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Developing vaccine strategies to generate high numbers of Ag-specific CD8 T cells may be necessary for protection against recalcitrant pathogens. Heterologous prime-boost-boost immunization has been shown to result in large quantities of functional memory CD8 T cells with protective capacities and long-term stability. Completing the serial immunization steps for heterologous prime-boost-boost can be lengthy,leaving the host vulnerable for an extensive period of time during the vaccination process. We show in this study that shortening the intervals between boosting events to 2 wk results in high numbers of functional and protective Ag-specific CD8 T cells. This protection is comparable to that achieved with long-term boosting intervals. Short-boosted Ag-specific CD8 T cells display a canonical memory T cell signature associated with long-lived memory and have identical proliferative potential to long-boosted T cells Both populations robustly respond to antigenic re-exposure. Despite this,short-boosted Ag-specific CD8 T cells continue to contract gradually over time,which correlates to metabolic differences between short- and long-boosted CD8 T cells at early memory time points. Our studies indicate that shortening the interval between boosts can yield abundant,functional Ag-specific CD8 T cells that are poised for immediate protection; however,this is at the expense of forming stable long-term memory. View Publication

Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However,natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that,on conjugation with CTL,human melanoma cells undergo an active late endosome/lysosome trafficking,which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking,pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance,we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. View Publication

Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis,the animal model for MS,myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby,the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently,B cells were found to participate in the pathogenesis of CNS autoimmunity,with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood-brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore,myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue. View Publication

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

Recent compelling evidence indicates that Th17 confer host immunity against a variety of microbes,including extracellular and intracellular pathogens. Therefore,understanding mechanisms for the induction and activation of Ag-specific Th17 is important for the rational design of vaccines against pathogens. To study this,we employed an in vitro system in which influenza hemagglutinin (HA) 1 was delivered to dendritic cells (DCs) via Dectin-1 using anti-human Dectin-1 (hDectin-1)-HA1 recombinant fusion proteins. We found that healthy individuals maintained broad ranges of HA1-specific memory Th17 that were efficiently activated by DCs targeted with anti-hDectin-1-HA1. Nonetheless,these DCs were not able to induce a significant level of HA1-specific Th17 responses even in the presence of the Th17-promoting cytokines IL-1? and IL-6. We further found that the induction of surface IL-1R1 expression by signals via TCRs and common ?-chain receptors was essential for naive CD4(+) T cell differentiation into HA1-specific Th17. This process was dependent on MyD88,but not IL-1R-associated kinase 1/4. Thus,interruptions in STAT3 or MyD88 signaling led to substantially diminished HA1-specific Th17 induction. Taken together,the de novo generation of pathogen-specific human Th17 requires complex,but complementary,actions of multiple signals. Data from this study will help us design a new and effective vaccine strategy that can promote Th17-mediated immunity against microbial pathogens. View Publication