shown in autoimmunity that CD3 antibodies cured type 1 diabetes and restored self-tolerance in a TGFβ-dependent manner (Belghith et al., 2003). The present results not only extend these findings to transplant tolerance but also provide new insights into the key regulatory role of this cytokine. First, the single cell PCR results showed that a large proportion of CD8+ T cells (as well as CD4+ T cells) present within the islet allografts produced TGFβ after CD3 antibody administration. This increased expression of TGFβ was restricted to the graft as it was not observed in the spleen. Secondly, we demonstrated that TGFβ signaling in T cells is mandatory for the induction of immune tolerance as CD3 Ab treatment failed to induce long-term islet graft survival in recipients presenting a T cell-selective mutated TGFβRII (Gorelik and Flavell, 2000). This result argues for an in situ autocrine/paracrine effect of TGFβ on allogeneic T cells. Third, we demonstrated a direct link between the TGFβ and the PD-1 pathways as TGFβ blockade, through the administration of neutralizing antibodies, downregulated PD-1 and PD-L1 expression on intragraft CD8+ T cells and abrogated the tolerogenic properties of CD3 Abs. This inhibitory effect was even more drastic on PD-L1 than PD-1 showing that CD3 Ab-induced PD-L1 expression on CD8+ T cells was highly dependent on TGFβ/TGFβR signaling.

In conclusion, our study highlighted new facets of immune mechanisms driving CD8+ T cell peripheral tolerance and permanent acceptance of fully mismatched allografts after CD3 Ab therapy. Tolerance was established through elimination of highly cytotoxic CD8+ T cells followed by the induction of CD8+ T cells anergy which depended on a cross-talk between the PD-1/PD-L1 and TGFβ/TGFβRII pathways acting in an autocrine and paracrine manner in the graft environment. These mechanistic findings support the therapeutic potential of CD3 Ab which, concerning clinical translation, have shown potential value in the treatment of patients with new onset type 1 diabetes (Herold et al., 2002; Keymeulen et al., 2005; Sherry et al., 2011).

Materials and methods

Mice

C57BL/6, RAG-/- C57BL/6, and BALB/c female mice were bred in our facility under specific pathogen-free conditions. DnTGFβRII female C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, USA). Blood glucose was measured using ACCU-CHECK Performa glucometer (Roche Diagnostics, Meylan, France). Experiments were conducted according to European Directive (2010/63/UE) and were approved by the Ethical Committee of Paris Descartes University (registered number: 14–075).

Pancreatic islet isolation and transplantation

Pancreatic islets were separated by density gradient centrifugation (Histopaque, Sigma-Aldrich, Lyon, France) after in situ digestion with collagenase P (Roche Diagnostics, Meylan, France) and transplanted (300 islets) under the kidney capsule of diabetic recipients. Diabetes was induced 3 to 4 days after a single injection of streptozotocin (Sigma-Aldrich) at 225 mg/kg. Diagnosis of graft rejection was made after three glucose measurements >250 mg/dl.

Antibodies and in vivo treatments

The murine myeloma cell line SP2/0 producing the genetically engineered F(ab’)2 fragments of the hamster anti-mouse CD3ε antibody 145-2C11 (Kostelny et al., 1992) was provided by J.A. Bluestone (UCSF, San Francisco, CA) The antibody was purified by protein G–Sepharose affinity chromatography. CD3 F(ab’)2 were injected i.v. at the dose of 50 µg/day for 5 consecutive days, starting on day 7 post-transplant. Anti-PD-L1 hybridoma (MIH5) was kindly provided by Pr. M. Azuma (Tokyo Medical and Dental University). Purified anti-PD-1 (RMP1-14) and anti-FasLigand antibodies (MLF4) were provided by Pr. H. Yagita (Juntendo University School of Medicine, Tokyo). TGFβ antibodies (2G.7) were produced in house. For flow cytometry, all antibodies were from BD Biosciences (Pharmingen, San Diego, CA, USA) except Foxp3 which was from eBioscience (San Diego, CA, USA).

Adoptive transfers

RAG-/- C57BL/6 mice were reconstituted with 3.106 CD8+ T cells isolated from the spleen of CD3 Ab-treated tolerant C57BL/6. The next day, recipients were transplanted with 300 BALB/c islets. PD-L1 antibodies were administered 100 days after transplantation. In another experiment, islet allografts were performed 4 weeks or 2 months after CD8+ T cell transfer.

