技术资料

Binding of ICAM-1 (intercellular adhesion molecule-1) to the β2-integrin LFA-1 (leukocyte function associated antigen-1) is known to induce crosstalk to the α4β1 integrin. Using different LFA-1 monoclonal antibodies we have been able to study the requirement and mechanism of action for the crosstalk in considerable detail. LFA-1 activating antibodies and those inhibitory antibodies that signal to α4β1 induce phosphorylation of Thr-758 on the β2-chain,which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the β1-chain of α4β1 and loss of binding to its ligand VCAM-1 (vascular cell adhesion molecule-1). The results show that with LFA-1 antibodies,we can either 1) activate LFA-1 and inhibit α4β1,2) inhibit both LFA-1 and α4β1,3) inhibit LFA-1 but not α4β1 or 4) not affect LFA-1 or α4β1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications. View Publication

Understanding how memory B cells are induced and relate to long-lived plasma cells is important for vaccine development. Immunity to oral vaccines has been considered short-lived because of a poor ability to develop IgA B-cell memory. Here we demonstrate that long-lived mucosal IgA memory is readily achieved by oral but not systemic immunization in mouse models with NP hapten conjugated with cholera toxin and transfer of B1-8(high)/GFP(+) NP-specific B cells. Unexpectedly,memory B cells are poorly related to long-lived plasma cells and less affinity-matured. They are α4β7-integrin(+)CD73(+)PD-L2(+)CD80(+) and at systemic sites mostly IgM(+),while 80% are IgA(+) in Peyer's patches. On reactivation,most memory B cells in Peyer's patches are GL7(-),but expand in germinal centres and acquire higher affinity and more mutations,demonstrating strong clonal selection. CCR9 expression is found only in Peyer's patches and appears critical for gut homing. Thus,gut mucosal memory possesses unique features not seen after systemic immunization. View Publication

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225,which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta-/- mice. In intact T cells,HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation,while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling. View Publication

Lipid rafts accumulate in the immunological synapse formed by an organized assembly of the TCR/CD3,LFA-1,and signaling molecules. However,the precise role of lipid rafts in the formation of the immunological synapse is unclear. In this study,we show that LFA-1 on CTL is constitutively active and mediates Ag-independent binding of CTL to target cells expressing its ligands. LFA-1 and CD3 on CTL,but not resting T cells,colocalize in lipid rafts. Binding of LFA-1 on CTL to targets initiates the formation of the immunological synapse,which is formed by LFA-1,CD3,and ganglioside GM1 distributed in the periphery of the cell contact site and cholesterol is more widely distributed. The formation of this synapse is Ag independent,but the recognition of Ag by the TCR induces accumulation of tyrosine phosphorylated proteins in the synapse as well as redistribution of the microtubule organization center toward the cell contact site. Our results suggest that LFA-1 recruits lipid rafts and the TCR/CD3 to the synapse,and facilitates efficient and rapid activation of CTL. View Publication

Experimental autoimmune encephalomyelitis (EAE) models,in animals,many characteristics of multiple sclerosis,for which there is no adequate therapy. We investigated whether lithium,an inhibitor of glycogen synthase kinase-3 (GSK3),can ameliorate EAE in mice. Pretreatment with lithium markedly suppressed the clinical symptoms of EAE induced in mice by myelin oligodendrocyte glycoprotein peptide (MOG35-55) immunization and greatly reduced demyelination,microglia activation,and leukocyte infiltration in the spinal cord. Lithium administered postimmunization,after disease onset,reduced disease severity and facilitated partial recovery. Conversely,in knock-in mice expressing constitutively active GSK3,EAE developed more rapidly and was more severe. In vivo lithium therapy suppressed MOG35-55-reactive effector T cell differentiation,greatly reducing in vitro MOG35-55- stimulated proliferation of mononuclear cells from draining lymph nodes and spleens,and MOG35-55-induced IFN-gamma,IL-6,and IL-17 production by splenocytes isolated from MOG35-55-immunized mice. In relapsing/remitting EAE induced with proteolipid protein peptide139-151,lithium administered after the first clinical episode maintained long-term (90 days after immunization) protection,and after lithium withdrawal the disease rapidly relapsed. These results demonstrate that lithium suppresses EAE and identify GSK3 as a new target for inhibition that may be useful for therapeutic intervention of multiple sclerosis and other autoimmune and inflammatory diseases afflicting the CNS. View Publication

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones,light and dark,with coordinated roles,controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells,but its functional role in the GC reaction has not been explored. In this study,we report high expression of LLT1 on GC-associated B cells,early plasmablasts,and GC-derived lymphomas. LLT1 expression was readily induced via BCR,CD40,and CpG stimulation on B cells. Unexpectedly,we found high expression of the LLT1 ligand,CD161,on follicular dendritic cells. Triggering of LLT1 supported B cell activation,CD83 upregulation,and CXCR4 downregulation. Overall,these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans. View Publication

