Gaëlle Beriou

IL-17-producing human peripheral regulatory T cells retain suppressive function

Although implicated in antagonistic functions, both regulatory T cells (Tregs) and Th17 effector cells play an important role in controlling autoimmune pathogenesis. Paradoxically, recent studies indicate that Tregs have the capacity to produce interleukin-17 (IL-17), although the ability of these cells to retain their suppressive function remains unknown. Here we report that human Tregs within the CD4(+)CD45RA(-)CD25(high)CCR6(+)HLA-DR(-)FoxP3(+) population produce IL-17 when activated in the presence of the proinflammatory cytokines IL-1beta and IL-6, whereas IL-17 secretion was inhibited by TGFbeta. To assess the ability of a single Treg to secrete IL-17 and to suppress in vitro immune function, we isolated clones from this population. We found that IL-17(+)/FoxP3(+) Treg clones retain suppressive function and exhibit the plasticity to secrete IL-17 or suppress depending on the nature of the stimulus provided. IL-17 production by these Treg clones was accompanied by sustained FoxP3 expression and concomitant, but reversible, loss of suppressive activity. Our data demonstrate that at the single cell level a subset of in vitro suppressive FoxP3(+) cells can be driven to secrete IL-17 under inflammatory conditions. These findings suggest a new mechanism by which inflammation can drive Tregs to secrete IL-17, thereby dampening suppression and promoting an inflammatory milieu.

TGF-β Induces IL-9 Production from Human Th17 Cells

The secretion of IL-9, initially recognized as a Th2 cytokine, was recently attributed to a novel CD4 T cell subset termed Th9 in the murine system. However, IL-9 can also be secreted by mouse Th17 cells and may mediate aspects of the proinflammatory activities of Th17 cells. Here we report that IL-9 is secreted by human naive CD4 T cells in response to differentiation by Th9 (TGF-β and IL-4) or Th17 polarizing conditions. Yet, these differentiated naive cells did not coexpress IL-17 and IL-9, unless they were repeatedly stimulated under Th17 differentiation-inducing conditions. In contrast to the naive cells, memory CD4 T cells were induced to secrete IL-9 by simply providing TGF-β during stimulation, as neither IL-4 nor proinflammatory cytokines were required. Furthermore, the addition of TGF-β to the Th17-inducing cytokines (IL-1β, IL-6, IL-21, IL-23) that induce memory cells to secrete IL-17, resulted in the marked coexpression of IL-9 in IL-17 producing memory cells. The proinflammatory cytokine mediating TGF-β–dependent coexpression of IL-9 and IL-17 was identified to be IL-1β. Moreover, circulating monocytes were potent costimulators of IL-9 production by Th17 cells via their capacity to secrete IL-1β. Finally, to determine whether IL-9/IL-17 coproducing CD4 cells were altered in an inflammatory condition, we examined patients with autoimmune diabetes and demonstrated that these subjects exhibit a higher frequency of memory CD4 cells with the capacity to transition into IL-9+IL-17+ cells. These data demonstrate the presence of IL-17+IL-9+ CD4 cells induced by IL-1β that may play a role in human autoimmune disease.

CD2 Costimulation Reveals Defective Activity by Human CD4+CD25hi Regulatory Cells in Patients with Multiple Sclerosis

Studying the activity of homogeneous regulatory T cell (Treg) populations will advance our understanding of their mechanisms of action and their role in human disease. Although isolating human Tregs exhibiting low expression of CD127 markedly increases purity, the resulting Treg populations are still heterogeneous. To examine the complexity of the Tregs defined by the CD127 phenotype in comparison with the previously described CD4+CD25hi subpopulations, we subdivided the CD25hi population of memory Tregs into subsets based on expression of CD127 and HLA-DR. These subsets exhibited differences in suppressive capacity, ability to secrete IL-10 and IL-17, Foxp3 gene methylation, cellular senescence, and frequency in neonatal and adult blood. The mature, short telomere, effector CD127loHLA-DR+ cells most strongly suppressed effector T cells within 48 h, whereas the less mature CD127loHLA-DR− cells required 96 h to reach full suppressive capacity. In contrast, whereas the CD127+HLA-DR− cells also suppressed proliferation of effector cells, they could alternate between suppression or secretion of IL-17 depending upon the stimulation signals. When isolated from patients with multiple sclerosis, both the nonmature and the effector subsets of memory CD127lo Tregs exhibited kinetically distinct defects in suppression that were evident with CD2 costimulation. These data demonstrate that natural and not induced Tregs are less suppressive in patients with multiple sclerosis.

