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The rising incidence of autoimmune diseases such as multiple sclerosis (MS) in developed countries might be due to a more hygienic environment,particularly during early life. To investigate this concept,we developed a model of neonatal exposure to a common pathogen-associated molecular pattern,LPS,and determined its impact on experimental autoimmune encephalomyelitis (EAE). Mice exposed to LPS at 2 wk of age showed a delayed onset and diminished severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE,induced at 12 wk,compared with vehicle-exposed animals. Spinal cord transcript levels of CD3epsilon and F4/80 were lower in LPS- compared with PBS-exposed EAE animals with increased IL-10 levels in the LPS-exposed group. Splenic CD11c(+) cells from LPS-exposed animals exhibited reduced MHC class II and CD83 expression but increased levels of CD80 and CD86 both before and during EAE. MOG-treated APC from LPS-exposed animals stimulated less T lymphocyte proliferation but increased expansion of CD4(+)FoxP3(+) T cells compared with APC from PBS-exposed animals. Neuropathological studies disclosed reduced myelin and axonal loss in spinal cords from LPS-exposed compared with PBS-exposed animals with EAE,and this neuroprotective effect was associated with an increased number of CD3(+)FoxP3(+) immunoreactive cells. Analyses of human brain tissue revealed that FoxP3 expression was detected in lymphocytes,albeit reduced in MS compared with non-MS patients' brains. These findings support the concept of early-life microbial exposure influencing the generation of neuroprotective regulatory T cells and may provide insights into new immunotherapeutic strategies for MS. View Publication

The Msh2 mismatch repair (MMR) protein is critical for class switch recombination (CSR) events that occur in mice that lack the Smu tandem repeat (SmuTR) region (SmuTR(-/-) mice). The pattern of microhomology among switch junction sites in Msh2-deficient mice is also dependent on the presence or absence of SmuTR sequences. It is not known whether these CSR effects reflect an individual function of Msh2 or the function of Msh2 within the MMR machinery. In the absence of the SmuTR sequences,Msh2 deficiency nearly ablates CSR. We now show that Mlh1 or Exo1 deficiencies also eliminate CSR in the absence of the SmuTR. Furthermore,in SmuTR(-/-) mice,deficiencies of Mlh1 or Exo1 result in increased switch junction microhomology as has also been seen with Msh2 deficiency. These results are consistent with a CSR model in which the MMR machinery is important in processing DNA nicks to produce double-stranded breaks,particularly in sequences where nicks are infrequent. We propose that double-stranded break paucity in MMR-deficient mice leads to increased use of an alternative joining pathway where microhomologies are important for CSR break ligation. Interestingly,when the SmuTR region is present,deficiency of Msh2 does not lead to the increased microhomology seen with Mlh1 or Exo1 deficiencies,suggesting that Msh2 might have an additional function in CSR. It is also possible that the inability to initiate MMR in the absence of Msh2 results in CSR junctions with less microhomology than joinings that occur when MMR is initiated but then proceeds abnormally due to Mlh1 or Exo1 deficiencies. View Publication

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections impair plasmacytoid dendritic cell (PDC) and natural killer (NK) cell subset numbers and functions,though little is known about PDC-NK cell interactions during these infections. We evaluated PDC-dependent NK cell killing and gamma interferon (IFN-gamma) and granzyme B production,using peripheral blood mononuclear cell (PBMC)-based and purified cell assays of samples from HCV- and HIV-infected subjects. CpG-enhanced PBMC killing and IFN-gamma and granzyme B activity (dependent on PDC and NK cells) were impaired in viremic HIV infection. In purified PDC-NK cell culture experiments,CpG-enhanced,PDC-dependent NK cell activity was cell contact and IFN-alpha dependent,and this activity was impaired in viremic HIV infection but not in HCV infection. In heterologous PDC-NK cell assays,impaired PDC-NK cell killing activity was largely attributable to an NK cell defect,while impaired PDC-NK cell IFN-gamma-producing activity was attributable to both PDC and NK cell defects. Additionally,the response of NK cells to direct IFN-alpha stimulation was defective in viremic HIV infection,and this defect was not attributable to diminished IFN-alpha receptor expression,though IFN-alpha receptor and NKP30 expression was closely associated with killer activity in viremic HIV infection but not in healthy controls. These data indicate that during uncontrolled HIV infection,PDC-dependent NK cell function is impaired,which is in large part attributable to defective IFN-alpha-induced NK cell activity and not to altered IFN-alpha receptor,NKP30,NKP44,NKP46,or NKG2D expression. View Publication