RT-qPCR

mRNA was isolated using the mMACS isolation Kit and reverse transcribed into cDNA using the mMACS One-step cDNA Kit (MACS Molecular, Miltenyi Biotec, Bergisch Gladbach, Germany). RT-qPCR was performed on an ABI 7900HT fast real-time PCR system using primers, probes and master mixes from Applied Biosystems (Life Technologies, Carlsbad, CA, USA). HPRT was the housekeeping gene.

Single cell PCR

Individual CD8+ T cells were FACS sorted from the spleen or the islet allografts of untreated or CD3 Ab-treated recipients. After cell lysis by heating/cooling steps, RNA was specifically retrotranscribed using MuLV Reverse Transcriptase (Applied Biosystems) and 3’ specific primers (Eurofins MWG, Ebersberg, Germany). The resulting cDNA was next amplified (first PCR with all primers). Product of this first PCR was then subjected to a second PCR using SYBR Green PCR Master Mix (Applied Biosystems) for each primers pairs. To ensure that each well contained a T cell, Cd3e mRNA was amplified simultaneously with the genes of interest. HPRT was the housekeeping gene. Multiplex single cell PCR was performed for the following genes: granzymes A and B (Gzma, Gzmb), perforin-1 (Prf1) and FasLigand (Fasl), which provide killing abilities, the transcription factors T-bet (Tbx21) and Eomesodermin (Eomes) which control IFNγ expression, and the killer-cell lectin like receptor G-1 (Klrg1) which marks terminally differentiated effector T cells.

Microarray hybridization and analysis

For each sample, 500 graft-infiltrating CD8+ T cells from individual tolerant CD3 Ab-treated recipients (n = 8) or untreated recipients (n = 8) were FACS sorted, lyzed using SuperAmpTMlysis buffer (Miltenyi Biotec) and stored at -80°C until all samples were collected. Amplification of RNA, sample hydridization (Agilent whole mouse genome oligo microarrays), scanning and data acquisition were performed by Miltenyi Biotec. Functional grouping analysis has been performed using Gene Ontology. Gene expression data have been deposited in the GEO database (accession number GSE68208).

Statistics

Cumulative graft survival was calculated using the Kaplan-Meier method. The statistical comparison was performed using the logrank (Mantel-Cox) test. When appropriate, the Student's t tests or Chi square (χ2) tests were used. A p value <0.05 was considered significant.

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Decision letter

  1. Satyajit Rath
    Reviewing Editor; National Institute of Immunology, India

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Revisiting CD8+ T cell anergy in transplant tolerance: a new role for TGF-β in regulating the PD-1/PD-L1 pathway" for peer review at eLife. Your submission has been favorably evaluated by Tadatsugu Taniguchi (Senior editor) and four reviewers, one of whom is a member of our Board of Reviewing Editors.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript shows a very interesting set of data implicating a TGFβ-PD1 axis in generating and maintaining CD8 T cell allotolerance, in the context of non-FcR-binding anti-CD3 mAb-mediated induction of CD8 T cell allotolerance of solid islet transplants in vivo. It also indicates roles for Fas/FasL and loss of perforin-based lytic mechanisms in CD8 T cells in the success of the anti-CD3 therapy. The authors identify, by single-cell gene profiling, two distinct granzyme B-expressing CD8 T cell populations in grafts, one perforin-positive and the other FasL-positive. Their data suggest that the perforin-expressing cells are selectively depleted in anti-CD3 therapy in a Fas-dependent process, and the other population is rendered non-responsive over the long term by active repression via the PD1-PDL1 pathway in a process requiring continuous antigen exposure. The molecular mechanisms that account for these interactions have not been studied but would be of great interest.

Essential revisions:

Despite enthusiasm, major concerns remain to be addressed, with either textual modification/s and/or additional information and/or evidence where appropriate.

1) No rationale is provided for the selection of seven genes for the characterization of CD8 T cells, such as experimental evidence correlating their expression patterns with CD8 T cell functionality. Further, the data assume 'anergy' of the graft-infiltrating CD8 T cells (as well as CD8 T cells in lymphoid organs), yet show no assays demonstrating such anergy in cellular function. This is a particular concern since 'anergy' refers to the failure of the cells to reject allografts, yet cellular non-responsiveness appears to be through active inhibition because anti-PD1 or anti-PDL1 can reverse it. The term 'anergy' could use either replacement or extensive clarification.

2) The data provide no estimates of cell numbers anywhere, either in grafts from non-tolerant versus tolerant mice, or in the cell transfer experiments into RAG-null mice, making it difficult to assess the relative impact of cell depletion versus anergy. Cell populations need better definition and statistical assessment of their numbers and the changes therein over time of therapy.

3) The manuscript needs to acknowledge that the evidence for Fas-dependence of selective depletion of CD8 T cells expressing granzyme B and perforin is correlative as yet, and would require approaches such as FasL blockade to be definitive.

