Matthieu Rouleau

A CD4 + T-cell subset inhibits antigen-specific T-cell responses and prevents colitis

Induction and maintenance of peripheral tolerance are important mechanisms to maintain the balance of the immune system. In addition to the deletion of T cells and their failure to respond in certain circumstances, active suppression mediated by T cells or T-cell factors has been proposed as a mechanism for maintaining peripheral tolerance. However, the inability to isolate and clone regulatory T cells involved in antigen-specific inhibition of immune responses has made it difficult to understand the mechanisms underlying such active suppression. Here we show that chronic activation of both human and murine CD4+T cells in the presence of interleukin (IL)-10 gives rise to CD4+T-cell clones with low proliferative capacity, producing high levels of IL-10, low levels of IL-2 and no IL-4. These antigen-specific T-cell clones suppress the proliferation of CD4+T cells in response to antigen, and prevent colitis induced in SCID mice by pathogenic CD4+CD45RBhighsplenic T cells. Thus IL-10 drives the generation of a CD4+T-cell subset, designated T regulatory cells 1 (Tr1), which suppresses antigen-specific immune responses and actively downregulates a pathological immune response in vivo .

Bone marrow Th17 TNFα cells induce osteoclast differentiation, and link bone destruction to IBD

Objective Under both physiological and pathological conditions, bone volume is determined by the rate of bone formation by osteoblasts and bone resorption by osteoclasts. Excessive bone loss is a common complication of human IBD whose mechanisms are not yet completely understood. Despite the role of activated CD4+ T cells in inflammatory bone loss, the nature of the T cell subsets involved in this process in vivo remains unknown. The aim of the present study was to identify the CD4+ T cell subsets involved in the process of osteoclastogenesis in vivo, as well as their mechanism of action. Design CD4+ T cells were studied in IL10−/− mice and Rag1−/− mice adoptively transferred with naive CD4+CD45RBhigh T cells, representing two well-characterised animal models of IBD and in patients with Crohns disease. They were phenotypically and functionally characterised by flow cytometric and gene expression analysis, as well as in in vitro cocultures with osteoclast precursors. Results In mice, we identified bone marrow (BM) CD4+ T cells producing interleukin (IL)-17 and tumour necrosis factor (TNF)-α as an osteoclastogenic T cell subset referred to as Th17 TNF-α+ cells. During chronic inflammation, these cells migrate to the BM where they survive in an IL-7-dependent manner and where they promote the recruitment of inflammatory monocytes, the main osteoclast progenitors. A population equivalent to the Th17 TNF-α+ cells was also detected in patients with Crohns disease. Conclusions Our results highlight the osteoclastogenic function of the Th17 TNF-α+ cells that contribute to bone loss in vivo in IBD.

Long-term persistence of transferred PPD-reactive T cells after allogeneic bone marrow transplantation.

T cells play an important role in protective immunity against tuberculosis. As patients are not reimmunized with BCG after BMT, the question arises as to whether PPD-specific memory T cells are transferred from the marrow donor to the recipient and persist in the long-term. We studied long-term survivors of non–T cell-depleted allogeneic bone marrow transplantation for in vitro PPD-induced proliferative responses (n=14), and delayed-type hypersensitivity after intradermal injection of tuberculin (n=20). We also studied 7 patients who received T cell-depleted bone marrow. Proliferative responses in the first group were low, but were increased by concentrating CD4+ T cells, the major responding cells in this system. In contrast, PBL from patients who received T cell–depleted marrow remained unresponsive to PPD, although they responded normally to CMV antigens. Of the 15 healthy patients in the first group who underwent tuberculin skin tests, 13 had positive reactions, while only two failed to react. (Five patients of the first group were suffering from chronic GVHD and 3 of them were negative.) All the patients in the second group had negative delayed-hypersensitivity responses. The difference between the two groups of patients was highly significant (P<0.003). These results show that transferred PPD-specific T cells persist in long-term survivors of non–T cell–depleted BMT, even in the absence of reimmunization.

Inflammatory osteoclasts prime TNFα‐producing CD4+ T cells and express CX3CR1

Bone destruction is a hallmark of chronic rheumatic diseases. Although the role of osteoclasts in bone loss is clearly established, their implication in the inflammatory response has not been investigated despite their monocytic origin. Moreover, specific markers are lacking to characterize osteoclasts generated in inflammatory conditions. Here, we have explored the phenotype of inflammatory osteoclasts and their effect on CD4+ T cell responses in the context of bone destruction associated with inflammatory bowel disease. We used the well‐characterized model of colitis induced by transfer of naive CD4+ T cells into Rag1–/– mice, which is associated with severe bone destruction. We set up a novel procedure to sort pure osteoclasts generated in vitro to analyze their phenotype and specific immune responses by FACS and qPCR. We demonstrated that osteoclasts generated from colitic mice induced the emergence of TNFα‐producing CD4+ T cells, whereas those generated from healthy mice induced CD4+FoxP3+ regulatory T cells, in an antigen‐dependent manner. This difference is related to the osteoclast origin from monocytes or dendritic cells, to their cytokine expression pattern, and their environment. We identified CX3CR1 as a marker of inflammatory osteoclasts and we demonstrated that the differentiation of CX3CR1+ osteoclasts is controlled by IL‐17 in vitro. This work is the first demonstration that, in addition to participating to bone destruction, osteoclasts also induce immunogenic CD4+ T cell responses upon inflammation. They highlight CX3CR1 as a novel dual target for antiresorptive and anti‐inflammatory treatment in inflammatory chronic diseases.

