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Immune tolerance is executed partly by Foxp3(+)regulatory T (Treg) cells,which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing β-cells. The development of autoantigen-specific vaccination strategies for Foxp3(+)Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here,using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice,we provide direct evidence for human autoantigen-specific Foxp3(+)Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4(+)T cells and demonstrate efficient human insulin-specific Foxp3(+)Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable,show increased expression of Treg signature genes such as Foxp3,CTLA4,IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3(+)Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3(+)Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D. View Publication

Leukocyte recruitment to inflammation sites progresses in a multistep cascade. Chemokines regulate multiple steps of the cascade,including arrest,transmigration,and chemotaxis. The most important chemokine receptor in mouse neutrophils is CXCR2,which couples through G$\alpha$i2- and G$\alpha$i3-containing heterotrimeric G proteins. Neutrophils arrest in response to CXCR2 stimulation. This is defective in G$\alpha$i2-deficient neutrophils. In this study,we show that G$\alpha$i3-deficient neutrophils showed reduced transmigration but normal arrest in mice. We also tested G$\alpha$i2- or G$\alpha$i3-deficient neutrophils in a CXCL1 gradient generated by a microfluidic device. G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils showed significantly reduced migration and directionality. This was confirmed in a model of sterile inflammation in vivo. G$\alpha$i2-,but not G$\alpha$i3-,deficient neutrophils showed decreased Ca(2+) flux in response to CXCR2 stimulation. Conversely,G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils exhibited reduced AKT phosphorylation upon CXCR2 stimulation. We conclude that G$\alpha$i2 controls arrest and G$\alpha$i3 controls transmigration and chemotaxis in response to chemokine stimulation of neutrophils. View Publication

We examined the role of NFκB1 in the homeostasis and function of peripheral follicular (Fo) B cells. Aging mice lacking NFκB1 (Nfκb1(-/-)) develop lymphoproliferative and multiorgan autoimmune disease attributed in large part to the deregulated activity ofNfκb1(-/-)Fo B cells that produce excessive levels of the proinflammatory cytokine interleukin 6 (IL-6). Despite enhanced germinal center (GC) B cell differentiation,the formation of GC structures was severely disrupted in theNfκb1(-/-)mice. Bone marrow chimeric mice revealed that the Fo B cell-intrinsic loss of NFκB1 led to the spontaneous generation of GC B cells. This was primarily the result of an increase in IL-6 levels,which promotes the differentiation of Fo helper CD4(+)T cells and acts in an autocrine manner to reduce antigen receptor and toll-like receptor activation thresholds in a population of proliferating IgM(+)Nfκb1(-/-)Fo B cells. We demonstrate that p50-NFκB1 repressesIl-6transcription in Fo B cells,with the loss of NFκB1 also resulting in the uncontrolled RELA-driven transcription ofIl-6.Collectively,our findings identify a previously unrecognized role for NFκB1 in preventing multiorgan autoimmunity through its negative regulation ofIl-6gene expression in Fo B cells. View Publication

FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically,FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly,administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely,mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis,increased cell survival and reduced proinflammatory chemokine expression. Hence,treatment with FNDC4 results in a state of dampened macrophage activity,while enhancing their survival. Thus,we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases. View Publication

IL-18 is a member of the IL-1 family involved in innate immunity and inflammation. Deregulated levels of IL-18 are involved in the pathogenesis of multiple disorders including inflammatory and metabolic diseases,yet relatively little is known regarding its regulation. Liver X receptors or LXRs are key modulators of macrophage cholesterol homeostasis and immune responses. Here we show that LXR ligands negatively regulate LPS-induced mRNA and protein expression of IL-18 in bone marrow-derived macrophages. Consistent with this being an LXR-mediated process,inhibition is abolished in the presence of a specific LXR antagonist and in LXR-deficient macrophages. Additionally,IL-18 processing of its precursor inactive form to its bioactive state is inhibited by LXR through negative regulation of both pro-caspase 1 expression and activation. Finally,LXR ligands further modulate IL-18 levels by inducing the expression of IL-18BP,a potent endogenous inhibitor of IL-18. This regulation occurs via the transcription factor IRF8,thus identifying IL-18BP as a novel LXR and IRF8 target gene. In conclusion,LXR activation inhibits IL-18 production through regulation of its transcription and maturation into an active pro-inflammatory cytokine. This novel regulation of IL-18 by LXR could be applied to modulate the severity of IL-18 driven metabolic and inflammatory disorders. View Publication

Viral infection triggers induction of antiviral cytokines and effectors,which are critical mediators of innate antiviral immune response. It has been shown that the processing body-associated protein LSm14A is involved in the induction of antiviral cytokines in cell lines but in vivo evidence is lacking. By generating LSm14A-deficient mice,in this study,we show that LSm14A plays a critical and specific role in the induction of antiviral cytokines in dendritic cells (DCs) but not in macrophages and fibroblasts. Induction of antiviral cytokines triggered by the DNA viruses HSV-1 and murid herpesvirus 68 and the RNA virus vesicular stomatitis virus but not Sendai virus was impaired in Lsm14a(-/-) DCs,which is correlated to the functions of the adaptor protein MITA/STING in the antiviral signaling pathways. LSm14A deficiency specifically downregulated MITA/STING level in DCs by impairing its nuclear mRNA precursor processing and subsequently impaired antiviral innate and adaptive immune responses. Our findings reveal a nuclear mRNA precursor processing and cell-specific regulatory mechanism of antiviral immune responses. View Publication

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here,we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen,while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner,while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells,a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack. View Publication

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here,we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes,where they trap T cells by releasing the CCR7 ligand,CCL19,and to inflamed central nervous system,where they locally limit immunopathogenesis through interleukin-10 production,thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment,where proB cells develop,is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition,these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases. View Publication

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However,the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum,blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus,RTM protects the normal arterial intima,and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions. View Publication

During adaptive immune responses,CD8(+) T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study,we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity,whereas one day later,the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor,ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation,as low affinity-primed T cells acquired cytotoxic activity earlier than high affinity-primed ones. After activation,low-affinity effector CD8(+) T cells accumulated at efferent lymphatic vessels for egress,whereas high affinity-stimulated CD8(+) T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8(+) T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. View Publication

CD4(+) T cells develop distinct and often contrasting helper,regulatory,or cytotoxic activities. Typically a property of CD8(+) T cells,granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4(+) T cells. However,the conditions that induce CD4(+) CTLs are not entirely understood. Using single-cell transcriptional profiling,we uncover a unique signature of Granzyme B (GzmB)(+) CD4(+) CTLs,which distinguishes them from other CD4(+) T helper (Th) cells,including Th1 cells,and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4(+) CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors Bcl6 and Tcf7 (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4(+) CTLs offers targets for their study,and its antagonism by the Tfh program separates CD4(+) T cells with either helper or killer functions. View Publication

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection,but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal β-glucans stimulate IFN-β production by dendritic cells (DCs) following detection by the Dectin-1 receptor,but the effects of β-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal β-glucan particle-stimulated DCs. We demonstrate that β-glucan-stimulated DCs induce CD8 T cell proliferation,activation marker (CD44 and CD69) expression,and production of IFN-γ,IL-2,and granzyme B. Moreover,we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by β-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically,type I IFNs promote Ag presentation on MHC I molecules,CD86 and CD40 expression,and the production of IL-12 p70,IL-2,IL-6,and TNF-α by β-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation,although,in the context of LPS stimulation,this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection,which may be useful in the rational design of vaccines directed against pathogens and tumors. View Publication