4) There seems an inconsistency between the amount of protein expression (Figure 1—figure supplement 2) and the frequency of protein-expressing cells (Figure 1). Thus, the amount of FasL normalized to CD3 is ~65% less in treated versus untreated mice at day 14 (Figure 1—figure supplement 2A) while the frequency of FasL+ cells is ~50% MORE (Figure 1A). The reduction of granzyme B is more drastic in amount (Figure 1—figure supplement 2A) than in frequency (Figure 1A). The criteria for the judgment of expression in Figure 1 should be carefully described, since if amounts of protein per cell are significantly different, the cells at different time points may have different origins or functions.

5) The data provide no evidence for the antigen-specificity of the tolerisation or of its maintenance in RAG-null mice, nor has the allospecificity of individual CD8 T cells been formally documented.

6) It is unclear from the description of the methods if the CD8 T cell transfers into RAG-null mice have used intragraft cells or splenic cells for the transfers. Since 3 million cells were transferred, it is likely that splenic T cells were used. The gene expression and phenotyping data show some differences between intragraft and splenic CD8 T cells from anti-CD3-tolerised mice. However, the intragraft CD8 T cells are extensively characterised, while splenic CD8 T cells allotolerise quite effectively. This makes it hard to interpret the mechanistic significance of the patterns observed in intragraft CD8 T cells.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "TGFβ-dependent expression of PD-1 and PD-L1 controls CD8+ T cell anergy in transplant tolerance" for further consideration at eLife. Your revised article has been favorably evaluated by Tadatsugu Taniguchi (Senior editor), a Reviewing editor, and three reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) In Figure 2B, the 'n' for the CD3+FasL abs appears to be 2. This is quite small. The related figure (2A) indicates that there were 4 animals studied. This needs to be clarified.

2) If there are data on the CD8+ cells from mice treated with the CD3+FasL abs, they would be useful to include.

3) CD3 antibody-induced upregulation of PD-L1 on intragraft CD8+ T cells was inhibited following in vivo FasL blockade; – it would be best to include Figure 3—figure supplement 3 in the main manuscript.

4) While Fas/FasL is a well-recognized pathway of cell death, it would be reasonable to address how this might be occurring – is it suicide or are neighboring cells providing the lethal ligand? Is Fas/FasL co-expression on the same cell functional? At the least, the issue needs to be acknowledged and discussed.

5) Clarification of Figure 4C: Should there be a point at d100 when the anti-PD-L1 was given? In other words were the grafts monitored before d100 after anti-PD-L1 injection? Since the number of mice is small, is it certain that those that really reject after anti-PD-L1 really been non-rejecting before d100 when the Ab was given?

6) In the experiment in Figure 5, presumably tolerance was lost during the 2 months in the absence of the BALB/c islets. Did RAG-/- recipients of spleen cells from the tolerant B6 mice not reject islets that were transplanted right after the transfer of cells? (Figure 5—figure supplement 1 does not address this control.)

7) Clarification of the implications of Figure 6: What explains the site differences in Tgfb1 expression, since TGFb was induced in culture, and since the soluble cytokine would probably have effects at all sites?

8) The last sentence of Results – EOMES has functions other than serving as a transcription factor associated with Th1 cell development. It would be useful to discuss the possible explanation for its increase in the tolerized treated cells.

9) In the interests of brevity, the Discussion could be shortened in text that is not necessarily supported by the data, such as the extended PD-L1 expression discussion when PD-L1 expression was not studied in the islet grafts.

https://doi.org/10.7554/eLife.08133.024

Author response

Essential revisions:

Despite enthusiasm, major concerns remain to be addressed, with either textual modification/s and/or additional information and/or evidence where appropriate. 1) No rationale is provided for the selection of seven genes for the characterization of CD8 T cells, such as experimental evidence correlating their expression patterns with CD8 T cell functionality.

We apologize for not having provided a clear rationale for the selection of the 7 genes analyzed. Indeed, these genes have been chosen for the following reasons:

First, in pancreatic islet allograft models, IFNγ is a key mediator of CD8++ T cell-mediated graft destruction (Diamond AS et al. J Immunol 2000; Sleater M Am J Transplant 2007). Single disruption of either pathway does not prolong graft survival. In contrast, simultaneous loss of both pathways inhibited graft rejection.

Second, our unpublished results show that expression of Gzmb, Prf1, Fasl, Ifnγ, Tbx21 and Eomes mRNAs increased with time within the islet allografts from day 7 to day 14 post-transplant in untreated mice (Author response image 1).

