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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

Th17 cells play a significant role in inflammatory and autoimmune responses. Although a number of molecular pathways that contribute to the lineage differentiation of T cells have been discovered,the mechanisms by which lineage commitment occurs are not fully understood. Transcription factors play a key role in driving T cells toward specific lineages. We have identified a role for the transcription factor Kruppel-like factor (KLF) 4 in the development of IL-17-producing CD4(+) T cells. KLF4 was required for the production of IL-17,and further,chromatin immunoprecipitation analysis demonstrated binding of KLF4 to the IL-17 promoter,indicating a direct effect on the regulation of IL-17. Further,KLF4-deficient T cells upregulated expression of retinoic acid-related orphan receptor γt similar to wild-type during the polarization process toward Th17,suggesting that these two transcription factors are regulated independently. View Publication

Adoptive cell therapy with engineered T cells to improve natural immune response and antitumor functions has shown promise for treating cancer. However,the requirement for extensive ex vivo manipulation of T cells and the immunosuppressive effects of the tumor microenvironment limit this therapeutic modality. In the present study,we investigated the possibility to circumvent these limitations by engineering Stat3 -deficient CD8(+) T cells or by targeting Stat3 in the tumor microenvironment. We show that ablating Stat3in CD8(+) T cells prior to their transfer allows their efficient tumor infiltration and robust proliferation,resulting in increased tumor antigen-specific T-cell activity and tumor growth inhibition. For potential clinical translation,we combined adoptive T-cell therapy with a Food and Drug Administration-approved tyrosine kinase inhibitor,sunitinib,in renal cell carcinoma and melanoma tumor models. Sunitinib inhibited Stat3 in dendritic cells and T cells and reduced conversion of transferred FoxP3(-) T cells to tumor-associated regulatory T cells while increasing transferred CD8(+) T-cell infiltration and activation at the tumor site,leading to inhibition of primary tumor growth. These data show that adoptively transferred T cells can be expanded and activated in vivo either by engineering Stat3-silenced T cells or by targeting Stat3 systemically with small-molecule inhibitors. View Publication

The T-cell functions of a proliferation-inducing ligand (APRIL,also known as TNFSF13) remain largely undefined. We previously showed that APRIL suppressed Th2 cytokine production in cultured CD4(+) T cells and Th2 antibody responses. Here we show that APRIL suppresses allergic lung inflammation,which is associated with diminished expression of the transcription factor c-maf. Mice deficient in the April gene (April(-/-) mice) had significantly aggravated lung inflammation compared with WT mice in the ovalbumin-induced allergic lung inflammation model. Likewise,blockade of APRIL in WT mice by the APRIL-receptor fusion protein,transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI)-Ig,enhanced lung inflammation. Transfer of APRIL-sufficient,ovalbumin-specific,TCR-transgenic CD4(+) T (OT-II) cells to April(-/-) mice restored the suppressive effect of APRIL on lung inflammation. Mechanistically,the expression of the Th2 cytokine transcription factor c-maf,but not GATA-3,was markedly enhanced in April(-/-) CD4(+) T cells at the RNA and protein level and under non-polarizing (Th neutral,ThN) and Th2-polarizing conditions. Since c-maf transactivates the IL-4 gene,the increased c-maf expression in April(-/-) mice readily explains increased Th2 cytokine production. Independent of its effect on IL-4,APRIL suppressed IL-13 expression. APRIL thus may regulate lung inflammation in a dual way,by acting on c-maf expression and by directly controlling IL-13 production. View Publication

B1 cells differ in many ways from conventional B cells,most prominently in the production of natural immunoglobulin,which is vitally important for protection against pathogens. B1 cells have also been implicated in the pathogenesis of autoimmune dyscrasias and malignant diseases. It has been impossible to accurately study B1 cells during health and illness because the nature of human B1 cells has not been successfully defined. This has produced controversy regarding the existence of human B1 cells. Here,we determined the phenotype of human B1 cells by testing sort-purified B cell fractions for three fundamental B1 cell functions based on mouse studies: spontaneous IgM secretion,efficient T cell stimulation,and tonic intracellular signaling. We found that a small population of CD20(+)CD27(+)CD43(+) cells present in both umbilical cord and adult peripheral blood fulfilled these criteria and expressed a skewed B cell receptor repertoire. These B cells express little or no surface CD69 and CD70,both of which are markedly up-regulated after activation of CD20(+)CD27(-)CD43(-) (naive) and CD20(+)CD27(+)CD43(-) (memory) B cells. This work identifies human B1 cells as CD20(+)CD27(+)CD43(+)CD70(-). We determined that the proportion of B1 cells declines with age,which may contribute to disease susceptibility. Identification of human B1 cells provides a foundation for future studies on the nature and role of these cells in human disease. View Publication