IL-6-dependent spontaneous proliferation is required for the induction of colitogenic IL-17-producing CD8+ T cells.
ABSTRACT: We propose a novel role for interleukin (IL) 6 in inducing rapid spontaneous proliferation (SP) of naive CD8(+) T cells, which is a crucial step in the differentiation of colitogenic CD8(+) T cells. Homeostasis of T cells is regulated by two distinct modes of cell proliferation: major histocompatibility complex/antigen-driven rapid SP and IL-7/IL-15-dependent slow homeostatic proliferation. Using our novel model of CD8(+) T cell-dependent colitis, we found that SP of naive CD8(+) T cells is essential for inducing pathogenic cytokine-producing effector T cells. The rapid SP was predominantly induced in mesenteric lymph nodes (LNs) but not in peripheral LNs under the influence of intestinal flora and IL-6. Indeed, this SP was markedly inhibited by treatment with anti-IL-6 receptor monoclonal antibody (IL-6R mAb) or antibiotic-induced flora depletion, but not by anti-IL-7R mAb and/or in IL-15-deficient conditions. Concomitantly with the inhibition of SP, anti-IL-6R mAb significantly inhibited the induction of CD8(+) T cell-dependent autoimmune colitis. Notably, the transfer of naive CD8(+) T cells derived from IL-17(-/-) mice did not induce autoimmune colitis. Thus, we conclude that IL-6 signaling is crucial for SP under lymphopenic conditions, which subsequently caused severe IL-17-producing CD8(+) T cell-mediated autoimmune colitis. We suggest that anti-IL-6R mAb may become a promising strategy for the therapy of colitis.
Project description:Oral administration of Ag induces regulatory T cells that express latent membrane-bound TGF-? (latency-associated peptide [LAP]) and have been shown to play an important role in the induction of oral tolerance. We developed an in vitro model to study modulation of LAP+ on CD4+ T cells. The combination of anti-CD3 mAb, anti-CD28 mAb, and recombinant IL-2 induced expression of LAP on naive CD4+ T cells, independent of Foxp3 or exogenous TGF-?. In vitro generated CD4+LAP+Foxp3- T cells were suppressive in vitro, inhibiting proliferation of naive CD4+ T cells and IL-17A secretion by Th17 cells. Assessing the impact of different cytokines and neutralizing Abs against cytokines, we found that LAP induction was decreased in the presence of IL-6 and IL-21, and to a lesser extent by IL-4 and TNF-?. IL-6 abrogated the in vitro induction of CD4+LAP+ T cells by STAT3-dependent inhibition of Lrrc32 (glycoprotein A repetitions predominant [GARP]), the adapter protein that tethers TGF-? to the membrane. Oral tolerance induction was enhanced in mice lacking expression of IL-6R by CD4+ T cells and by treatment of wild-type mice with neutralizing anti-IL-6 mAb. These results suggest that proinflammatory cytokines interfere with oral tolerance induction and that blocking the IL-6 pathway is a potential strategy for enhancing oral tolerance in the setting of autoimmune and inflammatory diseases.
Project description:The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.
Project description:Anti-CD3 mAb can modulate graft rejection and attenuate autoimmune diseases but their mechanism(s) of action remain unclear. CD8(+) T cells with regulatory function are induced in vitro by Teplizumab, a humanized anti-CD3 antibody and inhibit responses of autologous and allogeneic T cells. They inhibit CD4(+) T-cell proliferation by mechanisms involving TNF and CCL4, and by blocking target cell entry into G2/M phase of cell cycle but neither kill them, nor compete for IL-2. CD8(+) Treg can be isolated from peripheral blood following treatment of patients with Type 1 diabetes with Teplizumab, but not from untreated patients. The induction of CD8(+) Treg by anti-CD3 mAb requires TNF and signaling through the NF-?B cascade. The CD8(+) Treg express CD25, glucocorticoid-induced TNF receptor family, CTLA-4, Foxp3, and TNFR2, and the combined expression of TNFR2 and CD25 identifies a potent subpopulation of CD8(+) Treg. These studies have identified a novel mechanism of immune regulation by anti-CD3 mAb and markers that may be used to track inducible CD8(+) Treg in settings such as chronic inflammation or immune therapy.