Third, it has been shown by Dr. Benedita Rocha who set up the multiplex PCR on individual CD8+ T cells that the coexpression of effector genes, such as the ones we used, allows to identify CD8+ T cell subsets present at different phases of the immune responses and endowed with distinct inflammatory and/or killing ability (Peixoto A et al. Genome Research2004; Peixoto et al. J Exp Med 2007). We applied this methodology in the transplant setting to better dissect CD8+ T cell alloreactivity and behavior in response to CD3 antibody immunotherapy.

Therefore, we selected for our single cell PCR assays the transcription factors T-bet and Eomes which control IFNγ expression, granzymes A and B, perforin and FasLigand which provide killing capacities and KLRG1 which marks terminally differentiated effector T cells. These points and the corresponding references were added in the Results section (subsection “CD3 Ab therapy selectively depletes Gzmb+Perf+ CD8+ T cells and promotes anergy”) of the revised manuscript.

Author response image 1
Kinetics of intragraft effector gene expression in untreated recipients.

Pancreatic islets from BALB/c mice were transplanted under the kidney capsule of C57BL/6 recipients. Allografts were recovered on day 7 and 14 post-transplant and expression Gzmb, Prf1, Fasl Ifnγ, Tbx21 and Eomes mRNA was analyzed by RT-qPCR (n = 4-5) (*p<0.05).

https://doi.org/10.7554/eLife.08133.020

Further, the data assume 'anergy' of the graft-infiltrating CD8 T cells (as well as CD8 T cells in lymphoid organs), yet show no assays demonstrating such anergy in cellular function. This is a particular concern since 'anergy' refers to the failure of the cells to reject allografts, yet cellular non-responsiveness appears to be through active inhibition because anti-PD1 or anti-PDL1 can reverse it. The term 'anergy' could use either replacement or extensive clarification.

We understand the editor and reviewers’ concern on the term ‘anergy’ that we used to define the CD8+ T cells recovered from tolerant mice after CD3 antibody therapy. We apologize as should have better recalled and discussed data obtained from our previous works where functional assays were performed. Indeed, both in a pancreatic islet and in a cardiac allograft models published in 2012 and 2013 (You S et al. Am J Transplant 2012; Goto R et al. Am J Transplant 2013), we showed, using a 20 hr IFNγ Elispot assay, that total spleen cells or purified CD8+ T cells from CD3 antibody-treated recipients were unable to respond when stimulated with donor antigens (response to third-party antigens being conserved). In addition, our unpublished results show that graft infiltrating T cells from CD3 Ab-treated recipients were unable to mount IFNγ responses (20 hr Elispot assay) towards BALB/c antigens as opposed to that recovered from untreated recipients. From these results, we concluded that T cells, and notably CD8+ T cells, from tolerant mice were unresponsive to their cognate antigens.

Results gained in the present manuscript from the single cell PCR, the transcriptome and the phenotype analysis, reinforced the ‘anergic’ nature of CD8+ T cells in tolerant mice.

Finally, our data further acknowledge the role of inhibitory receptors in the regulation of T cell anergy as suggested by several reports in different contexts (Shrikant P et al. Immunity 1999; Wells AD et al. J Clin Invest 2001; Greenwald RJ Immunity 2001; Tsushima F et al. Blood 2007; Fife BT et al. Immunol Rev 2008; Bishop KD et al. Cell Immunol 2009; Chikuma S et al. J Immunol 2009).

For the sake of clarification, we have now better described our previous data in the Introduction, Results(subsection “CD3 Ab therapy selectively depletes Gzmb+Perf+ CD8+ T cells and promotes anergy”) and Discussionsections and results from the IFNγ Elispot assay using graft infiltrating T cells have been included as new Figure 1—figure supplement 1.

2) The data provide no estimates of cell numbers anywhere, either in grafts from non-tolerant versus tolerant mice, or in the cell transfer experiments into RAG-null mice, making it difficult to assess the relative impact of cell depletion versus anergy. Cell populations need better definition and statistical assessment of their numbers and the changes therein over time of therapy.

T cell numbers and proportions in CD3 Ab treated recipient mice versus untreated mice were presented in details in our previous publications reporting the tolerogenic effect of CD3 antibodies in the transplant setting (You S et al. Am J Transplant 2012; Baas M et al. Transplant Proc 2013). We apologize for having not enough recalled these results in the present study, which is of importance for interpreting the data.

