T cell Immunoglobulin and Mucin domain 3 (TIM-3) is an Ig superfamily member expressed on IFNγ-secreting Th1 and Tc1 cells identified as a negative regulator of immune tolerance. TIM-3 is expressed by a subset of activated CD4+ T cells, and anti-CD3/anti-CD28 stimulation increases both the level of expression and the number of TIM-3+ T cells. In mice, TIM-3 is constitutively expressed on natural regulatory T cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4+ T cell differentiation. Here, we examined TIM-3 expression on human Tregs to determine its role in T cell suppression. In contrast to mice, TIM-3 is not expressed on Tregs ex vivo but is upregulated after activation. While TIM-3+ Tregs with increased gene expression of lag3, ctla4 and foxp3 are highly efficient suppressors of effector T cells (Teff), TIM-3— Tregs poorly suppressed Th17 as compared to Th1 cells that was associated with decreased STAT-3 expression and phosphorylation with reduced gene expression of il10, ebi3, gzmB, prf1, il1Rα, and ccr6. Thus, our results suggest that TIM-3 expression on Tregs identifies a population highly effective in inhibiting pathogenic Th1 and Th17 responses.

Human blood CD14+ monocytes are bone marrow–derived white blood cells that sense and respond to pathogens. Although innate immune activation by RNA viruses preferentially occurs through intracellular RIG-I–like receptors, other nucleic acid recognition receptors, such as Toll-like receptors (TLRs), play a role in finely programming the final outcome of virus infection. Here, we dissected how human monocytes respond to infection with either Coxsackie (CV), encephalomyocarditis (EMCV), influenza A (IAV), measles (MV), Sendai (SV), or vesicular stomatitis (VSV) virus. We found that in monocytes, type I interferon (IFN) and cytokine responses to infection were RNA virus specific and differentially involved TLR7 and TLR8, which sense single-stranded RNA. These TLRs activated distinct signaling cascades in monocytes, which correlated with differences in the production of cytokines involved in the polarization of CD4+ T helper cells. Furthermore, we found that TLR7 signaling specifically increased expression of the transcription factor FOSL1, which reduced IL-27 and TNFα production by monocytes. TLR7, but not TLR8, activation of monocytes also stimulated Ca2+ flux that prevented type I IFN responses. Our work demonstrates that in human monocytes, TLR7 and TLR8 triggered different signaling pathways that contribute to distinct phenotypes during RNA virus infection. In addition, we defined individual targets within these pathways that promoted specific T helper and antiviral responses.

Pattern recognition of microbes by Toll-like receptors (TLR) is critical for innate immune system activation. Although TLRs are expressed in human CD4+ T cells, their function is not well understood. Here we demonstrate that engaging TLR7 in CD4+ T cells induces intracellular calcium flux with activation of an NFATc2-dependent anergic gene expression program and T cell non-responsiveness. As chronic infections with RNA viruses such as HIV-1 induce profound CD4+ T cell dysfunction, we examined the role of TLR7-induced anergy in HIV-1 infection. TLR7 gene silencing markedly decreases the frequency of HIV-1-infected CD4+ T cells and restores cell responsiveness in those HIV-1+ CD4+ T cells. These results elucidate a previously unknown function for microbial pattern recognition receptors to down-regulate immune responses.

Foxp3+ regulatory T cells (Tregs) exhibit plasticity, which dictates their function. Secretion of the inflammatory cytokine IFNc, together with the acquisition of a T helper 1 (Th1)-like effector phenotype as observed in cancer, infection, and autoimmune diseases, is associated with loss of Treg suppressor function through an unknown mechanism. Here, we describe the signaling events driving the generation of human Th1-Tregs. Using a genome-wide gene expression approach and pathway analysis, we identify the PI3K/AKT/Foxo1/3 signaling cascade as the major pathway involved in IFNc secretion by human Tregs. Furthermore, we describe the opposing roles of AKT isoforms in Th1-Treg generation ex vivo. Finally, we employ multiple sclerosis as an in vivo model with increased but functionally defective Th1-Tregs. We show that the PI3K/AKT/Foxo1/3 pathway is activated in ex vivo-isolated Tregs from untreated relapsing-remitting MS patients and that blockade of the pathway inhibits IFNc secretion and restores the immune suppressive function of Tregs. These data define a fundamental pathway regulating the function of human Tregs and suggest a novel treatment paradigm for autoimmune diseases.

Despite being a major cell population in blood, and one of the major cellular targets of many RNA virus infections in peripheral blood, the molecular consequences of interactions between human monocytes and RNA viruses, and the signaling pathways responsible for the activation of these cells during infection are not well understood. Toll-like receptors (TLR) are a major family of pattern recognition sensors that trigger specific activation pathways in cells from both the innate and adaptive arms of the immune system. There are at least 10 TLRs in humans, from which TLR7 and TLR8 recognize single-stranded RNA. Despite both recognizing the same general ligand, we and others have demonstrated different phenotypic outcomes on cells stimulated through either TLR7 or TLR8. Our laboratory has recently observed fundamental differences in the phenotype and function of monocytes stimulated via either TLR7 or TLR8, in the context of RNA virus infections, and specifically, in terms of type I IFN responses and effector cytokines they produce, as well as differences in cell surface markers. We have defined the molecular mediators that are responsible for these differences in phenotype, performing ex vivo experiments with human monocytes isolated from blood and we have shown the relevance of these data in common RNA virus infections, demonstrating that TLR7 and/or TLR8 stimulation by RNA viruses in human monocytes accounts for much of the phenotype the cells acquire upon virus interaction.