技术资料

Murine CMV (MCMV) establishes a systemic,low-level persistent infection resulting in the accumulation of CD8(+) T cells specific for a subset of viral epitopes,a process called memory inflation. Although replicating virus is rarely detected in chronically infected C57BL/6 mice,these inflationary cells display a phenotype suggestive of repeated Ag stimulation,and they remain functional. CD4(+) T cells have been implicated in maintaining the function and/or number of CD8(+) T cells in other chronic infections. Moreover,CD4(+) T cells are essential for complete control of MCMV. Thus,we wondered whether CD4(+) T cell deficiency would result in impaired MCMV-specific CD8(+) T cell responses. Here we show that CD4(+) T cell deficiency had an epitope-specific impact on CD8(+) T cell memory inflation. Of the three codominant T cell responses during chronic infection,only accumulation of the late-appearing IE3-specific CD8(+) T cells was substantially impaired in CD4(+) T cell-deficient mice. Moreover,the increased viral activity did not drive increased CD8(+) T cell division or substantial dysfunction in any MCMV-specific population that we studied. These data show that CD4(+) T cell help is needed for inflation of a response that develops only during chronic infection but is otherwise dispensable for the steady state maintenance and function of MCMV-specific CD8(+) 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

IL-12 is a potent proinflammatory cytokine. The effects of IL-12 are thought to be mediated by IFN-gamma production by NK,NKT,and T cells. In this study,we show that although IL-12 stimulates NK and NK1.1(+) T cells in bulk mouse splenocytes,it does not significantly stimulate purified NK cells,indicating that other cells are required. IL-12 stimulates T cell-deficient spleen cells and those depleted of macrophages. Unexpectedly,the depletion of dendritic cells also has little effect on the stimulation of spleen cells with IL-12. In contrast,B cell depletion almost completely inhibits IL-12-induced IFN-gamma production and B cell-deficient spleen cells are poorly stimulated with IL-12. Furthermore,purified NK cells are stimulated with IL-12 in the presence of purified B cells. Thus,B cells are necessary and also sufficient for the stimulation of purified NK cells with IL-12. Whereas spleen cells from IL-18-deficient mice are not stimulated with IL-12,NK cells purified from IL-18-deficient mice are stimulated with IL-12 in the presence of wild-type (WT) B cells,and WT NK cells are not stimulated with IL-12 in the presence of IL-18-deficient B cells. Cell contact between B and NK cells is also required for IL-12-induced IFN-gamma production. Finally,B cell-deficient mice injected with IL-12 produce significantly less IFN-gamma and IL-18 in the sera than WT mice do. Thus,stimulation of NK cells with IL-12 requires B cell cooperation in vitro as well as in vivo. View Publication

Because IL-1beta plays an important role in inflammation in human and murine arthritis,we investigated the contribution of the inflammasome components ASC,NALP-3,IPAF,and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA,decreased levels of synovial IL-1beta,and diminished serum amyloid A levels. In contrast,mice deficient in NALP-3,IPAF,or caspase-1 did not show any alteration of joint inflammation,thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes,we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro,as was the production of IFN-gamma,whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice,but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion,these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation. View Publication

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) share a developmental relationship with Foxp3(+) regulatory T cells (T(reg) cells). Here we show that a T(H)-17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-beta (TGF-beta). Like previously described T(H)-17 cells,the T(H)-17 cells that developed in the thymus expressed the transcription factor RORgamma t and the IL-23 receptor. These cells also expressed alpha(4)beta(1) integrins and the chemokine receptor CCR6 and were recruited to the lung,gut and liver. In the liver,these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus,T(H)-17 cells,like T(reg) cells,can be selected by self antigens in the thymus. 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