Project description:Signaling through IL-2/IL-15R? (CD122) is essential for the differentiation and function of T cells and NK cells. A mAb against CD122 has been implicated to suppress autoimmune type 1 diabetes (T1D) development in animal models. However, the mechanisms remain poorly understood. We find that in vivo administration of an anti-CD122 mAb (CD122 blockade) restores immune tolerance in nonobese diabetic (NOD) mice via multiple mechanisms. First, CD122 blockade selectively ablates pathogenic NK cells and memory phenotype CD8+ T cells from pancreatic islets. In contrast, islet CD4+Foxp3+ Tregs are only mildly affected. Second, CD122 blockade suppresses IFN-? production in islet immune cells. Third, CD122 blockade inhibits the conversion of islet Th17 cells into diabetogenic Th1 cells. Furthermore, a combination of anti-CD122 mAb and Treg-trophic cytokines (IL-2 or IL-33) enhances the abundance and function of islet Tregs. In summary, these data provide crucial mechanistic insights into CD122 blockade-mediated immunoregulation and support therapeutic benefits of this combinational treatment in T1D.
Project description:The graft-versus-tumor (GVT) effect is a potent form of immunotherapy against many hematologic malignancies and some solid tumors. The beneficial GVT effect after allogeneic bone marrow transplantation (BMT) is tightly linked to its most significant complication, graft-versus-host disease (GVHD). The role of interleukin-6 (IL-6) after allogeneic BMT is not well understood. This study used a series of complementary knockout and antibody blockade strategies to analyze the impact of IL-6 in multiple clinically relevant murine models of GVHD and GVT.We examined the effect of the source of IL-6 by analyzing the role IL-6 deficiency in donor T cells, donor bone marrow or in host tissues. We confirmed and extended the relevance of IL-6 deficiency on GVHD and GVT by treating BMT recipients with anti-mouse IL-6 receptor (IL-6R), MR16-1.Deficiency of IL-6 in donor T cells led to prolongation of survival. Total inhibition of IL-6 with MR16-1 caused an even greater reduction in GVHD-induced mortality. The reduction in GVHD was independent of the direct effects on T effector cell expansion or donor regulatory T cells. GVT responses were preserved after treatment with MR16-1.MR16-1 treatment reduced GVHD and preserved sufficient GVT. Tocilizumab, a humanized anti-IL-6R monoclonal antibody (mAb), is approved in several countries including the United States and European Union for the treatment of rheumatoid arthritis and other inflammatory diseases. Blockade of IL-6 with anti-IL-6R mAb therapy may be testable in clinical trials as an adjunct to prevent GVHD in BMT patients without a significant loss of GVT.
Project description:IL-6 is known to contribute to the differentiation of CD4+ T cells into different subsets of effector T helper cells. Less is known about the potential of IL-6 in regulating CD8+ T cell effector function. Here, we identify IL-6 as a master regulator of IL-21 in effector CD8+ T cells. IL-6 promotes the differentiation of a subset of naive CD8+ T cells that express IL-6R into a unique population of effector CD8+ T cells characterized by the production of high levels of IL-21 and low levels of IFN-γ. Similar to CD4+ T follicular helper (Tfh) cells, IL-21-producing CD8+ T cells generated in the presence of IL-6 directly provide help to B cells to induce isotype switching. CD8+ T cell-derived IL-21 contributes to the production of protective virus-specific IgG antibodies during influenza virus infection. Thus, this study reveals the presence of a new mechanism by which IL-6 regulates antibody production during viral infection, and a novel function of effector CD8+ T cells in the protection against viruses.