Retroviruses can establish persistent infection despite induction of a multipartite antiviral immune response. Whether collective failure of all parts of the immune response or selective deficiency in one crucial part underlies the inability of the host to clear retroviral infections is currently uncertain. We examine here the contribution of virus-specific CD4(+) T cells in resistance against Friend virus (FV) infection in the murine host. We show that the magnitude and duration of the FV-specific CD4(+) T-cell response is directly proportional to resistance against acute FV infection and subsequent disease. Notably,significant protection against FV-induced disease is afforded by FV-specific CD4(+) T cells in the absence of a virus-specific CD8(+) T-cell or B-cell response. Enhanced spread of FV infection in hosts with increased genetic susceptibility or coinfection with Lactate dehydrogenase-elevating virus (LDV) causes a proportional increase in the number of FV-specific CD4(+) T cells required to control FV-induced disease. Furthermore,ultimate failure of FV/LDV coinfected hosts to control FV-induced disease is accompanied by accelerated contraction of the FV-specific CD4(+) T-cell response. Conversely,an increased frequency or continuous supply of FV-specific CD4(+) T cells is both necessary and sufficient to effectively contain acute infection and prevent disease,even in the presence of coinfection. Thus,these results suggest that FV-specific CD4(+) T cells provide significant direct protection against acute FV infection,the extent of which critically depends on the ratio of FV-infected cells to FV-specific CD4(+) T cells. View Publication

The hypothesis that bystander inflammatory signals promote memory B cell (B(MEM)) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis,a detailed analysis is presented examining the response profiles of B-2 lineage B220(+)IgG(+) B(MEM) toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter,quiescent B(MEM) clonally expand. Surprisingly,proliferating B(MEM) do not acquire germinal center (GC) B cell markers before generating daughter B(MEM) and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen,inflammatory stimuli,including Toll-like receptor agonists or bystander T cell activation,fail to induce even low levels of B(MEM) proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection,no detectable bystander proliferation or differentiation of B(MEM) occurred. The absence of a B(MEM) response to nonspecific inflammatory signals clearly shows that B(MEM) proliferation and differentiation is a process tightly controlled by the availability of cognate antigen. View Publication

Mesenteric lymph node (mLN) CD103 (alphaE integrin)(+) dendritic cells (DCs) induce regulatory T cells and gut tolerance. However,the function of intestinal CD103(-) DCs remains to be clarified. CD47 is the ligand of signal regulatory protein alpha (SIRPalpha) and promotes SIRPalpha(+) myeloid cell migration. We first show that mucosal CD103(-) DCs selectively express SIRPalpha and that their frequency was augmented in the lamina propria and mLNs of mice that developed Th17-biased colitis in response to trinitrobenzene sulfonic acid. In contrast,the percentage of SIRPalpha(+)CD103(-) DCs and Th17 responses were decreased in CD47-deficient (CD47 knockout [KO]) mice,which remained protected from colitis. We next demonstrate that transferring wild-type (WT),but not CD47 KO,SIRPalpha(+)CD103(-) DCs in CD47 KO mice elicited severe Th17-associated wasting disease. CD47 expression was required on the SIRPalpha(+)CD103(-) DCs for efficient trafficking to mLNs in vivo,whereas it was dispensable on both DCs and T cells for Th17 polarization in vitro. Finally,administration of a CD47-Fc molecule resulted in reduced SIRPalpha(+)CD103(-) DC-mediated Th17 responses and the protection of WT mice from colitis. We thus propose SIRPalpha(+)CD103(-) DCs as a pathogenic DC subset that drives Th17-biased responses and colitis,and the CD47-SIRPalpha axis as a potential therapeutic target for inflammatory bowel disease. View Publication