CD4+ and CD8+ T cells were transiently depleted in the grafts and the spleen after the 5-day treatment with CD3 antibodies. At the periphery T cells were back to their normal levels within 4-5 weeks post-treatment. As illustrated in Author response image 2, in transplanted islets, numbers of CD8+ T cells remained low and stable up to day 100 after transplantation and treatment with CD3 antibodies (i.e. absence of significant de novoaccumulation after clearance of CD3 antibody from the blood circulation). Number of CD4+ T cells increased but remained significantly reduced over the long-term as compared to that detected in untreated recipients. This point has been clarified in the Introduction. In the RAG-/- mice, no CD3 antibody treatment was applied. Only cell infusion was performed and islet graft survival was assessed.

Author response image 2
Intragraft T cell counts.Pancreatic islet allografts were recovered from untreated (left) or CD3 antibody-treated (right) mice and CD4+ and CD8+ T cells were counted at different time-points post-transplant.

CD3 antibody treatment was applied on day +7 post-transplant for 5 days (gray area).

https://doi.org/10.7554/eLife.08133.021

3) The manuscript needs to acknowledge that the evidence for Fas-dependence of selective depletion of CD8 T cells expressing granzyme B and perforin is correlative as yet, and would require approaches such as FasL blockade to be definitive.

As requested, we have now included results showing pancreatic islet allografts in mice treated with anti-FasL antibodies in conjunction with CD3 antibodies. These are presented in a new Figure 2. In these recipients, CD3 antibody therapy did not induce long-term graft survival and thereby immune tolerance. Administration of FasL antibodies abrogated CD3 antibody-induced T cell depletion, notably that of CD8+ T cells infiltrating the islet allografts (new Figure 2B). As expected, FasL expression was inhibited on intragraft CD8+ T cells (new Figure 2C). Interestingly, anti-FasL administration also inhibited the CD3 antibody-induced upregulation of the inhibitory receptor PD-L1 on intragraft CD8+ T cells (new Figure 3—figure supplement 3). These points are now included in the Resultssection, at the end of the subsection “CD3 Ab therapy selectively depletes Gzmb+Perf+ CD8+ T cells and promotes anergy” and in the subsection “Graft infiltrating CD8+ T cells present an inhibitory phenotype after CD3 Ab therapy” as well as in the third paragraph of the Discussion.

4) There seems an inconsistency between the amount of protein expression (Figure 1—figure supplement 2) and the frequency of protein-expressing cells (Figure 1). Thus, the amount of FasL normalized to CD3 is ~65% less in treated versus untreated mice at day 14 (Figure 1—figure supplement 2A) while the frequency of FasL+ cells is ~50% MORE (Figure 1A). The reduction of granzyme B is more drastic in amount (Figure 1—figure supplement 2A) than in frequency (Figure 1A). The criteria for the judgment of expression in Figure 1 should be carefully described, since if amounts of protein per cell are significantly different, the cells at different time points may have different origins or functions.

We apologize for the apparent inconsistency. Figure 1 and Figure 1—figure supplement 2 show mRNA data obtained on distinct cell populations and using different methodologies which provide complementary results. Figure 1—figure supplement 2 represents PCR results on islet allografts as a whole and the analysis provided a global gene expression profile showing the downregulation of a cytotoxic/inflammatory signature after CD3 antibody therapy, including Gzmb and Fasl. Figure 1 focused on 72 individual intragraft CD8+ T cells subjected to multiplex PCR. As mRNAs were not quantified, we cannot exclude that although proportion of Fasl mRNA expressing CD8+ T cells increased after CD3 Ab treatment, the amount of Fasl mRNA per individual cells decreased. However, co-expression of inflammatory and cytotoxic genes provided important information on cell heterogeneity and on individual T cell behavior in the graft environment. Indeed, 2/3 of Fasl expressing CD8+ T cells from untreated recipients coexpressed Gzmb. In CD3 Ab treated hosts, only 1/3 coexpressed Gzmb and 1/3 did not coexpress any other inflammatory/cytotoxic genes. This suggests that Fasl mRNA expressing CD8+ T cells have distinct functional properties induced in response to the alloimmune stimulation and to CD3 antibody treatment.

The variable results between Figure 1 and Figure 1—figure supplement 2 further validate the benefit of performing single cell PCR experiments. Interestingly, flow cytometry analysis confirmed results obtained at the single cell level i.e. an increase of FasL protein expression on intragraft CD8+ T cells after CD3 antibody therapy (Figure 1D). These points have now been discussed in the Discussion of the revised manuscript (third paragraph).

5) The data provide no evidence for the antigen-specificity of the tolerisation or of its maintenance in RAG-null mice, nor has the allospecificity of individual CD8 T cells been formally documented.

