技术资料

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

Precise control of Wnt signaling is necessary for immune system development. In this study,we detected severely impaired development of all lymphoid lineages in mice,resulting from an N-ethyl-N-nitrosourea-induced mutation in the limb region 1-like gene (Lmbr1l),which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain-containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing destruction complex" proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/beta-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation acting as a negative regulator of the Wnt/beta-catenin pathway." 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

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC,the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming,low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo,as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice,but not in high affinity-primed mice. Mechanistically,low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge,low affinity-primed CD45RB(hi) cells became CD45RB(lo),demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus,these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency,low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge. View Publication

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics,we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo,LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3-/- CD4+ T cells,solidifying that LAG-3 controls these processes. Lag3-/- CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation,enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally,enhancement of STAT5 activation,as a result of LAG-3 deficiency,contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However,it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous,endogenously HIV-1-infected CD4+ T cells. Here,we stimulate primary CD4+ T cells,purified ex vivo from HIV-1-infected viremic patients,with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that,subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules,HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells,while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However,the degree of NK cell cytolytic activity against autologous,endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively,our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells. View Publication

The maintenance of homeostasis in the gut is a major challenge for the immune system. Here we demonstrate that the transcription factor MAF plays a central role in T cells for the prevention of gastro-intestinal inflammation. Conditional knock out mice lacking Maf in all T cells developed spontaneous late-onset colitis,correlating with a decrease of FOXP3+RORgammat+ T cells proportion,dampened IL-10 production in the colon and an increase of inflammatory TH17 cells. Strikingly,FOXP3+ specific conditional knock out mice for MAF did not develop colitis and demonstrated normal levels of IL-10 in their colon,despite the incapacity of regulatory T cells lacking MAF to suppress colon inflammation in Rag1-/- mice transferred with na{\{i}}ve CD4+ T cells. We showed that one of the cellular sources of IL-10 in the colon of these mice are TH17 cells. Thus MAF is critically involved in the maintenance of the gut homeostasis by regulating the balance between Treg and TH17 cells either at the level of their differentiation or through the modulation of their functions." View Publication

Upon infection,antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor,the core component of mammalian target of rapamycin complex 2 (mTORC2),regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover,mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation,repression of T-bet,enhanced mitochondrial spare respiratory capacity,and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore,mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions. View Publication

Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However,natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that,on conjugation with CTL,human melanoma cells undergo an active late endosome/lysosome trafficking,which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking,pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance,we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. View Publication