技术资料

In early HIV-1 infection,Vdelta1 T lymphocytes are increased in peripheral blood and this is related to chemokine receptor expression,chemokine response,and recirculation. Herein we show that,at variance with healthy donors,in HIV-1-infected patients ex vivo-isolated Vdelta1 T cells display cytoplasmic interferon-gamma (IFN-gamma). Interestingly,these cells coexpress cytoplasmic interleukin-17 (IL-17),and bear the CD27 surface marker of the memory T-cell subset. Vdelta1 T cells,isolated from either patients or healthy donors,can proliferate and produce IFN-gamma and IL-17 in response to Candida albicans in vitro,whereas Vdelta2 T cells respond with proliferation and IFN-gamma/IL-17 production to mycobacterial or phosphate antigens. These IFN-gamma/IL-17 double-producer gammadelta T cells express the Th17 RORC and the Th1 TXB21 transcription factors and bear the CCR7 homing receptor and the CD161 molecule that are involved in gammadelta T-cell transendothelial migration. Moreover,Vdelta1 T cells responding to C albicans express the chemokine receptors CCR4 and CCR6. This specifically equipped circulating memory gammadelta T-cell population might play an important role in the control of HIV-1 spreading and in the defense against opportunistic infections,possibly contributing to compensate for the impairment of CD4(+) T cells. View Publication

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971,rs17615,rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls,P=0.016,OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10,directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC),and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact. View Publication

Mutations in the RUNX1 gene are found at high frequencies in minimally differentiated acute myelogenous leukemia. In addition to null mutations,many of the mutations generate Runx1 DNA-binding (RDB) mutants. To determine if these mutants antagonize wild-type protein activity,cDNAs were transduced into murine bone marrow or human cord blood cells using retroviral vectors. Significantly,the RDB mutants did not act in a transdominant fashion in vivo to disrupt Runx1 activity in either T-cell or platelet development,which are highly sensitive to Runx1 dosage. However,RDB mutant expression impaired expansion and differentiation of the erythroid compartment in which Runx1 expression is normally down-regulated,showing that a RDB-independent function is incompatible with erythroid differentiation. Significantly,both bone marrow progenitors expressing RDB mutants or deficient for Runx1 showed increased replating efficiencies in vitro,accompanied by the accumulation of myeloblasts and dysplastic progenitors,but the effect was more pronounced in RDB cultures. Disruption of the interface that binds CBFbeta,an important cofactor of Runx1,did not impair RDB mutant replating activity,arguing against inactivation of Runx1 function by CBFbeta sequestration. We propose that RDB mutants antagonize Runx1 function in early progenitors by disrupting a critical balance between DNA-binding-independent and DNA-binding-dependent signaling. View Publication

Macrophages have a central role in the pathogenesis of cryptococcosis since they are an important line of defense,serve as a site for fungal replication,and also can contribute to tissue damage. The objective of this study was to investigate the interaction of macrophages with cells from smooth-colony variants (SM) and mucoid-colony variants (MC) arising from phenotypic switching of Cryptococcus neoformans. Alveolar macrophages (AMs) isolated from SM- and MC-infected mice exhibited differences in gene and surface expression of PD-L1,PD-L2,and major histocompatibility class II (MHC-II). PD-L1 and PD-L2 are the ligands for PD1 and are differentially regulated in Th1- and Th2-type cells. In addition,macrophage activation in SM- and MC-infected mice was characterized as alternatively activated. Flow cytometric and cytokine analysis demonstrated that MC infection was associated with the emergence of Th17 cells and higher levels of interleukin-17 (IL-17) in lung tissue,which were reduced by AM depletion. In conclusion,our results indicate that macrophages play a significant role in maintaining damage-promoting inflammation in the lung during MC infection,which ultimately results in death. View Publication

Coinfection of HIV-1 patients with Plasmodium falciparum,the etiological agent of malaria,results in a raise of viral load and an acceleration of disease progression. The primary objective of this study was to investigate whether the malarial pigment hemozoin (HZ),a heme by-product of hemoglobin digestion by malaria parasites,can affect HIV-1 transmission by monocytes-derived dendritic cells (DCs) to CD4(+) T cells when HZ is initially internalized in monocytes before their differentiation in DCs. We demonstrate in this study that HZ treatment during the differentiation process induces an intermediate maturation phenotype when compared with immature and fully mature DCs. Furthermore,the DC-mediated transfer of HIV-1 is enhanced in presence of HZ,a phenomenon that may be linked with the capacity of HZ-loaded cells to interact and activate CD4(+) T cells. Altogether our findings suggest a new mechanism that could partially explain the increased HIV-1 virus production during a coinfection with P. falciparum. Understanding the multifaceted interactions between P. falciparum and HIV-1 is an important challenge that could lead to the development of new treatment strategies. View Publication

The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-lambda1 (interleukin-29 [IL-29]),IFN-lambda2 (IL-28A),and IFN-lambda3 (IL-28B),each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-lambda producers among peripheral lymphocytes. Recently,it has been shown that IFN-lambda1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4(+) T cells and inhibiting the production of Th2-associated cytokines. Here,we asked whether Th2 cytokines exert reciprocal control over IFN-lambda production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-lambda1 transcription and secretion. However,pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments,we showed that IL-4-responsive monocytes are an intermediary cell,responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-lambda1 output. Thus,our experiments revealed a novel mechanism for regulation of both IFN-lambda1 production and pDC function,and suggests an expanded immunomodulatory role for Th2-associated cytokines. View Publication

