技术资料

Bone marrow-derived mesenchymal stromal cells (MSC) possess an immune plasticity manifested by either an immunosuppressive or,when activated with IFN-gamma,an APC phenotype. Herein,TLR expression by MSC and their immune regulatory role were investigated. We observed that human MSC and macrophages expressed TLR3 and TLR4 at comparable levels and TLR-mediated activation of MSC resulted in the production of inflammatory mediators such as IL-1beta,IL-6,IL-8/CXCL8,and CCL5. IFN-alpha or IFN-gamma priming up-regulated production of these inflammatory mediators and expression of IFNB,inducible NO synthase (iNOS),and TRAIL upon TLR activation in MSC and macrophages,but failed to induce IL-12 and TNF-alpha production in MSC. Nonetheless,TLR activation in MSC resulted in the formation of an inflammatory site attracting innate immune cells,as evaluated by human neutrophil chemotaxis assays and by the analysis of immune effectors retrieved from Matrigel-embedded MSC injected into mice after in vitro preactivation with cytokines and/or TLR ligands. Hence,TLR-activated MSC are capable of recruiting immune inflammatory cells. In addition,IFN priming combined with TLR activation may increase immune responses induced by Ag-presenting MSC through presentation of Ag in an inflammatory context,a mechanism that could be applied in a cell-based vaccine. View Publication

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases,although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology,we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus. View Publication

BACKGROUND: Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step,using a commercially available reagent designed for purifying cells from whole blood (RosetteSep). This technique relies on the presence of the large excess of red blood cells in whole blood for the formation of immunorosettes,whereas single cell suspensions from tonsils contain relatively few red blood cells. RESULTS: B cell enrichment from tonsils was achieved using RosetteSep with no modification to the whole blood procedure; however,the degree of purity depended on the extent of red blood cell contamination of the starting tonsil cell suspension. Addition of a 50-fold excess of allogeneic human red blood cells,but not sheep red blood cells,reproducibly resulted in high levels of purity. Depletion of mononuclear cells from the donor red blood cells eliminated potential contamination with allogeneic B cells. CONCLUSION: RosetteSep reagent can be used in combination with allogeneic human red blood cells to reproducibly isolate tonsil B lymphocytes to high levels of purity with no change in phenotype or loss of cells. This method provides considerable time and cost savings compared to other methods. View Publication

Immune dysfunction develops in patients with many cancer types and may contribute to tumor progression and failure of immunotherapy. Mechanisms underlying cancer-associated immune dysfunction are not fully understood. Efficient IFN signaling is critical to lymphocyte function; animals rendered deficient in IFN signaling develop cancer at higher rates. We hypothesized that altered IFN signaling may be a key mechanism of immune dysfunction common to cancer. To address this,we assessed the functional responses to IFN in peripheral blood lymphocytes from patients with 3 major cancers: breast cancer,melanoma,and gastrointestinal cancer. Type-I IFN (IFN-alpha)-induced signaling was reduced in T cells and B cells from all 3 cancer-patient groups compared to healthy controls. Type-II IFN (IFN-gamma)-induced signaling was reduced in B cells from all 3 cancer patient groups,but not in T cells or natural killer cells. Impaired-IFN signaling was equally evident in stage II,III,and IV breast cancer patients,and downstream functional defects in T cell activation were identified. Taken together,these findings indicate that defects in lymphocyte IFN signaling arise in patients with breast cancer,melanoma,and gastrointestinal cancer,and these defects may represent a common cancer-associated mechanism of immune dysfunction. View Publication

CD4+ and CD8+ T cell functions are rapidly aborted during chronic infection,preventing viral clearance. CD4+ T cell help is required throughout chronic infection so as to sustain CD8+ T cell responses; however,the necessary factor(s) provided by CD4+ T cells are currently unknown. Using a mouse model of chronic viral infection,we demonstrated that interleukin-21 (IL-21) is an essential component of CD4+ T cell help. In the absence of IL-21 signaling,despite elevated CD4+ T cell responses,CD8+ T cell responses are severely impaired. CD8+ T cells directly require IL-21 to avoid deletion,maintain immunity,and resolve persistent infection. Thus,IL-21 specifically sustains CD8+ T cell effector activity and provides a mechanism of CD4+ T cell help during chronic viral infection. View Publication

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G,which target HIV-1 capsid and viral infectivity factor (Vif),respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV),including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs),the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs,the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques,the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus,15 to 30% versus 1 to 5%; second rhesus,7 to 15% versus 0.5 to 2%,respectively) 3 to 7 months postinfusion. In summary,we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application. View Publication

CD8(+) T lymphocytes play a key role in host defense,in particular against important persistent viruses,although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this,we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation,including cytokines (e.g.,IL-17,IL-22),transcription factors (e.g.,retinoic acid-related orphan receptor gamma-t,P = 6 x 10(-9); RUNX2,P = 0.004),cytokine receptors (e.g.,IL-23R,P = 2 x 10(-7); IL-18 receptor,P = 4 x 10(-6)),and chemokine receptors (e.g.,CCR6,P = 3 x 10(-8); CXCR6,P = 3 x 10(-7); CCR2,P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man. View Publication

