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Background: Regulatory T cells (Treg) in diverse species include CD4 + and CD8 + T cells. In all species, CD8 + Treg have been only partially characterized and there is no rat model in which CD4 + and CD8 + FOXP3 + Treg are genetically tagged., Results: We generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP. CD4 + and CD8 + T cells were the only cells that expressed EGFP, in similar proportion as observed with anti-FOXP3 antibodies and co-labeled in the same cells. CD4 + EGFP + Treg were 5-10 times more frequent than CD8 + EGFP + Treg. The suppressive activity of CD4 + and CD8 + Treg was largely confined to EGFP + cells. RNAseq analyses showed similarities but also differences among CD4 + and CD8 + EGFP + cells and provided the first description of the natural FOXP3 + CD8 + Treg transcriptome. In vitro culture of CD4 + and CD8 + EGFP - cells with TGFbeta and IL-2 generated induced EGFP + Treg. CD4 + and CD8 + EGFP + Treg were expanded upon in vivo administration of a low dose of IL-2., Conclusions: This new and unique rat line constitutes a useful model to identify and isolate viable CD4 + and CD8 + FOXP3 + Treg. Additionally, it allows to identify molecules expressed in CD8 + Treg that may allow to better define their phenotype and function not only in rats but also in other species., (© 2023. The Author(s).)

Background: Immune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL-34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8 + Tregs in a rat model of transplantation tolerance and by activated FOXP3 + CD4 + and CD8 + Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remains to be determined., Methods: We generated Il34 -/- rats and using both Il34 -/- rats and mice, we investigated their phenotype under inflammatory conditions. Using Il34 -/- rats, we further analyzed the impact of the absence of expression of IL-34 for CD4 + Tregs suppressive function. We investigated the potential of IL-34 in human disease to prevent xenogeneic GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, taking advantage of a biocollection, we investigated the correlation between presence of IL-34 in the serum and kidney transplant rejection., Results: Here we report that the absence of expression of IL-34 in Il34 -/- rats and mice leads to an unstable immune phenotype, with production of multiple auto-antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS- and TNBS-colitis in Il34 -/- animals. Moreover, we revealed the striking inability of Il34 -/- CD4 + Tregs to protect Il2rg -/- rats from a wasting disease induced by transfer of pathogenic cells, in contrast to Il34 +/+ CD4 + Tregs. We also showed that IL-34 treatment delayed EAE in mice as well as GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, we show that presence of IL-34 in the serum is associated with a longer rejection-free period in kidney transplanted patients., Conclusion: Altogether, our data emphasize on the crucial necessity of IL-34 for immune homeostasis and for CD4 + Tregs suppressive function. Our data also shows the therapeutic potential of IL-34 in human transplantation and auto-immunity., Highlights: -Absence of expression of IL-34 in Il34 -/- rats and mice leads to an unstable immune phenotype, with a production of multiple auto-antibodies and exacerbated immune pathology under inflammatory conditions. -Il34 -/- CD4 + Tregs are unable to protect Il2rg -/- rats from colitis induced by transfer of pathogenic cells. -IL-34 treatment delayed EAE in mice, as well as acute GVHD and human skin allograft rejection in immune-humanized immunodeficient NSG mice., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Regulatory Tcells (Treg) are essential components of peripheral immune homeostasis. Adoptive Treg cell therapy has shown efficacy in a variety of immune-mediated diseases in preclinical studies and is now moving from phase I/IIa to larger phase II studies aiming to demonstrate efficacy. However, hurdles such as in vivo stability and efficacy remain to be addressed. Nevertheless, preclinical models have shown that Treg function and specificity can be increased by pharmacological substances or gene modifications, and even that conventional T cells can be converted to Treg potentially providing new sources of Treg and facilitating Treg cell therapy. The exponential growth in genetic engineering techniques and their application to T cells coupled to a large body of knowledge on Treg open numerous opportunities to generate Treg with "superpowers". This review summarizes the genetic engineering techniques available and their applications for the next-generation of Super-Treg with increased function, stability, redirected specificity and survival., Competing Interests: CG, IA, and SB have patents that have been licensed to TxCell S.A., a Sangamo Company. DW, LA, PR, H-DV, and MS-H have filed patent applications on Treg-related diagnostics and therapeutics. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amini, Greig, Schmueck-Henneresse, Volk, Bézie, Reinke, Guillonneau, Wagner and Anegon.)

