技术资料

The cytidine deaminase AID (encoded by Aicda in mice and AICDA in humans) is critical for immunoglobulin class-switch recombination (CSR) and somatic hypermutation (SHM). Here we show that AID expression was induced by the HoxC4 homeodomain transcription factor,which bound to a highly conserved HoxC4-Oct site in the Aicda or AICDA promoter. This site functioned in synergy with a conserved binding site for the transcription factors Sp1,Sp3 and NF-kappaB. HoxC4 was 'preferentially' expressed in germinal center B cells and was upregulated by engagement of CD40 by CD154,as well as by lipopolysaccharide and interleukin 4. HoxC4 deficiency resulted in impaired CSR and SHM because of lower AID expression and not some other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4(-/-) B cells fully restored CSR. Thus,HoxC4 directly activates the Aicda promoter,thereby inducing AID expression,CSR and SHM. View Publication

OBJECTIVE: T lymphocytes play an important role in systemic sclerosis (SSc),a connective tissue disease characterized by inflammation,fibrosis,and vascular damage. While their precise role and antigen specificity are unclear,T cell-derived cytokines likely contribute to the induction of fibrosis. The aim of this study was to establish the role of cytokine dysregulation by T cells in the pathogenesis of SSc. METHODS: To identify relationships between a specific cytokine,T cell subset,and the disease course,we studied a large cohort of patients with diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc). Using Luminex analysis and intracellular cytokine staining,we analyzed the intrinsic ability of CD4+ and CD8+ T cell subsets to produce cytokines following in vitro activation. RESULTS: High levels of the profibrotic type 2 cytokine interleukin-13 (IL-13) were produced following activation of peripheral blood effector CD8+ T cells from SSc patients as compared with normal controls or with patients with rheumatoid arthritis. In contrast,CD4+ T cells showed a lower and more variable level of IL-13 production. This abnormality correlated with the extent of fibrosis and was more pronounced in dcSSc patients than in lcSSc patients. CONCLUSION: Dysregulated IL-13 production by effector CD8+ T cells is important in the pathogenesis of SSc and is critical in the predisposition to more severe forms of cutaneous disease. Our study is the first to identify a specific T cell phenotype that correlates with disease severity in SSc and can be used as a marker of immune dysfunction in SSc and as a novel therapeutic target. View Publication

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation,which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo,antibody blockade of TNFSF15 (TL1A),which is the ligand for TNFR25,inhibits lung inflammation and production of Th2 cytokines such as IL-13,even when administered days after airway antigen exposure. Similarly,blockade of TNFR25 by a dominant-negative (DN) transgene,DN TNFR25,confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant,NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells,but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung,and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics. View Publication

Natural killer (NK) cells have been originally defined by their naturally occurring" effector function. However� View Publication

Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end,we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells,and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo,while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence,we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase,the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings,two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly,these cocktails were either deprived of thrombopoietin or of stem cell factor,two cytokines known to favor megakaryopoiesis and HPC expansion,respectively. Based on these results,a short resource-efficient two-phase culture protocol for the production of Mks near purity (textgreater95%) from human CD34+ CB cells has been established. View Publication

Complete T cell development requires postthymic maturation,and we investigated the influence of this ontological period on the CD8 T cell response to infection by comparing responses of mature CD8 T cells with those of recent thymic emigrants (RTEs). When activated with a noninflammatory stimulus or a bacterial or viral pathogen,CD8 RTEs generated a lower proportion of cytokine-producing effector cells and long-lived memory precursors compared with their mature counterparts. Although peripheral T cell maturation is complete within several weeks after thymic egress,RTE-derived memory cells continued to express inappropriate levels of memory cell markers and display an altered pattern of cytokine production,even 8 weeks after infection. When rechallenged,RTE-derived memory cells generated secondary effector cells that were phenotypically and functionally equivalent to those generated by their mature counterparts. The defects at the effector and memory stages were not associated with differences in the expression of T cell receptor-,costimulation-,or activation-associated cell surface markers yet were associated with lower Ly6C expression levels at the effector stage. This work demonstrates that the stage of postthymic maturation influences cell fate decisions and cytokine profiles of stimulated CD8 T cells,with repercussions that are apparent long after cells have progressed from the RTE compartment. View Publication

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76),hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly,cell death is triggered in macrophages cultured with GMME1,while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation,inhibition of p44/42,AKT and STAT3 phosphorylation,and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17,MOG-specific Ab titers,and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2. View Publication

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are,in part,controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis,we have compared gene expression profiles of human erythroblasts,megakaryocytes,B cells,cytotoxic and helper T cells,natural killer cells,granulocytes,and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors,immunoglobulin superfamily members,and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude,ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition,we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg,GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data,which are freely accessible,will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies. 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

Apoptosis is a key pathogenic mechanism in sepsis that induces extensive death of lymphocytes and dendritic cells,thereby contributing to the immunosuppression that characterizes the septic disorder. Numerous animal studies indicate that prevention of apoptosis in sepsis improves survival and may represent a potential therapy for this highly lethal disorder. Recently,novel cell-penetrating peptide constructs such as HIV-1 TAT basic domain and related peptides have been developed to deliver bioactive cargoes and peptides into cells. In the present study,we investigated the effects of sepsis-induced apoptosis in Bcl-x(L) transgenic mice and in wild-type mice treated with an antiapoptotic TAT-Bcl-x(L) fusion protein and TAT-BH4 peptide. Lymphocytes from Bcl-x(L) transgenic mice were resistant to sepsis-induced apoptosis,and these mice had a approximately 3-fold improvement in survival. TAT-Bcl-x(L) and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion,TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival. View Publication

The Wiskott-Aldrich syndrome protein (WASP) is a product of the gene defective in an Xid disorder,Wiskott-Aldrich syndrome. WASP expression is limited to hemopoietic cells,and WASP regulates the actin cytoskeleton. It has been reported that monocytes/macrophages from WASP-deficient Wiskott-Aldrich syndrome patients are severely defective in chemotaxis,resulting in recurrent infection. However,the molecular basis of such chemotactic defects is not understood. Recently,the WASP N-terminal region was found to bind to the three mammalian verprolin homologs: WASP interacting protein (WIP); WIP and CR16 homologous protein (WICH)/WIP-related protein (WIRE); and CR16. Verprolin was originally found to play an important role in the regulation of actin cytoskeleton in yeast. We have shown that WASP,WIP,and WICH/WIRE are expressed predominantly in the human monocyte cell line THP-1 and that WIP and WICH/WIRE are involved in monocyte chemotaxis. When WASP binding to verprolins was blocked,chemotactic migration of monocytes was impaired in both THP-1 cells and primary human monocytes. Increased expression of WASP and WIP enhanced monocyte chemotaxis. Blocking WASP binding to verprolins impaired cell polarization but not actin polymerization. These results indicate that a complex of WASP with mammalian verprolins plays an important role in chemotaxis of monocytes. Our results suggest that WASP and mammalian verprolins function as a unit in monocyte chemotaxis and that the activity of this unit is critical to establish cell polarization. In addition,our results also indicate that the WASP-verprolin complex is involved in other functions such as podosome formation and phagocytosis. 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