As for points #1 and 2, the antigenic specificity of the immune tolerance induced by CD3 antibodies towards pancreatic islet allografts have been demonstrated in our previous work (You S et al. Am J Transplant 2012). Recipient mice, that showed long-term islet graft survival after treatment with CD3 antibodies on day +7 after transplantation, were rendered diabetic again on day +100 post-transplant either by injecting a single high dose of streptozotocin or after the nephrectomy of the kidney bearing the islets grafts. All recipients showed hyperglycemia as assessed by blood glucose levels > 400 mg/dL. Second islet grafts from the original donors (BALB/c) or third party donors (C3H) were then performed on the controlateral kidney. BALB/c islets survived indefinitely in contrast to C3H islets that were rejected within 20-30 days, thereby demonstrating that antigen-specific tolerance had indeed been induced in CD3 antibody-treated recipients. This point has been added in the Introduction(fourth paragraph).

In addition, ex-vivoexperiments (IFNγ Elispot) showed that spleen CD8+ T cells from CD3 Ab-treated mice were unable to mount donor-specific responses while responses to third-party antigens were normal (You S et al. Am J Transplant 2012).

Finally, in contrast to BALB/c allogeneic pancreatic islets, syngeneic islets were not rejected in RAG-/- recipients infused 4 weeks earlier with CD8+ T cells, highlighting the allospecificity of the CD8+ T cells recovered from CD3 Ab-treated mice. This new result is now illustrated on Figure 5—figure supplement 1.

6) It is unclear from the description of the methods if the CD8 T cell transfers into RAG-null mice have used intragraft cells or splenic cells for the transfers. Since 3 million cells were transferred, it is likely that splenic T cells were used. The gene expression and phenotyping data show some differences between intragraft and splenic CD8 T cells from anti-CD3-tolerised mice. However, the intragraft CD8 T cells are extensively characterised, while splenic CD8 T cells allotolerise quite effectively. This makes it hard to interpret the mechanistic significance of the patterns observed in intragraft CD8 T cells.

This point is well taken and we apologize for not having sufficiently detailed the design of the experiments. We used CD8+ T cells from the spleen of tolerant CD3 antibody-treated mice for cell transfer into RAG-/- mice. This point has been clarified in the Results(subsection “Continuous presence of alloantigen and the PD-1/PD-L1 pathway are mandatory for the induction and maintenance of CD3 Ab-induced CD8+ T cell tolerance”) and Materials and methods(subsection “Adoptive transfers”). We used spleen cells for the following reasons. First, technically, it was not possible to recover CD8+ T cells from the islet allografts in sufficient number to perform transfer experiments. Secondly and more importantly, as mentioned in points #1 and 5, we showed in our previous work that spleen purified CD8+ T cells were unable to respond to donor antigens after CD3 antibody therapy (You S et al. Am J Transplant 2012). Thus, CD8+ T cell unresponsiveness was evidenced at the periphery, which reflected what observed within the islet allografts (i.e. an absence of graft destruction). Lastly, PD-1 and PD-L1 expressions were also upregulated on spleen CD8+ T cells after CD3 antibody therapy and up to day 100 post-transplant as compared to untreated recipients (Figure 3—figure supplement 2). Therefore, spleen CD8+ T cells exhibited key functional features that reflected what observed within the islet allografts and we decided to use them to investigate CD8+ T cell tolerance in transfer experiments.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) In Figure 2B, the 'n' for the CD3+FasL abs appears to be 2. This is quite small. The related figure (2A) indicates that there were 4 animals studied. This needs to be clarified.

We understand the reviewers’ concern. We have treated a total of 6 recipient mice with CD3 Abs combined to FasL Abs. Two of them were sacrificed on day 14 post- transplant, i.e. right after the last Ab injection, to evaluate the effect of FasL blockade on the survival and phenotype of intragraft CD8+ T cells (Figure 2B-C and new panel Figure 3C). The remaining four were used to monitor graft survival or rejection illustrated on Figure 2A. This point has been clarified in the legend of revised Figure 2B-C and of Figure 3C.

2) If there are data on the CD8+ cells from mice treated with the CD3+FasL abs, they would be useful to include.

As requested, we have included new data on the phenotype of CD8+ T cells recovered from the islet allografts after the combined CD3+FasL Ab treatment. We now show that these CD8+ T cells express increased levels of CD25 and T-bet as compared to those issued from CD3 Ab alone-treated recipients, highlighting the recovery of effector functions after FasL blockade. These results are shown on revised Figure 3C (new panel) and are described in the subsection “Graft infiltrating CD8+ T cells present an inhibitory phenotype after CD3 Ab therapy.

3) CD3 antibody-induced upregulation of PD-L1 on intragraft CD8+ T cells was inhibited following in vivo FasL blockade;it would be best to include Figure3—figure supplement 3 in the main manuscript.