Cytokines are major players regulating immune responses toward inflammatory and tolerogenic results. In organ and bone marrow transplantation, new reagents are needed to inhibit tissue destructive mechanisms and eventually induce immune tolerance without overall immunosuppression. IL-34 is a cytokine with no significant homology with any other cytokine but that acts preferentially through CSF-1R, as CSF-1 does, and through PTPζ and CD138. Although IL-34 and CSF-1 share actions, a detailed analysis of their effects on immune cells needs further research. We previously showed that both CD4 + and CD8 + FOXP3 + Tregs suppress effector T cells through the production of IL-34, but not CSF-1, and that this action was mediated through antigen-presenting cells. We showed here by single-cell RNAseq and cytofluorimetry that different subsets of human monocytes expressed different levels of CSF-1R, CD138, and PTPζ and that both CD4 + and CD8 + FOXP3 + Tregs expressed higher levels of CSF-1R than conventional T cells. The effects of IL-34 differed in the survival of these different subpopulations of monocytes and RNAseq analysis showed several genes differentially expressed between IL-34, CSF-1, M0, M1, and also M2 macrophages. Acute graft-vs.-host disease (aGVHD) in immunodeficient NSG mice injected with human PBMCs was decreased when treated with IL-34 in combination with an anti-CD45RC mAb that depleted conventional T cells. When IL-34-differentiated monocytes were used to expand Tregs in vitro , both CD4 + and CD8 + FOXP3 + Tregs were highly enriched and this effect was superior to the one obtained with CSF-1. Human CD8 + Tregs expanded in vitro with IL-34-differentiated allogeneic monocytes suppressed human immune responses in an NSG mouse aGVHD model humanized with hPBMCs. Overall, we showed that IL-34 induced the differentiation of human monocytes with a particular transcriptional profile and these cells favored the development of potent suppressor FOXP3 + Tregs., (Copyright © 2020 Bézie, Freuchet, Sérazin, Salama, Vimond, Anegon and Guillonneau.)

Polyclonal CD8+CD45RClow/- 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-PrkdcscidIl2rgtm1Wjl/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., (© 2019 by The American Society of Hematology.)

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., (© 2019 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Although cluster of differentiation (CD)8 regulatory T (Treg) cells have been in the last 20 years more studied since evidences of their role in tolerance as been demonstrated in transplantation, autoimmune diseases and cancer, their characteristics are still controversial. In this review, we will focus on recent advances on CD8 Treg cells and description of a role for CD8 Treg cells in tolerance in both solid organ transplantation and graft-versus-host disease and their potential for clinical trials.

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., Competing Interests: CG and IA have submitted a patent (WO2016016442) that is pending.

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+CD45RClo 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+CD45RClo Tregs and human FOXP3+CD45RCloCD8+ 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.

In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function.

Besides their therapeutic benefit as cell source, neural stem/progenitor cells (NSPCs) exhibit immunosuppressive properties of great interest for modulating immune response in the central nervous system. To decipher the mechanisms of NSPC-mediated immunosuppression, activated T cells were exposed to NSPCs isolated from fetal rat brains. Analyses revealed that NSPCs inhibited T-cell proliferation and interferon-gamma production in a dose-dependent manner. A higher proportion of helper T cells (CD4+ T cells) was found in the presence of NSPCs, but analyses of FoxP3 population indicated that T-cell suppression was not secondary to an induction of suppressive regulatory T cells (FoxP3+ CD4+ CD25+). Conversely, induction of the high affinity interleukin-2 (IL-2) receptor (CD25) and the inability of IL-2 to rescue T-cell proliferation suggest that NSPCs display immunosuppressive activity without affecting T-cell activation. Cultures in Transwell chambers or addition of NSPC-conditioned medium to activated T cells indicated that part of the suppressive activity was not contact dependent. We therefore searched for soluble factors that mediate NSPC immunosuppression. We found that NSPCs express several immunosuppressive molecules, but the ability of these cells to inhibit T-cell proliferation was only counteracted by heme oxygenase (HO) inhibitors in association or not with nitric oxide synthase inhibitors. Taken together, our findings highlight a dynamic crosstalk between NSPCs and T lymphocytes and provide the first evidence of an implication of HO-1 in mediating the immunosuppressive effects of the NSPCs., (Copyright © 2012 AlphaMed Press.)

Recent studies in the field of CD8(+) Tregs have allowed a better identification and characterization of this subset of regulatory cells. Their key role in the regulation of allogeneic responses is now well established. To take full advantage of CD8(+) Treg cells in future therapeutic applications, a better knowledge is required, particularly concerning the contribution of the T-cell receptor (TCR) in cell function as well as the role and importance of its antigenic specificity. Here, we focused on the CD8(+)CD45RC(low) Tregs, which in rats induce an indefinite long-term allograft acceptance. We summarized recent findings on their interaction properties with antigen-presenting cells. Identification of the antigenic targets and TCR repertoire of CD8(+) Tregs will allow a better understanding of their recognition properties and will highlight the potential of such of specific population in cell-based treatment.