C57BL/6 (B6) mice are genetically highly susceptible to chronic type II Toxoplasma gondii infections that invariably cause lethal toxoplasmic encephalitis. We examined the ability of an attenuated type I vaccine strain to elicit long-term immunity to lethal acute or chronic type II infections in susceptible B6 mice. Mice immunized with the type I cps1-1 vaccine strain were not susceptible to a lethal (100-cyst) challenge with the type II strain ME49. Immunized mice challenged with 10 ME49 cysts exhibited significant reductions in brain cyst and parasite burdens compared to naive mice,regardless of the route of challenge infection. Remarkably,cps1-1 strain-immunized B6 mice chronically infected with ME49 survived for at least 12 months without succumbing to the chronic infection. Potent immunity to type II challenge infections persisted for at least 10 months after vaccination. While the cps1-1 strain-elicited immunity did not prevent the establishment of a chronic infection or clear established brain cysts,cps1-1 strain-elicited CD8(+) immune T cells significantly inhibited recrudescence of brain cysts during chronic ME49 infection. In addition,we show that uracil starvation of the cps1-1 strain induces early markers of bradyzoite differentiation. Collectively,these results suggest that more effective immune control of chronic type II infection in the genetically susceptible B6 background is established by vaccination with the nonreplicating type I uracil auxotroph cps1-1 strain. View Publication

The activating NK cell receptor DNAX accessory molecule-1 (DNAM-1) contributes to tumor immune surveillance and plays a crucial role in NK cell-mediated recognition of several types of human tumors,including ovarian carcinoma. Here,we have analyzed the receptor repertoire and functional integrity of NK cells in peritoneal effusions from patients with ovarian carcinoma. Relative to autologous peripheral blood NK cells,tumor-associated NK cells expressed reduced levels of the DNAM-1,2B4,and CD16 receptors and were hyporesponsive to HLA class I-deficient K562 cells and to coactivation via DNAM-1 and 2B4. Moreover,tumor-associated NK cells were also refractory to CD16 receptor stimulation,resulting in diminished Ab-dependent cellular cytotoxicity against autologous tumor cells. Coincubation of NK cells with ovarian carcinoma cells expressing the DNAM-1 ligand CD155 led to reduction of DNAM-1 expression. Therefore,NK cell-mediated rejection of ovarian carcinoma may be limited by perturbed DNAM-1 expression on tumor-associated NK cells induced by chronic ligand exposure. Thus,these data support the notion that tumor-induced alterations of activating NK cell receptor expression may hamper immune surveillance and promote tumor progression. View Publication

Effector cells derived from central memory CD8(+) T cells were reported to engraft and survive better than those derived from effector memory populations,suggesting that they are superior for use in adoptive immunotherapy studies. However,previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve,rather than central memory T cells,gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells,but did not acquire the expression of KLRG-1,a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1(-) phenotype,naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8(+) T cells may allow superior efficacy upon adoptive transfer. View Publication

The active form of vitamin D,1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)),has potent immunomodulatory properties that have promoted its potential use in the prevention and treatment of infectious disease and autoimmune conditions. A variety of immune cells,including macrophages,dendritic cells,and activated T cells express the intracellular vitamin D receptor and are responsive to 1,25(OH)(2)D(3.) Despite this,how 1,25(OH)(2)D(3) regulates adaptive immunity remains unclear and may involve both direct and indirect effects on the proliferation and function of T cells. To further clarify this issue,we have assessed the effects of 1,25(OH)(2)D(3) on human CD4(+)CD25(-) T cells. We observed that stimulation of CD4(+)CD25(-) T cells in the presence of 1,25(OH)(2)D(3) inhibited production of proinflammatory cytokines including IFN- gamma,IL-17,and IL-21 but did not substantially affect T cell division. In contrast to its inhibitory effects on inflammatory cytokines,1,25(OH)(2)D(3) stimulated expression of high levels of CTLA-4 as well as FoxP3,the latter requiring the presence of IL-2. T cells treated with 1,25(OH)(2)D(3) could suppress proliferation of normally responsive T cells,indicating that they possessed characteristics of adaptive regulatory T cells. Our results suggest that 1,25(OH)(2)D(3) and IL-2 have direct synergistic effects on activated T cells,acting as potent anti-inflammatory agents and physiologic inducers of adaptive regulatory T cells. 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