ABSTRACT Although described in the 70’s, CD8 + regulatory T cells (Tregs) remain incompletely understood and to date, although several markers are used to define them, they remain poorly defined. The identification of reliable and consistent markers, as it was done for CD4 + Tregs, remains an urgent task and a challenge to advance our understanding. Herein, we analyzed total CD8 + T cells using single cell CITEseq and VDJ T cell receptor sequencing utilizing markers used previously to identify Tregs, in particular CD45RC described by our team and others to divide pro-inflammatory (CD45RC high ) and pro-regulatory (CD45RC low/- ) CD8 + T cells in rat, mice and human. 7000 freshly isolated, non-stimulated CD8 + T lymphocytes of four healthy volunteers were analyzed. Combining at a single cell level transcriptome and protein expression data led for the first time to the characterization and definition of three subsets of regulatory CD8 + T cells. Further in vitro functional analysis based on three markers highlighted the superior suppressive activity of the CD8 + CD45RC low/- TNFR2 + CD29 low Tregs subset. To our knowledge, this is the largest characterization of human CD8 + Tregs to date. This data resource will help improve our understanding of CD8 + T cells heterogeneity and will help to translate CD8 + Tregs to the clinic.

CD8+ regulatory T cells (Tregs) were the first suppressive cells reported in 1970, but they were put aside for years due to a lack of markers to properly define them. Our team demonstrated that CD8+ Tregs identified by low and/or negative expression of CD45RC, one of the isoforms of the CD45 molecule, show potent suppressive activity in vitro and in vivo, while cells expressing high levels of CD45RC do not. Herein, we addressed the heterogeneity within CD8+CD45RClow/- Tregs and identified new markers. We performed single cell RNA-sequencing on more than 10000 non-stimulated CD8+CD45RClow/- Tregs of 3 healthy volunteers (HV) and a CD screen analysis of 360 membrane molecules on different cells subsets of 4 HV (CD8+CD45RChigh; non-stimulated, stimulated or expanded CD4+CD25highCD127low and CD8+CD45RClow/- Tregs). Using these technologies, we were able to characterize the transcriptomic heterogeneity at a single cell level and the proteomic heterogeneity from non-stimulated CD8+CD45RClow/- Tregs. We thus identified sub-populations of cells and one cluster of particular interest expressing critical markers and molecules involved in tolerance. Further work on two of these promising membrane markers, i.e., TNFR2 and CD29, using cell sorting and suppressive assays highlighted CD8+CD45RClow/-TNFR2+CD29low Tregs subset as the most suppressive subsets within CD8+CD45RClow/- Tregs. Here, we reveal the importance of the combination of TNFR2 and CD29 as highly specific membrane markers for human CD8+ Tregs and highlighted their potential roles as targets for treatments in tolerance. Other new promising markers identified in our study showed an interesting role in CD8+ Tregs function; their precise roles are still under investigation. To date, to our knowledge, this is the largest characterization study of human CD8+ Tregs, this huge data resource will help in the current revival of CD8+ Tregs in research.

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.

Immunotherapy using primary T cells has revolutionized medical care in some pathologies in recent years, but limitations associated to challenging cell genome edition, insufficient cell number production, the use of only autologous cells, and the lack of product standardization have limited its clinical use. The alternative use of T cells generated in vitro from human pluripotent stem cells (hPSCs) offers great advantages by providing a self-renewing source of T cells that can be readily genetically modified and facilitate the use of standardized universal off-the-shelf allogeneic cell products and rapid clinical access. However, despite their potential, a better understanding of the feasibility and functionality of T cells differentiated from hPSCs is necessary before moving into clinical settings. In this study, we generated human-induced pluripotent stem cells from T cells (T-iPSCs), allowing for the preservation of already recombined TCR, with the same properties as human embryonic stem cells (hESCs). Based on these cells, we differentiated, with high efficiency, hematopoietic progenitor stem cells (HPSCs) capable of self-renewal and differentiation into any cell blood type, in addition to DN3a thymic progenitors from several T-iPSC lines. In order to better comprehend the differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had very similar profiles to cord blood hematopoietic stem cells (HSCs). Furthermore, differentiated T-cell progenitors had a similar profile to thymocytes at the DN3a stage of thymic lymphopoiesis. Therefore, utilizing this approach, we were able to regenerate precursors of therapeutic human T cells in order to potentially treat a wide range of diseases.