Increasing evidence shows the presence and significance of CD8+ T regulatory cells (CD8+ Tregs) in both human and rodent transplant recipients, as well as in autoimmune disease models. We, hereafter, review all available data on the phenotypic and functional characterization of CD8+ Tregs, and we also provide detailed protocols to purify them and analyze their suppressive function. Different subsets of dendritic cells (DCs) and CD4+ effector T cells may modulate the suppression mediated by CD8+ Tregs. By analyzing the proliferation of CFSE-labeled naïve CD4+CD25- T cells in coculture MLR and transwell experiments, we explored the mutual modulation of CD8+ Tregs, DC subsets, and CD4+ T effector cells. The suppressive function of CD8+ Tregs was mediated by both cell-contact-dependent and -independent mechanisms.

Despite accumulating evidence for the importance of allospecific CD8(+) regulatory T cells (Tregs) in tolerant rodents and free immunosuppression transplant recipients, mechanisms underlying CD8(+) Treg-mediated tolerance remain unclear. By using a model of transplantation tolerance mediated by CD8(+) Tregs following CD40Ig treatment in rats, in this study, we show that the accumulation of tolerogenic CD8(+) Tregs and plasmacytoid dendritic cells (pDCs) in allograft and spleen but not lymph nodes was associated with tolerance induction in vascularized allograft recipients. pDCs preferentially induced tolerogenic CD8(+) Tregs to suppress CD4(+) effector cells responses to first-donor Ags in vitro. When tolerogenic CD8(+) Tregs were not in contact with CD4(+) effector cells, suppression was mediated by IDO. Contact with CD4(+) effector cells resulted in alternative suppressive mechanisms implicating IFN-gamma and fibroleukin-2. In vivo, both IDO and IFN-gamma were involved in tolerance induction, suggesting that contact with CD4(+) effector cells is crucial to modulate CD8(+) Tregs function in vivo. In conclusion, CD8(+) Tregs and pDCs interactions were necessary for suppression of CD4(+) T cells and involved different mechanisms modulated by the presence of cell contact between CD8(+) Tregs, pDCs, and CD4(+) effector cells.

We have previously shown that human monocyte-derived dendritic cells (DC) express indoleamine 2,3-dioxygenase (IDO), as well as several other enzymes of the kynurenine pathway at the mRNA level upon maturation. The tolerogenic mechanisms of this pathway remain unclear. Here we show that LPS-treated DC metabolize tryptophan as far as quinolinate. We found that IDO contributes to LPS and TNF-alpha + poly(I:C)-induced DC maturation since IDO inhibition using two different inhibitors impairs DC maturation. IDO knock-down using short-hairpin RNA also led to diminished LPS-induced maturation. In line with these results, the tryptophan-derived catabolites 3-hydroxyanthranilic acid and 3-hydroxykynurenine increased maturation of LPS-treated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS-induced production of reactive oxygen species and NF-kappaB activation, two processes that lead to DC maturation. Finally, we show that mature DC expand CD4(+)CD25(high) regulatory T cells in an IDO-dependent manner. In conclusion, we show that IDO constitutes an intermediate pathway in DC maturation leading to expansion of CD4(+)CD25(high) regulatory T cells.

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3(-) CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNgamma-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNgamma or IDO, and anti-IFNgamma reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNgamma and IDO in this process.

Background: We have previously demonstrated that a short-course treatment with LF15-0195, a 15-deoxyspergualin analogue, induces donor-specific tolerance of cardiac allografts in rats and expansion of splenic CD4CD25 regulatory T cells., Methods: To further characterize long-term tolerance in this model, we have analyzed the phenotype, regulatory properties and TCR-Vbeta usage of the T cells infiltrating the tolerated allografts., Results: We demonstrate that the tolerated allografts express high levels of FoxP3 transcripts and contain a large number of CD4 T cells, half of which express CD25. Moreover, T cells from these tolerated allografts are very powerful at transferring tolerance to a subsequent allograft recipient, demonstrating the presence of potent regulatory T cells at the site of the graft. Interestingly, the T cells infiltrating the tolerated allografts systematically display restricted Vbeta7 TCR rearrangements., Conclusion: These results demonstrate in this model of tolerance, a specific accumulation of T cells with potent regulatory properties and exhibiting restricted Vbeta7-TCR rearrangements at the graft site.