Mtb influences DC activity and T cell-mediated immune responses. We show that the treatment of immature monocyte-derived DC with Mtb elicited the formation of mature DC,producing TNF-alpha,IL-1beta,IL-6,and IL-23 and instructing CD4(+) cells to secrete IFN-gamma and IL-17. Mtb-induced cytokine release by DC depended on dectin-1 receptor engagement,whereas MR or DC-SIGN stimulation inhibited this process. A selective dectin-1 binding by the receptor agonist glucan was sufficient to enable DC to generate Th1/Th17 lymphocytes,showing features comparable with those induced by Mtb-treated DC. Interestingly,DC-SIGN or MR engagement inhibited Th17 and increased Th1 generation by glucan- or Mtb-treated DC. Our results indicate that Mtb modulates the lymphocyte response by affecting DC maturation and cytokine release. Dectin-1 engagement by Mtb enables DC to promote a Th1/Th17 response,whereas DC-SIGN and MR costimulation limits dectin-1-dependent Th17 generation and favors a Th1 response,probably by interfering with release of cytokines. View Publication

TNF-like ligand 1A (TL1A),a member of the TNF superfamily,is the ligand of DR3 and DcR3. Several types of cells,such as endothelial cells,monocytes/macrophages,dendritic cells,and CD4 and CD8 T cells,are capable of producing this cytokine. In present study,we demonstrated that TL1A aggravated collagen-induced arthritis in mice. It increased collagen-induced arthritis penetrance and clinical scores as well as the severity of the pathological findings. TL1A administration led to the occurrence of multiple enlarged germinal centers in the spleen,and it boosted serum anti-collagen Ab titers in vivo. In vitro,TL1A augmented TNF-alpha production by T cells upon TCR ligation,and it greatly enhanced Th17 differentiation and IL-17 production. We further showed that human rheumatoid arthritis (RA) synovial fluids had elevated TL1A titers,and human chrondrocytes and synovial fibroblasts were capable of secreting TL1A upon TNF-alpha or IL-1beta stimulation. Taken together,these data suggest that TL1A secretion in lymphoid organs might contribute to RA initiation by promoting autoantibody production,and TL1A secretion stimulated by inflammatory cytokines in RA joints might be a part of a vicious circle that aggravates RA pathogenesis. View Publication

Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17),which are able to indirectly induce the recruitment of neutrophils. Recently,human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors,including CCR2 and CCR6. Herein,we show that highly purified neutrophils cultured with interferon-gamma plus lipopolysaccharide produce the CCL2 and CCL20 chemokines,the known ligands of CCR2 and CCR6,respectively. Accordingly,supernatants from activated neutrophils induced chemotaxis of Th17 cells,which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment,neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally,we report that,although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor,tumor necrosis factor-alpha,and interferon-gamma release,these latter cells cannot be activated by IL-17A and IL-17F,because of their lack of IL-17RC expression. Collectively,our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells,which may represent a useful target for the treatment of chronic inflammatory diseases. View Publication