Tolerogenic dendritic cells: applications for solid organ transplantation.

Purpose of reviewWe discuss the use of tolerogenic dendritic cells (TolDCs) as a therapeutic tool in solid organ transplantation, with particular emphasis on recent experimental and preclinical data supporting the clinical translation of TolDC therapy. Recent findingsTolDC have been successfully used in rodents to promote long-term allograft survival. Although most studies have focused on donor dendritic cells or donor antigen-pulsed dendritic cells, our group investigated a strategy based on the administration of autologous dendritic cells (not pulsed with donor antigens). We discuss the therapeutic efficacy, mechanisms, and potential risks and advantages of each strategy. We also highlight recent findings on the generation of clinical grade human TolDC from blood monocytes. Finally, we discuss preliminary experience with dendritic cells in humans and critical issues regarding the implementation of TolDC therapy to clinical organ transplantation. SummaryTolDC hold therapeutic promise for the treatment of transplanted patients. Cell therapy with unpulsed, autologous dendritic cells appears as a well tolerated, clinically relevant approach that might help in improving long-term allograft survival and limit the harmful effects of immunosuppressive treatments.

Superiority of bone marrow-derived dendritic cells over monocyte-derived ones for the expansion of regulatory T cells in the macaque.

Regulatory T cells (Treg) have been identified as playing a pivotal role in the control of tolerance and in the suppression of pathologic immune responses in autoimmune diseases, transplantation, and graft-versus-host disease. Treg expanded ex vivo by dendritic cells could be potential reagents to promote antigen-specific tolerance in vivo. However, in vivo studies have been carried out mostly in rodents and will need validation in primates before clinical application. We characterized macaque dendritic cell derived either from bone marrow with and without prior CD34+ cell selection (BMDC), or from CD14+ peripheral blood mononuclear cells (Mo-DC). We demonstrate that with a semi-mature phenotype, BMDC are superior to Mo-DC in their capacity to expand freshly isolated allogeneic macaque CD4+ CD25+ CD127- Foxp3+ Treg in vitro in the presence of interleukin-2. Moreover, the expanded Treg maintain their phenotype and suppressive activity. These data provide a step toward the use of macaque dendritic cell to expand Treg for future preclinical testing.

RNA Interference Screen in Primary Human T Cells Reveals FLT3 as a Modulator of IL-10 Levels

Functional studies of human primary immune cells have been hampered by the lack of tools to silence gene functions. In this study, we report the application of a lentiviral RNA interference library in primary human T cells. Using a subgenomic short hair RNA library targeting ∼1000 signaling genes, we identified novel genes that control the levels of IL-10 produced. IL-10 is a potent anti-inflammatory cytokine secreted by several cell types, including T regulatory type 1 cells, a subset of T regulatory cells that exert their suppressive activity through IL-10 secretion. FLT3, a known hematopoeitic growth factor, was found to be a negative regulator of IL-10 levels in activated T cells. This was based on several observations. First, FLT3 and its ligand (FL) were both induced by T cell activation. Second, silencing of FLT3 led to increased IL-10 levels, whereas addition of FL suppressed IL-10 secretion and increased FLT3 surface levels. Third, engagement of CD46, a known inducer of T regulatory type 1 cells, upregulated surface FLT3, and secreted FL, which then inhibited IL-10 production by T cells. Hence, FL and FLT3 form a novel regulatory feedback loop that limits IL-10 production in T cells. Our results identified FLT3 as a new regulator of T cell function and offer a strategy to genetically dissect specific pathways in T cells.

Evaluation of the Therapeutic Potential of Bone Marrow-Derived Myeloid Suppressor Cell (MDSC) Adoptive Transfer in Mouse Models of Autoimmunity and Allograft Rejection

