参考文献

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells,when isolated and cultured in vitro,were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition,Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor. View Publication

Although studies with primary lymphocytes are almost always conducted in CO(2) incubators maintained at atmospheric oxygen levels (atmosO(2); 20%),the physiological oxygen levels (physO(2); 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO(2) significantly alters the intracellular redox state (decreases intracellular glutathione,increases oxidized intracellular glutathione),whereas culturing at physO(2) maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore,we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO(2) than at physO(2). This apparently paradoxical finding,we suggest,may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO(2) than at atmosO(2); and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO(2) that at atmosO(2). Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo,the lower proliferative T cell responses at physO(2) likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus,we suggest caution in culturing primary lymphocytes at atmosO(2) because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses,particularly after several days in culture. View Publication

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins,as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells,by enzyme-linked immunosorbent assay,immunoprecipitation followed by Western blotting,and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system,and that all forms of the recombinant proteins have in vitro functional activity. Furthermore,we find that in addition to the homodimers of IL-17F and IL-17A,activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses. View Publication

IL-12 is an immunoregulatory cytokine,which promotes Th1 cell differentiation and is a major inducer of IFN-gamma. IFN-beta,a Type I IFN used in the treatment of multiple sclerosis,has been shown to significantly increase the expression of the anti-inflammatory cytokine IL-10,a major suppressor of Th1 cytokines. The beneficial immunomodulatory effects of IFN-beta may in part be a result of its ability to suppress IL-12. However,IL-12 and IFN-beta signal via the STAT4 pathway. Our aim was to investigate the relationship between IL-12 and IFN-beta by observing the effect of prior exposure to IL-12 or IFN-beta on the ability of T cells to subsequently respond to the other cytokine. We report that IFN-beta increases IL-12-induced STAT4 phosphorylation and up-regulates IL-12 receptor beta1 and beta2 expression. However,despite this up-regulation,IFN-beta suppressed IL-12-induced IFN-gamma expression. Our results suggest that this may be a result of the parallel induction of IL-10 by IFN-beta. View Publication

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders,including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules,we investigated whether ASM can present SAg to resting CD4(+) T cells,and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg,staphylococcal enterotoxin A (SEA),elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface,indicative of immunological synapse formation,in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine,IL-13,into the coculture medium,which was MHC class II dependent. Moreover,when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues,the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively,these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells,and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that,in turn,evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus,a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and,accordingly,may underlie the reported association between microbial SAg exposure,T cell activation,and severe asthma. View Publication

NK and T cells are important for combating CMV infection. Some NK and T cells express leukocyte Ig-like receptor-1 (LIR-1),an inhibitory receptor recognizing MHC class I and the CMV-encoded homolog UL18. We previously demonstrated an early increase in LIR-1-expressing blood lymphocytes in lung-transplanted patients later developing CMV disease. We now show that NK and T cells account for the observed LIR-1 augmentation. Coincubation of PBMC from CMV-seropositive donors with virus-infected lung fibroblasts led to a T cell-dependent secretion of IFN-gamma,produced mainly by LIR-1(+) T cells and by NK cells. Cytokine production during coculture with fibroblasts infected with virus containing the UL18 gene was augmented compared with the UL18 deletion virus,suggesting a stimulatory role for UL18. However,purified UL18Fc proteins inhibited IFN-gamma production of LIR-1(+) T cells. We propose that cytokine production in the transplant induces NK and T cells to express LIR-1,which may predispose to CMV disease by MHC/LIR-1-mediated suppression. Although the UL18/LIR-1 interaction could inhibit T cell responses,this unlikely plays a role in response to infected cells. Instead,our data point to an activating role for viral UL18 during infection,where indirect intracellular effects cannot be excluded. View Publication

