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.

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.

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 CD8⁺ T 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 CD44^highCD62L^lowCD69⁺CD45RB^low 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).

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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.

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