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 remain to be determined. Here we report that the absence of expression of IL-34 in Il34 -/- rodents leads to an unstable phenotype, with production of multiple auto-antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS- and TNBS-colitis in deficient animals. Moreover, we strikingly revealed the 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. 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.

Targeted monoclonal antibody (mAb) therapies show great promise for the treatment of transplant rejection and autoimmune diseases by inducing more specific immunomodulatory effects than broadly immunosuppressive drugs routinely used. We recently described the therapeutic advantage of targeting CD45RC, expressed at high levels by conventional T (Tconv) cells (CD45RChi), their precursors, and terminally differentiated T (TEMRA) cells, but not by regulatory T cells (Tregs; CD45RClo/–). We demonstrated efficacy of anti-CD45RC mAb treatment in transplantation, but its potential has not been examined in autoimmune diseases. Autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) is a rare genetic syndrome caused by loss-of-function mutations of autoimmune regulator (AIRE), a key central tolerance mediator, leading to abnormal autoreactive T cell responses and autoantibody production. Herein, we show that, in a rat model of APECED syndrome, anti-CD45RC mAb was effective for both prevention and treatment of autoimmune manifestations and inhibited autoantibody development. Anti-CD45RC mAb intervention depleted CD45RChi T cells, inhibited CD45RChi B cells, and restored the Treg/Tconv cell ratio and the altered Treg transcriptomic profile. In APECED patients, CD45RC was significantly increased in peripheral blood T cells, and lesioned organs from APECED patients were infiltrated by CD45RChi cells. Our observations highlight the potential role for CD45RChi cells in the pathogenesis of experimental and human APECED syndrome and the potential of anti-CD45RC antibody treatment.

Background and Objectives Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Studies of immune dysfunction in MS have mostly focused on CD4+ Tregs, but the role of CD8+ Tregs remains largely unexplored. We previously evidenced the suppressive properties of rat and human CD8+CD45RClow/neg Tregs from healthy individuals, expressing Forkhead box P3 (FOXP3) and acting through interferon-gamma (IFN-γ), transforming growth factor beta (TGFβ), and interleukin-34 (IL-34). secretions to regulate immune responses and control diseases such as transplant rejection. To better understand CD8+CD45RClow/neg Tregs contribution to MS pathology, we further investigated their phenotype, function, and transcriptome in patients with MS. Methods We enrolled adults with relapsing-remitting MS and age-matched and sex-matched healthy volunteers (HVs). CD8+ T cells were segregated based on low or lack of expression of CD45RC. First, the frequency in CSF and blood, phenotype, transcriptome, and function of CD8+CD45RClow and neg were investigated according to exacerbation status and secondarily, according to clinical severity based on the MS severity score (MSSS) in patients with nonexacerbating MS. We then induced active MOG35-55 EAE in C57Bl/6 mice and performed adoptive transfer of fresh and expanded CD8+CD45RCneg Tregs to assess their ability to mitigate neuroinflammation in vivo. Results Thirty-one untreated patients with relapsing-remitting MS were compared with 40 age-matched and sex-matched HVs. We demonstrated no difference of CSF CD8+CD45RClow and CD8+CD45RCneg proportions, but blood CD8+CD45RClow frequency was lower in patients with MS exacerbation when compared with that in HVs. CD8+CD45RCneg Tregs but not CD8+CD45RClow showed higher suppressive capacities in vitro in MS patients with exacerbation than in patients without acute inflammatory attack. In vitro functional assays showed a compromised suppression capacity of CD8+CD45RClow Tregs in patients with nonexacerbating severe MS, defined by the MSSS. We then characterized murine CD8+CD45RCneg Tregs and demonstrated the potential of CD45RCneg cells to migrate to the CNS and mitigate experimental autoimmune encephalomyelitis in vivo. Discussion Altogether, these results suggest a defect in the number and function of CD8+CD45RClow Tregs during MS relapse and an association of CD8+CD45RClow Tregs dysfunction with MS severity. Thus, CD8+CD45RClow/neg T cells might bring new insights into the pathophysiology and new therapeutic approaches of MS.

