B-Cells induce regulatory T cells through TGF-β/IDO production in A CTLA-4 dependent manner☆
Introduction
The bio-pathological processes that underpin susceptibility to auto-immune diseases have been extensively explored by many investigators but remain largely undefined. The vast majority of these studies focused on mechanisms that trigger effector T lymphocytes since these cells were considered as the initiators of harmful immunological responses, including the production of pathogenic antibodies by B lymphocytes. However, targeting T cells as therapeutic approaches for treating such diseases have proved to be largely ineffective. In recent years, B lymphocyte functions have been reconsidered, and it is now accepted that these cells play much more diverse functions than simply just producing antibodies [1]. The key role played by B-cells in autoimmune diseases is further supported by therapeutic efficacy of B cell-ablation strategies for treatment [2]. Interestingly, a number of studies identified potential adverse effects in total B cell ablation suggesting that some B cell subsets with regulatory functions may provide ameliorating effects in many pathological settings [3].
Regulatory B lymphocytes were discovered in the past decade, principally, in studies involving murine models of autoimmune diseases. The cells were termed Breg cells, based on their ability to dampen or even suppress effector immune processes [4]. The most widely-studied immunosuppressive mechanism through which Breg cells exert their immune regulatory functions is generally-believed to be interleukin (IL)-10 based [5]. IL-10 production is one of the most important anti-inflammatory cytokines capable of down-regulating inflammation [6]. Although stimulated B cells produce IL-10, the importance of regulatory B cell-derived IL-10 in immunosuppression was only recently revealed. In 2002, Mizoguchi et al. identified a subset of IL-10-producing CD1d+ B cells in gut-associated lymphoid tissues during the course of chronic intestinal inflammation [7]. When these Breg cells were enriched or transferred into recipient mice with intestinal inflammation the cells mediated an IL-10-dependent suppression of the inflammation. In a murine model of autoimmune encephalomyelitis (EAE), a key role for IL-10-producing splenic B cells was highlighted as important for controlling the disease [8]. In humans, Blair and colleagues subsequently demonstrated that CD24high CD38high Breg cells inhibited interferon (IFN)γ and tumor necrosis factor (TNF)α production by autologous co-cultured T-cells through the production of IL-10 and that Breg function is deficient in patients with systemic lupus erythematosus (SLE) [9]. The existence of Breg cells in humans was subsequently confirmed in various in vitro models [4], [10], [11]. However, beyond the IL-10-mediated suppressive effects, strong evidence exist that part of the immunosuppressive effects of Breg cells is dependent on interactions with other regulatory cell lineages. Thus, repeated nasal antigenic challenge induced a Breg population that suppressed lung inflammation and asthma in recipient OVA-sensitized mice following adoptive transfer. The effectiveness of these Breg cells was linked to the generation of CD4+ CD25+ Foxp3+ regulatory T cell (Treg cells) that produced transforming growth factor-β (TGF-β) [12]. Other studies revealed that human Breg cells can inhibit T cell proliferation through cell-to-cell contact, leading to anergy, or T cell apoptosis [13]. Our own studies revealed that activated human CD19+ IgD+ CD38high CD5+ B cells strongly suppressed autologous T cell proliferation independently of IL-10 production [14]. Although effectiveness of induced Breg cells (iBregs) has been clearly demonstrated, the precise mechanisms through which these cells control immune responses remain to be elucidated.
Indoleamine 2,3-dioxygenase (IDO) enzyme degrades the essential amino-acid tryptophan and promotes immune tolerance by inhibiting T cell activation [15]. Inhibition of IDO with a competitive inhibitor, 1-methyl-tryptophan (1 MT) resulted in rejection of pancreatic islet allograft and aggravated autoimmunity [16], [17]. The production of IDO is transitory induced in dendritic cells (DC) by IFN-γ whereas TGF-β mediates durable IDO-dependent regulatory functions [18]. Cytotoxic T lymphocyte associated antigen-4 (CTLA-4), is a central inhibitory regulator of T-cell proliferation and expansion [19]. Mice genetically deficient in CTLA-4 develop a massive lymphoproliferative disorder and an early lethality [20], [21]. CTLA-4 pathway through ligation to CD80 and CD86 on antigen presenting cells (APC) has been shown to upregulate Foxp3 expression induced by TGFβ leading to induction of adaptative CD4+ CD25+ regulatory T cells [22]. Moreover CTLA-4 engagement of B7 ligands on DCs can induce release of IDO involved in maintenance of peripheral tolerance [23].
These findings strengthen the need for better characterize the mechanisms involved in Breg cell functions. Such insights would be an essential step to therapeutically exploit the function of Breg cells in diseases. In this context, we have presently investigated, in in vitro models, the mechanisms by which human iBreg cells regulate T cell functions. We reveal a novel regulatory pathway in B cells, that is mediated by the TGF-β/IDO axis in a CTLA-4 dependent manner. This novel regulatory pathway in Breg cells provides news perspectives for future treatment strategies in autoimmune diseases.
Section snippets
Cell isolation
Peripheral blood from the healthy controls (HC) was collected, layered onto Ficoll-Hypaque and fractionated by centrifugation. Mononuclear cells were incubated with neuraminidase-treated sheep red blood cells and T cells depleted by a second 30-min round of centrifugation. B cells were further enriched by negative selection with a B cell enrichment kit (CD19+CD43+ Stem Cell Technologies) according to the manufacturer's instructions. All preparations were >97% B cells (CD19+CD11c−HLADR+)
Peripheral blood B cells exert regulatory properties
To demonstrate the regulatory properties of peripheral B cells, a co-culture system was set up as previously described [24]. Briefly, CpG-stimulated B cells were incubated with autologous T cells upon CD3/CD28 stimulation. As shown in Fig. 1A (left panel), when B cells were added to the co-culture system, the proliferative T cell response at day 4 decreased. B cells were able to regulate T cell proliferation with time (Fig. 1A, right panel). The proliferative T cell response was reduced by
Discussion
This study explored mechanisms by which B cells induce immune suppression. A number of reports have provided evidence that activated B cells (iBregs) induce and/or expand regulatory Foxp3+ T cells. In mice, splenic B cells were shown in vitro to induce the proliferation of Foxp3+ T cells in co-culture through TGF-β [29]. In a model of tolerance to self-antigens, naïve B cells induce peripheral expansion of regulatory T cells by presenting Ag [30]. In humans, CD40L-activated B cells drive the
Acknowledgments
The authors thank Professor Rizgar Mageed for editorial help and for critically reading the manuscript. They also thank Simone Forest and Geneviève Michel for secretarial assistance.
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This study was supported by a grant from “Investissements d’Avenir” program ANR11-LABX-0013-01.
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P.P. and S.Q contributed equally to this work.