As requested, we have included this result in the revised manuscript, Figure 3C (new panel).

4) While Fas/FasL is a well-recognized pathway of cell death, it would be reasonable to address how this might be occurringis it suicide or are neighboring cells providing the lethal ligand? Is Fas/FasL co-expression on the same cell functional? At the least, the issue needs to be acknowledged and discussed.

As requested, we have discussed this issue in the third paragraph of the Discussion. We have demonstrated that T cells expressed both Fas and FasL after CD3 Ab treatment. Our results suggest, together with previous reports using T cell hybridomas or clones, that apoptosis has occurred by triggering of Fas by FasL-expressing neighboring T cells (Brunner T et al. Nature 1995, Ayroldi E et al. Blood 1997).

5) Clarification of Figure 4C: Should there be a point at d100 when the anti-PD-L1 was given? In other words were the grafts monitored before d100 after anti-PD-L1 injection? Since the number of mice is small, is it certain that those that really reject after anti-PD-L1 really been non-rejecting before d100 when the Ab was given?

We apologize for not having provided a clear experimental design for Figure 4C. Short course treatment (5 days) with CD3 antibodies induced permanent survival of BALB/c pancreatic islets transplanted under the kidney capsule of C57BL/6 mice. On day 100 post-transplant, i.e. once tolerance was established, recipient mice were treated with anti- PD-L1 antibodies or IgG2a isotype control. Graft rejection occurred 2-3 weeks later. For the sake of clarification, we have modified the text in the subsection “Continuous presence of alloantigen and triggering of the PD-1/PD-L1 pathway are mandatory for the induction and maintenance of CD3 Ab-induced CD8+ T cell tolerance” and the legend of Figure 4C.

6) In the experiment in Figure 5, presumably tolerance was lost during the 2 months in the absence of the BALB/c islets. Did RAG-/- recipients of spleen cells from the tolerant B6 mice not reject islets that were transplanted right after the transfer of cells? (Figure 5—figure supplement 1 does not address this control.)

As requested, we now show on one single figure (Figure 5) the graft survival rate obtained when islet transplantation was performed either on the day of CD8+ T cell transfer (D0) or 4 weeks (4wks) or 2 months after cell transfer (2mo). Survival of control syngeneic islet graft is also included on Figure 5. Therefore, Figure 5—figure supplement 1 has been removed. The text describing the results in the subsection “Continuous presence of alloantigen and triggering of the PD-1/PD-L1 pathway are mandatory for the induction and maintenance of CD3 Ab-induced CD8+ T cell tolerance”, has been modified.

7) Clarification of the implications of Figure 6: What explains the site differences in Tgfb1 expression, since TGFb was induced in culture, and since the soluble cytokine would probably have effects at all sites?

Figure 6B shows ex-vivo Tgfb mRNA expression by individual CD8+ and CD4+ T cells sorted from the spleen or the islet allografts of untreated or CD3 Ab-treated mice. Results showed that a large proportion of T cells present within the islet allografts, but not in the spleen, produced TGFβ after CD3 Ab administration. No culture was performed in these experiments. For the sake of clarity, we have modified the text in the subsection “Efficient TGFβ/TGFβR signaling promotes PD-1 and PD-L1 expression on graft infiltrating CD8+ T cells and is required for CD3 Ab-induced transplant tolerance.”

8) The last sentence of ResultsEOMES has functions other than serving as a transcription factor associated with Th1 cell development. It would be useful to discuss the possible explanation for its increase in the tolerized treated cells.

The increased Eomes expression in CD3 Ab-induced tolerant CD8+ T cells is indeed one novel message of our work. Eomes was mostly shown to play key role in CD8+ T cell differentiation and acquisition of an effector memory phenotype (Intlekofer M et al. Nat Immunol 2005). However, investigations in models of chronic infections in human and mouse (HIV, LCMV) have demonstrated that expression of Eomes by virus-specific CD8+ T cells was associated with high expression of inhibitory receptors and impaired functions (Doering TA et al. Immunity 2012; Paley MA et al. Science 2012; Buggert M et al. Plos Pathogens 2014). In our transplant model, as detailed below, the PD-1/PD-L1 pathway is mandatory for CD8+ T cell tolerance. Thus, increased expression of Eomes in conjunction with PD-1/PD-L1 may characterize anergized CD8+ T cells induced after CD3 Ab therapy. This finding highlights the issue of a molecular link between Eomes and PD-1/PD-L1. Lastly, one important feature common to our model and the infectious setting is the continuous presence of the cognate antigens. Therefore, we may hypothesize that chronic antigenic stimulation delivers signals inducing a preferential and sustained expression of Eomes. This point has been included in the Discussion section, sixth paragraph.