Pattern of cytokine expression in circulation CD57+ T cells from long-term renal allograft recipients.

We made a quantitative analysis of the lymphokine mRNA and of proteins produced by CD57+ and CD57- circulating T cells isolated from long-term kidney-transplanted patients with expanded CD4+/CD57+ and CD8+/CD57+ T cells, and from normal individuals. We concentrated on IL-2 and IFN-gamma, which define a Th1-like type of lymphokine production, and on IL-4 which defines a Th-2-like type. We also analysed the production of IL-10 which is endowed with inhibitory effects on IL-2 and IFN-gamma synthesis, and of TNF-alpha, a pleiotropic inflammatory cytokine. On ionomycin + PMA stimulation, which reveals the intrinsic potential of lymphokine production by T cells, the CD57+ T cell subsets from all individuals produced high amounts of IFN-gamma and TNF-alpha mRNA and protein. They also produced IL-2, but to a much lesser extend than their CD57- counterparts, and little IL-4 and IL-10. They were no more capable of producing IL-2 when stimulated through the CD3/TCR in the presence of monocytes, yet still synthesized IFN-gamma. Our data suggest that the in vivo expansion of CD57+ T cells in stable allograft renal recipients might correspond to Th1 energized cells which on triggering of cell surface receptors hardly secrete lymphokines involved in cell cycle progression, but can still exert some effector functions, including IFN-gamma secretion.

Roles of osteoclasts in the control of medullary hematopoietic niches

Bone marrow is the major site of hematopoiesis in mammals. The bone marrow environment plays an essential role in the regulation of hematopoietic stem and progenitor cells by providing specialized niches in which these cells are maintained. Many cell types participate to the composition and regulation of hematopoietic stem cell (HSC) niches, integrating complex signals from the bone, immune and nervous systems. Among these cells, the bone-resorbing osteoclasts (OCLs) have been described as main regulators of HSC niches. They are not limited to carving space for HSCs, but they also provide signals that affect the molecular and cellular niche components. However, their exact role in HSC niches remains unclear because of the variety of models, signals and conditions used to address the question. The present review will discuss the importance of the implication of OCLs focusing on the formation of HSC niches, the maintenance of HSCs in these niches and the mobilization of HSCs from the bone marrow. It will underline the importance of OCLs in HSC niches.

Induction of cytolytic function in resting peripheral blood CD8+/Leu-7+ T cells through IL2/p 75 IL2-receptor interaction: a study in the allogeneic human bone marrow transplantation model.

CD8+/Leu-7+ T cells which circulate in increased proportions in the blood of long-term surviving BMT patients are for the most part high-density resting lymphocytes lacking IL2R-alpha (p55) expression. We show that they can be induced by IL2 to manifest cytolytic function after 24-48 hr stimulation by using rather high concentrations of IL2 (at least 50 U/ml). This function was much more readily induced in high-density CD8+/Leu-7+ T cells than in high-density CD8+/Leu-7+ T cells and occurred in the presence of minimal cell proliferation. Other cytokines involved in primary CTL differentiation (IFN-gamma, IL4 and IL6) were without effect suggesting that CD8+/Leu-7+ T cells are, in the BMT model, in vivo preactivated CTL ready to differentiate into cytolytic effectors under the sole IL2 stimulus. TU27 Mab directed at IL2R-beta (p75) subunit almost completely prevented IL2-induced cytolytic function of CD8+ T cells while 33B3.1 Mab directed at IL2R-alpha (p55) subunit was ineffective, suggesting that the signal for this function has its origin in IL2R-beta chains constitutively expressed by these cells.

microRNA-184 Induces a Commitment Switch to Epidermal Differentiation

Summary miR-184 is a highly evolutionary conserved microRNA (miRNA) from fly to human. The importance of miR-184 was underscored by the discovery that point mutations in miR-184 gene led to corneal/lens blinding disease. However, miR-184-related function in vivo remained unclear. Here, we report that the miR-184 knockout mouse model displayed increased p63 expression in line with epidermal hyperplasia, while forced expression of miR-184 by stem/progenitor cells enhanced the Notch pathway and induced epidermal hypoplasia. In line, miR-184 reduced clonogenicity and accelerated differentiation of human epidermal cells. We showed that by directly repressing cytokeratin 15 (K15) and FIH1, miR-184 induces Notch activation and epidermal differentiation. The disease-causing miR-184C57U mutant failed to repress K15 and FIH1 and to induce Notch activation, suggesting a loss-of-function mechanism. Altogether, we propose that, by targeting K15 and FIH1, miR-184 regulates the transition from proliferation to early differentiation, while mis-expression or mutation in miR-184 results in impaired homeostasis.

Osteoimmune Interactions in Inflammatory Bowel Disease: Central Role of Bone Marrow Th17 TNFα Cells in Osteoclastogenesis

Osteoimmunology is an interdisciplinary research field dedicated to the study of the crosstalk between the immune and bone systems. CD4+ T cells are central players in this crosstalk. There is an emerging understanding that CD4+ T cells play an important role in the bone marrow (BM) under physiological and pathological conditions and modulate the differentiation of bone-resorbing osteoclasts. However, identification of the mechanisms that maintain CD4+ T cells in the BM is still a matter of investigation. This article describes the CD4+ T cell populations of the BM and reviews their role as osteoclastogenic population in inflammatory bowel disease.

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4+ FoxP3+ regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3+-Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.