Ras activation is crucial for lymphocyte development and effector function. Both T and B lymphocytes contain two types of Ras activators: ubiquitously expressed SOS and specifically expressed Ras guanyl nucleotide-releasing protein (RasGRP). The need for two activators is enigmatic since both are activated following antigen receptor stimulation. In addition,RasGRP1 appears to be dominant over SOS in an unknown manner. The crystal structure of SOS provides a clue: an unusual allosteric Ras-GTP binding pocket. Here,we demonstrate that RasGRP orchestrates Ras signaling in two ways: (i) by activating Ras directly and (ii) by facilitating priming of SOS with RasGTP that binds the allosteric pocket. Priming enhances SOS' in vivo activity and creates a positive RasGTP-SOS feedback loop that functions as a rheostat for Ras activity. Without RasGRP1,initiation of this loop is impaired because SOS' catalyst is its own product (RasGTP)-hence the dominance of RasGRP1. Introduction of an active Ras-like molecule (RasV12C40) in T- and B-cell lines can substitute for RasGRP function and enhance SOS' activity via its allosteric pocket. The unusual RasGRP-SOS interplay results in sensitive and robust Ras activation that cannot be achieved with either activator alone. We hypothesize that this mechanism enables lymphocytes to maximally respond to physiologically low levels of stimulation. View Publication

In this study,we analyzed IL-2-activated polyclonal natural killer (NK) cells derived from 2 patients affected by leukocyte adhesion deficiency type I (LAD1),an immunodeficiency characterized by mutations of the gene coding for CD18,the beta subunit shared by major leukocyte integrins. We show that LAD1 NK cells express normal levels of various triggering NK receptors (and coreceptors) and that mAb-mediated engagement of these receptors results in the enhancement of both NK cytolytic activity and cytokine production. Moreover,these activating NK receptors were capable of recognizing their specific ligands on target cells. Thus,LAD1 NK cells,similarly to normal NK cells,were capable of killing most human tumor cells analyzed and produced high amounts of IFN-gamma when cocultured in presence of target cells. Murine target cells represented a common exception,as they were poorly susceptible to LAD1 NK cells. Finally,LAD1 NK cells could efficiently kill or induce maturation of monocyte-derived immature dendritic cells (DCs). Altogether our present study indicates that in LAD1 patients,3 important functions of NK cells (eg,cytotoxicity,IFN-gamma production,and DC editing) are only marginally affected and provides new insight on the cooperation between activating receptors and LFA-1 in the induction of NK cell activation and function. View Publication

Systemic lupus erythematosus (SLE) is an autoimmune/inflammatory disease characterized by autoantibody production and abnormal T cells that infiltrate tissues through not well-known mechanisms. We report that SLE T lymphocytes display increased levels of CD44,ezrin,radixin,and moesin (ERM) phosphorylation,stronger actin polymerization,higher polar cap formation,and enhanced adhesion and chemotactic migration compared with T cells from patients with rheumatoid arthritis and normal individuals. Silencing of CD44 by CD44 small interfering RNA in SLE T cells inhibited significantly their ability to adhere and migrate as did treatment with Rho kinase and actin polymerization inhibitors. Forced expression of T567D-ezrin,a phosphorylation-mimic form,enhanced remarkably the adhesion and migration rate of normal T cells. Anti-CD3/TCR autoantibodies present in SLE sera caused increased ERM phosphorylation,adhesion,and migration in normal T cells. pERM and CD44 are highly expressed in T cells infiltrating in the kidneys of patients with lupus nephritis. These data prove that increased ERM phosphorylation represents a key molecular abnormality that guides T cell adhesion and migration in SLE patients. View Publication

Costimulation by the chemokine,stromal cell-derived factor-1 (SDF-1)/CXCL12,has been shown to increase the amount of IL-10 secreted by TCR-stimulated human T cells; however,the molecular mechanisms of this response are unknown. Knowledge of this signaling pathway may be useful because extensive evidence indicates that deficient IL-10 secretion promotes autoimmunity. The human IL-10 locus is highly polymorphic. We report in this study that SDF-1 costimulates IL-10 secretion from T cells containing all three of the most common human IL-10 promoter haplotypes that are identified by single-nucleotide polymorphisms at -1082,-819,and -592 bp (numbering is relative to the transcription start site). We further show that SDF-1 primarily costimulates IL-10 secretion by a diverse population of CD45RA(-) (memory") phenotype T cells that includes cells expressing the presumed regulatory T cell marker� View Publication