9) In the interests of brevity, the Discussion could be shortened in text that is not necessarily supported by the data, such as the extended PD-L1 expression discussion when PD-L1 expression was not studied in the islet grafts.

As requested, the Discussion has been shortened to better focus the message of our work.

https://doi.org/10.7554/eLife.08133.025

Article and author information

Author details

  1. Marije Baas

    1. University Paris Descartes, Sorbonne Paris Cité, Paris, France
    2. Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, Paris, France
    3. Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Paris, France
    Present address
    Department of Nephrology, Radboud University Medical center, Nijmegen, The Netherlands
    Contribution
    MB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Alix Besançon
    Competing interests
    The authors declare that no competing interests exist.
  2. Alix Besançon

    1. University Paris Descartes, Sorbonne Paris Cité, Paris, France
    2. Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, Paris, France
    3. Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Paris, France
    Contribution
    AB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Marije Baas
    Competing interests
    The authors declare that no competing interests exist.
  3. Tania Goncalves

    1. University Paris Descartes, Sorbonne Paris Cité, Paris, France
    2. Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, Paris, France
    3. Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Paris, France
    Contribution
    TG, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  4. Fabrice Valette

    1. University Paris Descartes, Sorbonne Paris Cité, Paris, France
    2. Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, Paris, France
    3. Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Paris, France
    Contribution
    FV, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  5. Hideo Yagita

    Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
    Contribution
    HY, Provided purified anti-PD1 and anti-FasLigand antibodies in large amounts for in vivo experiments. These tools were essential to our research project. He provided advices on the dose and timing of injection. He also contributed to the discussion of the manuscript, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  6. Birgit Sawitzki

    Institute of Medical Immunology, Charité University Medicine, Berlin, Germany
    Contribution
    BS, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  7. Hans-Dieter Volk

    1. Institute of Medical Immunology, Charité University Medicine, Berlin, Germany
    2. Berlin-Brandenburg Center for Regenerative Therapies, Charité University Medicine, Berlin, Germany
    Contribution
    H-DV, Provided critical advices on the direction of some experiments and the discussion of the results. Conception and design, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  8. Emmanuelle Waeckel-Enée

    1. University Paris Descartes, Sorbonne Paris Cité, Paris, France
    2. Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, Paris, France
    3. Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Paris, France
    Contribution
    EWE, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  9. Benedita Rocha

    1. University Paris Descartes, Sorbonne Paris Cité, Paris, France
    2. Lymphocyte Population Biology Unit, Pasteur Institute, Paris, France
    Contribution
    BR, Provided critical advices on the multiplex PCR performed on individual CD8+ T cells, from the conception of the experiments to the interpretation of the results. She also provided reagents for these single cell PCR., Conception and design, Analysis and interpretation of data, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  10. Lucienne Chatenoud

    1. University Paris Descartes, Sorbonne Paris Cité, Paris, France
    2. Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, Paris, France
    3. Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Paris, France
    Contribution
    LC, Conception and design, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  11. Sylvaine You

    1. University Paris Descartes, Sorbonne Paris Cité, Paris, France
    2. Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, Paris, France
    3. Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Paris, France
    Contribution
    SY, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    sylvaine.you@inserm.fr
    Competing interests
    The authors declare that no competing interests exist.

Funding

European Commission (RISET, 512090 IP)

  • Lucienne Chatenoud
  • Sylvaine You

Juvenile Diabetes Research Foundation (R11119KK)

  • Lucienne Chatenoud
  • Sylvaine You

Institut National de la Santé et de la Recherche Médicale (Institutional funding)

  • Lucienne Chatenoud
  • Sylvaine You

Université (Institutional funding)

  • Lucienne Chatenoud
  • Sylvaine You

Nierstichting (KFB 10.003)

  • Marije Baas

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank Claire Mangez for technical assistance and Jérome Megret for single cell sorting. We are also grateful to Pr. Azuma (Tokyo Medical and Dental University) for providing the anti-PD-L1 hybridoma.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommandations of the European Directive 2010/63/UE. All of the animals were handled according to approved institutional animal care and use committee protocols of the University of Paris Descartes.The protocol was approved by the Ethical Committee of Paris Descartes University (registered number: 14-075). All surgery was performed under ketamine/xylasine anesthesia, and every effort was made to minimize suffering.

Reviewing Editor

  1. Satyajit Rath, National Institute of Immunology, India

Publication history

  1. Received: April 15, 2015
  2. Accepted: January 2, 2016
  3. Version of Record published: January 29, 2016 (version 1)

Copyright

© 2016, Baas et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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