The success of inducing human pluripotent stem cells (hIPSC) offers new opportunities for cell-based therapy. Since B cells exert roles as effector and as regulator of immune responses in different clinical settings, we were interested in generating B cells from hIPSC. We differentiated human embryonic stem cells (hESC) and hIPSC into B cells onto OP9 and MS-5 stromal cells successively. We overcame issues in generating CD34(+)CD43(+) hematopoietic progenitors with appropriate cytokine conditions and emphasized the difficulties to generate proper hematopoietic progenitors. We highlight CD31(int)CD45(int) phenotype as a possible marker of hematopoietic progenitors suitable for B cell differentiation. Defining precisely proper lymphoid progenitors will improve the study of their lineage commitment and the signals needed during the in vitro process.

One major goal in transplantation is to develop novel specific and non-toxic anti-rejection immunotherapies. Strategies based on regulatory T cells (Tregs) are promising to prevent graft rejection. Our group has shown that rat and human CD8+CD45RClow/- Tregs display significant suppressive function. The team has also shown that cell therapy using human CD8+CD45RClow/- Tregs was efficient to prevent graft rejection and GVHD in humanized NSG mice models. However, the heterogeneity of the CD8+CD45RClow/- Tregs population is important from a phenotypic point of view, suggesting that either a fraction of the population is tolerogenic, or the induction of tolerance is due to a combination of cells forming an “immunological niche”. In order to be able to discriminate between these two hypotheses, we explored heterogeneity inside the CD8+CD45RClow/- T cell population. We sorted CD8+CD45RClow/- Tregs from blood of healthy volunteers and sequenced their transcriptomes by single cell RNA sequencing methods using two technologies: one based on microfluidic systems (Fluidigm C1) and one based on droplets (10x Genomics). To our knowledge those are the first single cell RNA-seq datasets of human CD8+ Tregs. The cell transcriptomes analysis with Seurat package in R highlighted the heterogeneity inside the population with the identification of 4 distinct clusters with a specific signature. We identified one cluster of particular interest based on its immune and regulatory gene signature. However, other genes such as TGFβ were expressed by all clusters inside CD8+CD45RClow/- T cells. We then focused our analysis on genes that encode for membrane proteins that have never been used for CD8+ Tregs isolation yet such as TNFR2 and ITGB1. We confirmed their expression at protein level on human PBMC from healthy volunteers by a subset of CD8+CD45RClow/- T cells. Finally, we sorted cell subsets according to the expression of membrane markers to test their suppressive function in vitro. In conclusion, this project characterized the heterogeneity inside CD8+CD45RClow/- Tregs both at single cell level and proteomic level. This project will help us to better define a consensus phenotype for CD8+ Tregs and will also improve the use of human CD8+ Tregs as a cell therapy for treating transplanted patients.