Thrombopoietin (Tpo) is the primary cytokine regulating megakaryocyte development and platelet production. Tpo signaling through its receptor,c-mpl,activates multiple pathways including signal transducer and activator of transcription (STAT)3,STAT5,phosphoinositide 3-kinase-Akt,and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor and immunoreceptor signaling. Here,we show that Lnk overexpression negatively regulates Tpo-mediated cell proliferation and endomitosis in hematopoietic cell lines and primary hematopoietic cells. Lnk attenuates Tpo-induced S-phase progression in 32D cells expressing mpl,and Lnk decreases Tpo-dependent megakaryocyte growth in bone marrow (BM)-derived megakaryocyte culture. Consistent with this result,we found that in both BM and spleen,Lnk-deficient mice exhibited increased numbers of megakaryocytes with increased ploidy compared with wild-type mice. In addition,Lnk-deficient megakaryocytes derived from BM and spleen showed enhanced sensitivity to Tpo during culture. The absence of Lnk caused enhanced and prolonged Tpo induction of STAT3,STAT5,Akt,and MAPK signaling pathways in CD41+ megakaryocytes. Furthermore,the Src homology 2 domain of Lnk is essential for Lnk's inhibitory function. In contrast,the conserved tyrosine near the COOH terminus is dispensable and the pleckstrin homology domain of Lnk contributes to,but is not essential for,inhibiting Tpo-dependent 32D cell growth or megakaryocyte development. Thus,Lnk negatively modulates mpl signaling pathways and is important for Tpo-mediated megakaryocytopoiesis in vivo. View Publication

Our recent studies have shown that immune cell-produced complement provides costimulatory and survival signals to naive CD4(+) T cells. Whether these signals are similarly required during effector cell expansion and what molecular pathways link locally produced complement to T-cell survival were not clarified. To address this,we stimulated monoclonal and polyclonal T cells in vitro and in vivo with antigen-presenting cells (APCs) deficient in the complement regulatory protein,decay accelerating factor (DAF),and/or the complement component C3. We found that T-cell expansion induced by DAF-deficient APCs was augmented with diminished T-cell apoptosis,whereas T-cell expansion induced by C3(-/-) APCs was reduced because of enhanced T-cell apoptosis. These effects were traced to locally produced C5a,which through binding to T cell-expressed C5aR,enhanced expression of Bcl-2 and prevented Fas up-regulation. The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion,as well as to maintain naive cell viability,and does so by suppressing programmed cell death. View Publication

Costimulatory signals are critical to T cell activation,but how their effects are mediated remains incompletely characterized. Here,we demonstrate that locally produced C5a and C3a anaphylatoxins interacting with their G protein-coupled receptors (GPCRs),C5aR and C3aR,on APCs and T cells both upstream and downstream of CD28 and CD40L signaling are integrally involved in T cell proliferation and differentiation. Disabling these interactions reduced MHC class II and costimulatory-molecule expression and dramatically diminished T cell responses. Importantly,impaired T cell activation by Cd80-/-Cd86-/- and Cd40-/- APCs was reconstituted by added C5a or C3a. C5aR and C3aR mediated their effects via PI-3 kinase-gamma-dependent AKT phosphorylation,providing a link between GPCR signaling,CD28 costimulation,and T cell survival. These local paracrine and autocrine interactions thus operate constitutively in naive T cells to maintain viability,and their amplification by cognate APC partners thus is critical to T cell costimulation. View Publication

Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation,which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time,attesting to a selective advantage of transduced progenitors. Notably,a high transgene copy number was detected in all lymphoid organs,and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether,these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases. 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

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and remains a major cause of mortality in children with recurrent disease and in adults. Despite observed graft-versus-leukemia effects after stem cell transplantation,successful immune therapies for ALL have proven elusive. We previously reported immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG ODN) enhance allogeneic T(h)1 responses and reduce leukemic burden of primary human ALL xenografts. To further the development of CpG ODN as a novel ALL therapy,we investigated the antileukemia activity induced by CpG ODN in a transplantable syngeneic pre-B ALL model. CpG ODN induced early killing of leukemia by innate immune effectors both in vitro and in vivo. Mice were treated with CpG ODN starting 7 days after injection with leukemia to mimic a minimal residual disease state and achieved T cell-dependent remissions of more than 6 months. In addition,mice in remission after CpG ODN treatment were protected from leukemia rechallenge,and adoptive transfer of T cells from mice in remission conferred protection against leukemia growth. To our knowledge,this is the first demonstration that CpG ODN induce a durable remission and ongoing immune-mediated protection in ALL,suggesting this treatment may have clinical utility in patients with minimal residual disease. View Publication