Project description:Polymorphisms in NOD2 confer risk for Crohn's disease, characterized by intestinal inflammation. How NOD2 regulates both inflammatory and regulatory intestinal T cells, which are critical to intestinal immune homeostasis, is not well understood. Anti-CD3 mAb administration is used as therapy in human autoimmune diseases, as well as a model of transient intestinal injury. The stages of T cell activation, intestinal injury, and subsequent T tolerance are dependent on migration of T cells into the small intestinal (SI) lamina propria. Upon anti-CD3 mAb treatment of mice, we found that NOD2 was required for optimal small intestinal IL-10 production, in particular from CD8(+) T cells. This requirement was associated with a critical role for NOD2 in SI CD8(+) T cell accumulation and induction of the CXCR3 ligands CXCL9 and CXCL10, which regulate T cell migration. NOD2 was required in both the hematopoietic and nonhematopoietic compartments for optimal expression of CXCR3 ligands in intestinal tissues. NOD2 synergized with IFN-? to induce CXCL9 and CXCL10 secretion in dendritic cells, macrophages, and intestinal stromal cells in vitro. Consistent with the in vitro studies, during anti-CD3 mAb treatment in vivo, CXCR3 blockade, CD8(+) T cell depletion, or IFN-? neutralization each inhibited SI CD8(+) T cell recruitment, and reduced chemokine expression and IL-10 expression. Thus, NOD2 synergizes with IFN-? to promote CXCL9 and CXCL10 expression, thereby amplifying CXCR3-dependent SI CD8(+) T cell migration during T cell activation, which, in turn, contributes to induction of both inflammatory and regulatory T cell outcomes in the intestinal environment.
Project description:Cytokine release syndrome (CRS) is known to be a factor in morbidity and mortality associated with acute viral infections including those caused by filoviruses and coronaviruses. IL-6 has been implicated as a cytokine negatively associated with survival after filovirus and coronavirus infection. However, IL-6 has also been shown to be an important mediator of innate immunity and important for the host response to an acute viral infection. Clinical studies are now being conducted by various researchers to evaluate the possible role of IL-6 blockers to improve outcomes in critically ill patients with CRS. Most of these studies involve the use of anti-IL-6R monoclonal antibodies (?-IL-6R mAbs). We present data showing that direct neutralization of IL-6 with an ?-IL-6 mAb in a BALB/c Ebolavirus (EBOV) challenge model produced a statistically significant improvement in outcome compared with controls when administered within the first 24 h of challenge and repeated every 72 h. A similar effect was seen in mice treated with the same dose of ?-IL-6R mAb when the treatment was delayed 48 h post-challenge. These data suggest that direct neutralization of IL-6, early during the course of infection, may provide additional clinical benefits to IL-6 receptor blockade alone during treatment of patients with virus-induced CRS.
Project description:Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+?/?+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]-like CLL cells) produce high amounts of IL-10 and transforming growth factor ? (TGF-?) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-?-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-? and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL-patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.
Project description:In addition to CD4(+) regulatory T cells (Tregs), CD8(+) suppressor T cells are emerging as an important subset of regulatory T cells. Diverse populations of CD8(+) T cells with suppressive activities have been described. Among them, a small population of CD8(+)CD25(+)FOXP3(+) T cells is found both in mice and humans. In contrast to thymic-derived CD4(+)CD25(+)FOXP3(+) Tregs, their origin and their role in the pathophysiology of autoimmune diseases (AIDs) are less understood. We report here the number, phenotype, and function of CD8(+) Tregs cells in mice and humans, at the steady state and in response to low-dose interleukin-2 (IL-2). CD8(+) Tregs represent approximately 0.4 and 0.1% of peripheral blood T cells in healthy humans and mice, respectively. In mice, their frequencies are quite similar in lymph nodes (LNs) and the spleen, but two to threefold higher in Peyer patches and mesenteric LNs. CD8(+) Tregs express low levels of CD127. CD8(+) Tregs express more activation or proliferation markers such as CTLA-4, ICOS, and Ki-67 than other CD8(+) T cells. In vitro, they suppress effector T cell proliferation as well as or even better than CD4(+) Tregs. Owing to constitutive expression of CD25, CD8(+) Tregs are 20- to 40-fold more sensitive to in vitro IL-2 stimulation than CD8(+) effector T cells, but 2-4 times less than CD4(+) Tregs. Nevertheless, low-dose IL-2 dramatically expands and activates CD8(+) Tregs even more than CD4(+) Tregs, in mice and humans. Further studies are warranted to fully appreciate the clinical relevance of CD8(+) Tregs in AIDs and the efficacy of IL-2 treatment.