The activating receptor NKG2D,expressed by natural killer (NK) cells and CD8(+) T cells,has a role in the specific killing of transformed cells. We examined NKG2D expression in patients with glioblastoma multiforme and found that NKG2D was downregulated on NK cells and CD8(+) T cells. Expression of NKG2D on lymphocytes significantly increased following tumor resection and correlated with an increased ability to kill NKG2D ligand-positive tumor targets. Despite the presence of soluble NKG2D ligands in the sera of glioblastoma patients,NKG2D downregulation was primarily caused by tumor-derived tumor growth factor-beta,suggesting that blocking of this cytokine may have therapeutic benefit. View Publication

The ability of NK cells to directly recognize pathogens and be activated via Toll-like receptors (TLR) is increasingly recognized. Nevertheless,controversial results on the NK cell ability to be directly activated by lipopolysaccharide (LPS),the ligand of TLR4,have been recently reported. To start elucidating the reasons explaining the contrasting observations of the literature,we focused on the potential role of currently used NK cell purification procedures to condition putative NK cell responsiveness to LPS. To do so,human NK cells were isolated by negative selection,using three different commercial kits,to be comparatively evaluated for the production of IFNgamma in response to ultra-pure LPS and/or IL-2. Despite the lack of surface TLR4,we found that two out of the three NK cell-enriched populations released IFNgamma (and one of the two,IL-12p70 as well) in response to the LPS plus IL-2 combination,whereas the last one did not. However,the two LPS plus IL-2-responsive NK cell populations were found variably contaminated with 6-sulfo LacNAc(+) dendritic cells (slanDC),demonstrated responsible for triggering,via the production of IL-12p70 in response to LPS,the release of IFNgamma by IL-2-stimulated NK cells. Accordingly,slanDC depletion completely abrogated the capacity to produce both IL-12p70 and IFNgamma in response to LPS plus IL-2 by slanDC-containing NK cells. Taken together,our data uncover that two commercially available kits,specifically designed to isolate NK cells by negative selection,also co-purify variable amounts of slanDC. The latter cells may dramatically affect the outcome of experiments carried on to evaluate NK cell responsiveness to TLR agonists such as LPS. View Publication

RATIONALE: Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However,the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated. OBJECTIVES: We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells. METHODS: The study was performed in a cohort of 21 subjects with allergic asthma,21 healthy control subjects,and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells. MEASUREMENTS AND MAIN RESULTS: Dendritic cells cultured with natural regulatory T cells up-regulated IL-10,down-regulated costimulatory molecules,and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore,this defective function of natural regulatory T cells was associated with their decreased IL-10 expression,disease severity,and could be reversed by oral corticosteroid therapy. CONCLUSIONS: These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma. View Publication

Because IL-1beta plays an important role in inflammation in human and murine arthritis,we investigated the contribution of the inflammasome components ASC,NALP-3,IPAF,and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA,decreased levels of synovial IL-1beta,and diminished serum amyloid A levels. In contrast,mice deficient in NALP-3,IPAF,or caspase-1 did not show any alteration of joint inflammation,thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes,we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro,as was the production of IFN-gamma,whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice,but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion,these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation. View Publication

The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T-cell responses has been associated with the immunological control of HIV-1 replication; however,the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4(+) T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells,each of the individuals in the present study exhibited progressive disease,with one individual showing rapid progression. In this rapid progressor,three IL-2-producing HIV-1 Gag-specific CD4(+) T-cell responses were identified and mapped to the following optimal epitopes: HIVWASRELER,REPRGSDIAGT,and FRDYVDRFYKT. Responses to these epitopes in peripheral blood mononuclear cells were monitored longitudinally to textgreater1 year postinfection,and contemporaneous circulating plasma viruses were sequenced. A variant of the FRDYVDRFYKT epitope sequence,FRDYVDQFYKT,was observed in 1/21 plasma viruses sequenced at 5 months postinfection and 1/10 viruses at 7 months postinfection. This variant failed to stimulate the corresponding CD4(+) T-cell clone and thus constitutes an escape mutant. Responses to each of the three Gag epitopes were rapidly lost,and this loss was accompanied by a loss of antigen-specific cells in the periphery as measured by using an FRDYVDRFYKT-presenting major histocompatibility complex class II tetramer. Highly active antiretroviral therapy was associated with the reemergence of FRDYVDRFYKT-specific cells by tetramer. Thus,our data support that IL-2-producing HIV-1-specific CD4(+) T-cell responses can exert immune pressure during early HIV-1 infection but that the inability of these responses to enforce enduring control of viral replication is related to the deletion and/or dysfunction of HIV-1-specific CD4(+) T cells rather than to the fixation of escape mutations at high frequencies. View Publication