There is no licensed vaccine against the intracellular pathogen Francisella tularensis. The use of conventional mouse strains to screen protective vaccine antigens may be problematic,given the differences in the major histocompatibility complex (MHC) binding properties between murine and human antigen-presenting cells. We used engineered humanized mice that lack endogenous MHC class II alleles but that express a human HLA allele (HLA-DR4 transgenic [tg] mice) to identify potential subunit vaccine candidates. Specifically,we applied a biochemical and immunological screening approach with bioinformatics to select putative F. tularensis subsp. novicida T-cell-reactive antigens using humanized HLA-DR4 tg mice. Cell wall- and membrane-associated proteins were extracted with Triton X-114 detergent and were separated by fractionation with a Rotofor apparatus and whole-gel elution. A series of proteins were identified from fractions that stimulated antigen-specific gamma interferon (IFN-gamma) production,and these were further downselected by the use of bioinformatics and HLA-DR4 binding algorithms. We further examined the validity of this combinatorial approach with one of the identified proteins,a 19-kDa Francisella tularensis outer membrane protein (designated Francisella outer membrane protein B [FopB]; FTN0119). FopB was shown to be a T-cell antigen by a specific IFN-gamma recall assay with purified CD4(+) T cells from F. tularensis subsp. novicida DeltaiglC-primed HLA-DR4 tg mice and cells of a human B-cell line expressing HLA-DR4 (DRB1*0401) functioning as antigen-presenting cells. Intranasal immunization of HLA-DR4 tg mice with the single antigen FopB conferred significant protection against lethal pulmonary challenge with an F. tularensis subsp. holarctica live vaccine strain. These results demonstrate the value of combining functional biochemical and immunological screening with humanized HLA-DR4 tg mice to map HLA-DR4-restricted Francisella CD4(+) T-cell epitopes. View Publication

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases,their prevalence among tumor-infiltrating lymphocytes (TILs) and function in human tumor immunity remains largely unknown. We have recently demonstrated high percentages of Th17 cells in TILs from ovarian cancer patients,but the mechanisms of accumulation of these Th17 cells in the tumor microenvironment are still unclear. In this study,we further showed elevated Th17 cell populations in the TILs obtained from melanoma and breast and colon cancers,suggesting that development of tumor-infiltrating CD4(+) Th17 cells may be a general feature in cancer patients. We then demonstrated that tumor microenvironmental RANTES and MCP-1 secreted by tumor cells and tumor-derived fibroblasts mediate the recruitment of Th17 cells. In addition to their recruitment,we found that tumor cells and tumor-derived fibroblasts produce a proinflammatory cytokine milieu as well as provide cell-cell contact engagement that facilitates the generation and expansion of Th17 cells. We also showed that inflammatory TLR and nucleotide oligomerization binding domain 2 signaling promote the attraction and generation of Th17 cells induced by tumor cells and tumor-derived fibroblasts. These results identify Th17 cells as an important component of human TILs,demonstrate mechanisms involved in the recruitment and regulation of Th17 cells in tumor microenvironments,and provide new insights relevant for the development of novel cancer immunotherapeutic approaches. View Publication

Increased proportions of CD8 T lymphocytes lacking expression of the CD28 costimulatory receptor have been documented during both aging and chronic infection with HIV-1,and their abundance correlates with numerous deleterious clinical outcomes. CD28-negative cells also arise in cell cultures of CD8(+)CD28(+) following multiple rounds of Ag-driven proliferation,reaching the end stage of replicative senescence. The present study investigates the role of a second T cell costimulatory receptor component,adenosine deaminase (ADA),on the process of replicative senescence. We had previously reported that CD28 signaling is required for optimal telomerase upregulation. In this study,we show that the CD8(+)CD28(+) T lymphocytes that are ADA(+) have significantly greater telomerase activity than those that do not express ADA and that ADA is progressively lost as cultures progress to senescence. Because ADA converts adenosine to inosine,cells lacking this enzyme might be subject to prolonged exposure to adenosine,which has immunosuppressive effects. Indeed,we show that chronic exposure of CD8 T lymphocytes to exogenous adenosine accelerates the process of replicative senescence,causing a reduction in overall proliferative potential,reduced telomerase activity,and blunted IL-2 gene transcription. The loss of CD28 expression was accelerated,in part due to adenosine-induced increases in constitutive caspase-3,known to act on the CD28 promoter. These findings provide the first evidence for a role of ADA in modulating the process of replicative senescence and suggest that strategies to enhance this enzyme may lead to novel therapeutic approaches for pathologies associated with increases in senescent CD8 T lymphocytes. View Publication