Therapeutic use of immunoregulatory cells represents a promising approach for the treatment of uncontrolled immunity. During the last decade, myeloid-derived suppressor cells (MDSC) have emerged as novel key regulatory players in the context of tumor growth, inflammation, transplantation or autoimmunity. Recently, MDSC have been successfully generated in vitro from naive mouse bone marrow cells or healthy human PBMCs using minimal cytokine combinations. In this study, we aimed to evaluate the potential of adoptive transfer of such cells to control auto- and allo-immunity in the mouse. Culture of bone marrow cells with GM-CSF and IL-6 consistently yielded a majority of CD11b+Gr1hi/lo cells exhibiting strong inhibition of CD8+ T cell proliferation in vitro. However, adoptive transfer of these cells failed to alter antigen-specific CD8+ T cell proliferation and cytotoxicity in vivo. Furthermore, MDSC could not prevent the development of autoimmunity in a stringent model of type 1 diabetes. Rather, loading the cells prior to injection with a pancreatic neo-antigen peptide accelerated the development of the disease. Contrastingly, in a model of skin transplantation, repeated injection of MDSC or single injection of LPS-activated MDSC resulted in a significant prolongation of allograft survival. The beneficial effect of MDSC infusions on skin graft survival was paradoxically not explained by a decrease of donor-specific T cell response but associated with a systemic over-activation of T cells and antigen presenting cells, prominently in the spleen. Taken together, our results indicate that in vitro generated MDSC bear therapeutic potential but will require additional in vitro factors or adjunct immunosuppressive treatments to achieve safe and more robust immunomodulation upon adoptive transfer.

Characterization of Schlafen-3 expression in effector and regulatory T cells

Members of the Slfn protein family have been implicated in the regulation of cell growth, hematopoietic cell differentiation, and T cell development/differentiation in the thymus. Ten members of this family have been described in the mouse, and they have been divided into three subgroups based on the overall sequence homology and the size of the encoded proteins. We have identified Slfn3, a member of Subgroup II, as an overexpressed gene in CD4+ CD25+ T cells in the periphery. Interestingly, we demonstrate that upon activation and proliferation, Slfn3 mRNA is down‐regulated in CD4+ CD25+ Tregs and up‐regulated in CD4+ CD25– Teffs. Moreover, TGF‐β inhibits the expression of Slfn3 in anti‐CD3/CD28‐activated CD4+ T cells, and the same conditions induce FoxP3 mRNA. Our results suggest that Slfn3 could have a role in T cell differentiation and activation.

LF 15-0195 Treatment Protects against Central Nervous System Autoimmunity by Favoring the Development of Foxp3-Expressing Regulatory CD4 T Cells

Experimental autoimmune encephalomyelitis (EAE) is an instructive model for the human demyelinating disease multiple sclerosis. Lewis (LEW) rats immunized with myelin-basic protein (MBP) develop EAE characterized by a single episode of paralysis, from which they recover spontaneously and become refractory to a second induction of disease. LF 15-0195 is a novel molecule that has potent immunosuppressive effects in several immune-mediated pathological manifestations, including EAE. In the present study, we show that a 30-day course of LF 15-0195 treatment not only prevents MBP-immunized LEW rats from developing EAE but also preserves their refractory phase to reinduction of disease. This effect is Ag driven since it requires priming by the autoantigen during the drug administration. In contrast to other immunosuppressive drugs, short-term treatment with this drug induces a persistent tolerance with no rebound of EAE up to 4 mo after treatment withdrawal. This beneficial effect of LF 15-0195 on EAE does not result from the deletion of MBP-specific Vβ8.2 encephalitogenic T cells. In contrast, this drug favors the differentiation of MBP-specific CD4 T cells into Foxp3-expressing regulatory T cells that, upon adoptive transfer in syngeneic recipients, prevent the development of actively induced EAE. Finally, we demonstrate that the tolerance induced by LF 15-0195 treatment is not dependent on the presence of TGF-β. Together, these data demonstrate that short-term treatment with LF 15-0195 prevents MBP-immunized LEW rats from EAE by favoring the development of Foxp-3-expressing regulatory CD4 T cells.

RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus

Retinoid-related orphan receptor gamma t (RORγt) is a master transcription factor central to type 17 immunity involving cells such as T helper 17, group 3 innate lymphoid cells or IL-17-producing γδ T cells. Here we show that the intracellular ion channel TMEM176B and its homologue TMEM176A are strongly expressed in these RORγt+ cells. We demonstrate that TMEM176A and B exhibit a similar cation channel activity and mainly colocalise in close proximity to the trans-Golgi network. Strikingly, in the mouse, the loss of Tmem176b is systematically associated with a strong upregulation of Tmem176a. While Tmem176b single-deficiency has no effect on the course of experimental autoimmune encephalomyelitis, T cell or DSS-induced colitis, it significantly reduces imiquimod-induced psoriasis-like skin inflammation. These findings shed light on a potentially novel specific process linked to post-Golgi trafficking for modulating the function of RORγt+ cells and indicate that both homologues should be simultaneously targeted to clearly elucidate the role of this intracellular ion flow.