Tumors that recur following surgical resection of melanoma are typically metastatic and associated with poor prognosis. Using the murine B16F10 melanoma and a robust antimelanoma vaccine,we evaluated immunization as a tool to improve tumor-free survival following surgery. We investigated the utility of vaccination in both neoadjuvant and adjuvant settings. Surprisingly,neoadjuvant vaccination was far superior and provided approximately 100% protection against tumor relapse. Neoadjuvant vaccination was associated with enhanced frequencies of tumor-specific T cells within the tumor and the tumor-draining lymph nodes following resection. We also observed increased infiltration of antigen-specific T cells into the area of surgery. This method should be amenable to any vaccine platform and can be readily extended to the clinic. View Publication

Many cellular responses,such as autoimmunity and cytotoxicity,are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here,we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk,concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore,Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity,providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex,observed here with two types of inhibitory receptors,expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism. View Publication

Recent studies have shown that NK-dendritic cell (DC) interaction plays an important role in the induction of immune response against tumors and certain viruses. Although the effect of this interaction is bidirectional,the mechanism or molecules involved in this cross-talk have not been identified. In this study,we report that coculture with NK cells causes several fold increase in IL-12 production by Toxoplasma gondii lysate Ag-pulsed DC. This interaction also leads to stronger priming of Ag-specific CD8+ T cell response by these cells. In vitro blockade of NKG2D,a molecule present on human and murine NK cells,neutralizes the NK cell-induced up-regulation of DC response. Moreover,treatment of infected animals with Ab to NKG2D receptor compromises the development of Ag-specific CD8+ T cell immunity and reduces their ability to clear parasites. These studies emphasize the critical role played by NKG2D in the NK-DC interaction,which apparently is important for the generation of robust CD8+ T cell immunity against intracellular pathogens. To the best of our knowledge,this is the first work that describes in vivo importance of NKG2D during natural infection. View Publication

NOD.B10-H2(b) and NOD/LtJ mice manifest,respectively,many features of primary and secondary Sjögren's syndrome (SjS),an autoimmune disease affecting primarily the salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). B lymphocytes play a central role in the onset of SjS with clinical manifestations dependent on the appearance of autoantibodies reactive to multiple components of acinar cells. Previous studies with NOD.IL4(-/-) and NOD.B10-H2(b).IL4(-/-) mice suggest that the Th2 cytokine,IL-4,plays a vital role in the development and onset of SjS-like disease in the NOD mouse model. To investigate the molecular mechanisms by which IL-4 controls SjS development,a Stat6 gene knockout mouse,NOD.B10-H2(b).C-Stat6(-/-),was constructed and its disease profile was defined and compared with that of NOD.B10-H2(b).C-Stat6(+/+) mice. As the NOD.B10-H2(b).C-Stat6(-/-) mice aged from 4 to 24 wk,they exhibited leukocyte infiltration of the exocrine glands,produced anti-nuclear autoantibodies,and showed loss and gain of saliva-associated proteolytic enzymes,similar to NOD.B10-H2(b).C-Stat6(+/+) mice. In contrast,NOD.B10-H2(b).C-Stat6(-/-) mice failed to develop glandular dysfunction,maintaining normal saliva flow rates. NOD.B10-H2(b).C-Stat6(-/-) mice were found to lack IgG1 isotype-specific anti-muscarinic acetylcholine type-3 receptor autoantibodies. Furthermore,the IgG fractions from NOD.B10-H2(b).C-Stat6(-/-) sera were unable to induce glandular dysfunction when injected into naive recipient C57BL/6 mice. NOD.B10-H2(b).C-Stat6(-/-) mice,like NOD.B10-H2(b).IL4(-/-) mice,are unable to synthesize IgG1 Abs,an observation that correlates with an inability to develop end-stage clinical SjS-like disease. These data imply a requirement for the IL-4/STAT6-pathway for onset of the clinical phase of SjS-like disease in the NOD mouse model. View Publication

Aberrant T cell responses during T cell activation and immunological synapse (IS) formation have been described in systemic lupus erythematosus (SLE). Kv1.3 potassium channels are expressed in T cells where they compartmentalize at the IS and play a key role in T cell activation by modulating Ca(2+) influx. Although Kv1.3 channels have such an important role in T cell function,their potential involvement in the etiology and progression of SLE remains unknown. This study compares the K channel phenotype and the dynamics of Kv1.3 compartmentalization in the IS of normal and SLE human T cells. IS formation was induced by 1-30 min exposure to either anti-CD3/CD28 Ab-coated beads or EBV-infected B cells. We found that although the level of Kv1.3 channel expression and their activity in SLE T cells is similar to normal resting T cells,the kinetics of Kv1.3 compartmentalization in the IS are markedly different. In healthy resting T cells,Kv1.3 channels are progressively recruited and maintained in the IS for at least 30 min from synapse formation. In contrast,SLE,but not rheumatoid arthritis,T cells show faster kinetics with maximum Kv1.3 recruitment at 1 min and movement out of the IS by 15 min after activation. These kinetics resemble preactivated healthy T cells,but the K channel phenotype of SLE T cells is identical to resting T cells,where Kv1.3 constitutes the dominant K conductance. The defective temporal and spatial Kv1.3 distribution that we observed may contribute to the abnormal functions of SLE T cells. 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