Cytokines and metabolic pathway–controlling enzymes regulate immune responses and have potential as powerful tools to mediate immune tolerance. Blockade of the interaction between CD40 and CD40L induces long-term cardiac allograft survival in rats through a CD8⁺CD45RC^lo Treg potentiation. Here, we have shown that the cytokine IL-34, the immunoregulatory properties of which have not been previously studied in transplantation or T cell biology, is expressed by rodent CD8⁺CD45RC^lo Tregs and human FOXP3⁺CD45RC^loCD8⁺ and CD4⁺ Tregs. IL-34 was involved in the suppressive function of both CD8⁺ and CD4⁺ Tregs and markedly inhibited alloreactive immune responses. Additionally, in a rat cardiac allograft model, IL-34 potently induced transplant tolerance that was associated with a total inhibition of alloantibody production. Treatment of rats with IL-34 promoted allograft tolerance that was mediated by induction of CD8⁺ and CD4⁺ Tregs. Moreover, these Tregs were capable of serial tolerance induction through modulation of macrophages that migrate early to the graft. Finally, we demonstrated that human macrophages cultured in the presence of IL-34 greatly expanded CD8⁺ and CD4⁺ FOXP3⁺ Tregs, with a superior suppressive potential of antidonor immune responses compared with non–IL-34–expanded Tregs. In conclusion, we reveal that IL-34 serves as a suppressive Treg–specific cytokine and as a tolerogenic cytokine that efficiently inhibits alloreactive immune responses and mediates transplant tolerance.

CD8⁺ Tregs have been long described and significant progresses have been made about their phenotype, their functional mechanisms, and their suppressive ability compared to conventional CD4⁺ Tregs. They are now at the dawn of their clinical use. In this review, we will summarize their phenotypic characteristics, their mechanisms of action, the similarities, differences and synergies between CD8⁺ and CD4⁺ Tregs, and we will discuss the biology, development and induction of CD8⁺ Tregs, their manufacturing for clinical use, considering open questions/uncertainties and future technically accessible improvements notably through genetic modifications.

Both CD4⁺ and CD8⁺ Tregs play a critical role in the control of immune responses and immune tolerance; however, our understanding of CD8⁺ Tregs is limited while they are particularly promising for therapeutic application. We report here existence of highly suppressive human CD8⁺CD45RC^low/− Tregs expressing Foxp3 and producing IFNγ, IL-10, IL-34, and TGFβ to mediate their suppressive activity. We demonstrate that total CD8⁺CD45RC^low/− Tregs can be efficiently expanded in the presence of anti-CD3/28 mAbs, high-dose IL-2 and IL-15 and that such expanded Tregs efficiently delay GVHD and human skin transplantation rejection in immune humanized mice. Robustly expanded CD8⁺ Tregs displayed a specific gene signature, upregulated cytokines and expansion in the presence of rapamycin greatly improved proliferation and suppression. We show that CD8⁺CD45RC^low/− Tregs are equivalent to canonical CD4⁺CD25^highCD127^low/− Tregs for suppression of allogeneic immune responses in vitro. Altogether, our results open new perspectives to tolerogenic strategies in human solid organ transplantation and GVHD.

Polyclonal CD8⁺CD45RC^low/− Tregs are potent regulatory cells able to control solid organ transplantation rejection and even induce tolerance. However, donor major histocompatibility complex (MHC)–specific Tregs are more potent than polyclonal Tregs in suppressing T-cell responses and preventing acute as well as chronic rejection in rodent models. The difficulty of identifying disease-relevant antigens able to stimulate Tregs has reduced the possibility of obtaining antigen-specific Tregs. To bypass this requirement and gain the advantage of antigen specificity, and thus improve the therapeutic potential of CD8⁺ Tregs, we stably introduced a chimeric antigen receptor (CAR) derived from a HLA-A*02 antigen-specific antibody (A2-CAR) in human CD8⁺ Tregs and developed a clinically compatible protocol of transduction and expansion. We demonstrated that A2-CAR CD8⁺ Tregs were not phenotypically altered by the process, were specifically activated, and did not exhibit cytotoxic activity toward HLA-A*02⁺ kidney endothelial cells (ECs). We showed that A2-CAR CD8⁺ Tregs were more potent suppressors of immune responses induced by HLA-A*02 mismatch than control-CAR CD8⁺ Tregs, both in vitro and in vivo, in models of human skin graft rejection and graft-versus-host disease (GVHD) in NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice. We showed that integrity of human skin graft was preserved with A2-CAR CD8⁺ Tregs at least 100 days in vivo after administration, and that interaction between the A2-CAR CD8⁺ Tregs and HLA-A*02⁺ kidney ECs resulted in a fine-tuned and protolerogenic activation of the ECs without cytotoxicity. Together, our results demonstrated the relevance of the CAR engineering approach to develop antigen-specific CAR-CD8⁺ Tregs for clinical trials in transplantation, and potentially in other diseases.

Rat and human CD4 ⁺ and CD8 ⁺ Tregs expressing low levels of CD45RC have strong immunoregulatory properties. We describe here that human CD45 isoforms are nonredundant and identify distinct subsets of cells. We show that CD45RC is not expressed by CD4 ⁺ and CD8 ⁺ Foxp3 ⁺ Tregs, while CD45RA/RB/RO are. Transient administration of a monoclonal antibody (mAb) targeting CD45RC in a rat cardiac allotransplantation model induced transplant tolerance associated with inhibition of allogeneic humoral responses but maintained primary and memory responses against cognate antigens. Anti-CD45RC mAb induced rapid death of CD45RC ^high T cells through intrinsic cell signaling but preserved and potentiated CD4 ⁺ and CD8 ⁺ CD45RC ^low/– Tregs, which are able to adoptively transfer donor-specific tolerance to grafted recipients. Anti-CD45RC treatment results in distinct transcriptional signature of CD4 ⁺ and CD8 ⁺ CD45RC ^low/– Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-host disease (GVHD) in immune-humanized NSG mice. Thus, short-term anti-CD45RC is a potent therapeutic candidate to induce transplantation tolerance in human.