Cell therapy using T cells has revolutionized medical care in recent years but limitations are associated with the difficulty of genome editing of the cells, the production of a sufficient number of cells and standardization of the product. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into T cells to provide a standardized homogenous product of defined origin in indefinite quantity, therefore they are of great potential to alleviate limitations of therapeutic T cell production. The differentiation of hPSCs takes place in two steps: first the induction of hematopoietic stem/progenitor cells (HSPCs), then the induction of lymphopoiesis by Notch signaling. However, the differentiation of T cells from hPSCs can be difficult and lack reproducibility. One parameter that needs to be better assessed is the potential of DLL1 vs. DLL4 ligands of the Notch pathway to induce T cells. In addition, culture of hPSCs is labor-intensive and not compatible with GMP production, especially when they are cultured on feeder cells. Thus, the definition of a robust GMP-compatible differentiation protocol from hPSCs cultured in feeder-free conditions would increase the accessibility to off-the-shelf hematopoietic and T cell progenitors derived from hPSCs. In this article, we describe an efficient, rapid and reproducible protocol for the generation of hematopoietic and T cell progenitors in two steps: (1) generation of HSPCs from embryoid bodies (EB) in serum free medium and GMP-compatible feeder-free systems, (2) directed differentiation of hPSC-derived HSPCs into T-cell progenitors in the presence of bone marrow stromal cells expressing Notch-ligands OP9-DLL1 vs. OP9-DLL4.

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.

Induced pluripotent stem cells (iPSCs) have considerably impacted human developmental biology and regenerative medicine, notably because they circumvent the use of cells of embryonic origin and offer the potential to generate patient-specific pluripotent stem cells. However, conventional reprogramming protocols produce developmentally advanced, or primed, human iPSCs (hiPSCs), restricting their use to post-implantation human development modeling. Hence, there is a need for hiPSCs resembling preimplantation naive epiblast. Here, we develop a method to generate naive hiPSCs directly from somatic cells, using OKMS overexpression and specific culture conditions, further enabling parallel generation of their isogenic primed counterparts. We benchmark naive hiPSCs against human preimplantation epiblast and reveal remarkable concordance in their transcriptome, dependency on mitochondrial respiration and X-chromosome status. Collectively, our results are essential for the understanding of pluripotency regulation throughout preimplantation development and generate new opportunities for disease modeling and regenerative medicine.

Over a decade after their discovery, induced pluripotent stem cells (iPSCs) have become a major biological model. The iPSC technology allows generation of pluripotent stem cells from somatic cells bearing any genomic background. The challenge ahead of us is to translate human iPSCs (hiPSCs) protocols into clinical treatment. To do so, we need to improve the quality of hiPSCs produced. In this study we report the reprogramming of multiple patient urine-derived cell lines with mRNA reprogramming, which, to date, is one of the fastest and most faithful reprogramming method. We show that mRNA reprogramming efficiently generates hiPSCs from urine-derived cells. Moreover, we were able to generate feeder-free bulk hiPSCs lines that did not display genomic abnormalities. Altogether, this reprogramming method will contribute to accelerating the translation of hiPSCs to therapeutic applications.

Autoimmune regulator (AIRE) deficiency in humans induces a life-threatening generalized autoimmune disease called autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), and no curative treatments are available. Several models of AIRE-deficient mice have been generated, and although they have been useful in understanding the role of AIRE in central tolerance, they do not reproduce accurately the APECED symptoms, and thus there is still a need for an animal model displaying APECED-like disease. We assessed, in this study, the potential of the rat as an accurate model for APECED. In this study, we demonstrate that in rat, AIRE is expressed by MHC class II (MCH-II)(+) and MHC-II- medullary thymic epithelial cells in thymus and by CD4(int) conventional dendritic cells in periphery. To our knowledge, we generated the first AIRE-deficient rat model using zinc-finger nucleases and demonstrated that they display several of the key symptoms of APECED disease, including alopecia, skin depigmentation, and nail dystrophy, independently of the genetic background. We observed severe autoimmune lesions in a large spectrum of organs, in particular in the pancreas, and identified several autoantibodies in organs and cytokines such as type I IFNs and IL-17 at levels similar to APECED. Finally, we demonstrated a biased Ab response to IgG1, IgM, and IgA isotypes. Altogether, our data demonstrate that AIRE-deficient rat is a relevant APECED animal model, opening new opportunity to test curative therapeutic treatments.