技术资料

NLRC5 is a transcriptional regulator of MHC class I (MHCI),which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK-T-cell crosstalk,raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly,NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice,we show that NK cells surprisingly break tolerance even towards 'self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore,during chronic LCMV infection,the total CD8(+) T-cell population is severely decreased in these mice,a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets,having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions. View Publication

Plant lectins displaying similar single sugar-binding specificity and identical molecular structure might present various biological effects. To explore this possibility,the effects on human lymphocytes of two mannose-specific and structurally closely related lectins,Morniga M from Morus nigra and artocarpin from Artocarpus integrifolia were investigated. In silico analysis revealed that Morniga M presents a more largely open carbohydrate-binding cavity than artocarpin,probably allowing interactions with a broader spectrum of carbohydrate moieties. In vitro,Morniga M interacted strongly with the lymphocyte surface and was uptaken quickly by cells. Morniga M and artocarpin triggered the proliferation and activation of human T and NK lymphocytes. A minority of B lymphocytes was activated in artocarpin-treated culture,whereas Morniga M favored the emergence of CD4+ CD8+ T lymphocytes. Moreover,cell death occurred in activated PBMC,activated T lymphocytes,and Jurkat T leukemia cells incubated with Morniga M only. The biological effects of both lectins were dependent on carbohydrate recognition. The Morniga M-induced cell death resulted,at least in part,from caspase-dependent apoptosis and FADD-dependent receptor-mediated cell death. Finally,Morniga M,but not artocarpin,triggered AICD of T lymphocytes. In conclusion,both lectins trigger lymphocyte activation,but only Morniga M induces cell death. In spite of similar in vitro mannose-binding specificities and virtually identical structure,only Morniga M probably interacts with carbohydrate moieties bound to molecules able to induce cell death. The present data suggest that subtle alterations in N-glycans can distinguish activation and cell death molecules at the lymphocyte surface. View Publication

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor,including ULBP-1,-2,and -3,but not MICA or MICB,in infected cells both in vitro and in vivo. However,the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G(2) cell-cycle arrest,conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4(+) T lymphocytes by a process that is Vpr dependent. Importantly,Vpr enhanced the susceptibility of HIV-1-infected cells to NK cell-mediated killing. Strikingly,Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell-mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells,suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall,these results indicate that Vpr is a key determinant responsible for HIV-1-induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced CD4(+) T-lymphocyte depletion but may also take part in HIV-1-induced NK-cell dysfunction. View Publication

Chemokine receptor CX3CR1(+) dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103(+) DCs in the mucosa,which,through their ability to synthesize retinoic acid (RA),appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103(+) DC,CX3CR1(+) cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103(+) DCs,but not CX3CR1-expressing cells,migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover,CX3CR1(+) cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103(+) DC. These findings indicate that selectively CD103(+) DCs serve classical DC functions and initiate adaptive immune responses in local LNs,whereas CX3CR1(+) populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens. View Publication

Tandem pleckstrin homology domain proteins (TAPPs) are recruited to the plasma membrane via binding to phosphoinositides produced by phosphoinositide 3-kinases (PI3Ks). Whereas PI3Ks are critical for B-cell activation,the functions of TAPP proteins in B cells are unknown. We have identified 40 potential interaction partners of TAPP2 in B cells,including proteins involved in cytoskeletal rearrangement,signal transduction and endocytic trafficking. The association of TAPP2 with the cytoskeletal proteins utrophin and syntrophin was confirmed by Western blotting. We found that TAPP2,syntrophin,and utrophin are coexpressed in normal human B cells and B-chronic lymphocytic leukemia (B-CLL) cells. TAPP2 and syntrophin expression in B-CLL was variable from patient to patient,with significantly higher expression in the more aggressive disease subset identified by zeta-chain-associated protein kinase of 70 kDa (ZAP70) expression and unmutated immunoglobulin heavy chain (IgH) genes. We examined whether TAPP can regulate cell adhesion,a known function of utrophin/syntrophin in other cell types. Expression of membrane-targeted TAPP2 enhanced B-cell adhesion to fibronectin and laminin,whereas PH domain-mutant TAPP2 inhibited adhesion. siRNA knockdown of TAPP2 or utrophin,or treatment with PI3K inhibitors,significantly inhibited adhesion. These findings identify TAPP2 as a novel link between PI3K signaling and the cytoskeleton with potential relevance for leukemia progression. View Publication

IFN-beta-1a has been used over the past 15 years as a primary therapy for relapsing-remitting multiple sclerosis (MS). However,the immunomodulatory mechanisms that provide a therapeutic effect against this CNS inflammatory disease are not yet completely elucidated. The effect of IFN-beta-1a on Th17 cells,which play a critical role in the development of the autoimmune response,has not been extensively studied in humans. We have investigated the effect of IFN-beta-1a on dendritic cells (DCs) and naive CD4(+)CD45RA(+) T cells derived from untreated MS patients and healthy controls in the context of Th17 cell differentiation. We report that IFN-beta-1a treatment down-regulated the expression of IL-1beta and IL-23p19 in DCs,whereas it induced the gene expression of IL-12p35 and IL-27p28. We propose that IFN-beta-1a-mediated up-regulation of the suppressor of cytokine signaling 3 expression,induced via STAT3 phosphorylation,mediates IL-1beta and IL-23 down-regulation,while IFN-beta-1a-induced STAT1 phosphorylation induces IL-27p28 expression. CD4(+)CD45RA(+) naive T cells cocultured with supernatants from IFN-beta-1a-treated DCs exhibited decreased gene expression of the Th17 cell markers retinoic acid-related orphan nuclear hormone receptor c (RORc),IL-17A,and IL-23R. A direct IFN-beta-1a treatment of CD45RA(+) T cells cultured in Th17-polarizing conditions also down-regulated RORc,IL-17A,and IL-23R,but up-regulated IL-10 gene expression. Studies of the mechanisms involved in the Th17 cell differentiation suggest that IFN-beta-1a inhibits IL-17 and induces IL-10 secretion via activated STAT1 and STAT3,respectively. IFN-beta's suppression of Th17 cell differentiation may represent its most relevant mechanism of selective suppression of the autoimmune response in MS. View Publication

Here,we describe a procedure and first-in-man clinical effects of adoptive transfer of ex vivo expanded CD4+CD25+CD127- T regulatory cells (Tregs) in the treatment of graft versus host disease (GvHD). The cells were sorted from buffy coats taken from two family donors,expanded ex vivo and transferred to respective recipients who suffered from either acute or chronic GvHD. The therapy allowed for significant alleviation of the symptoms and reduction of pharmacologic immunosuppression in the case of chronic GvHD,while in the case of grade IV acute GvHD it only transiently improved the condition,for the longest time within all immunosuppressants used nonetheless. View Publication

BACKGROUND AIMS Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products,which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities,there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment,and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS This study compared cryopreservation of PBSC in a novel intracellular-like,fully defined,serum- and protein-free preservation solution,CryoStor (BioLife Solutions Inc.),with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly,human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard,Normosol-R,5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed,and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC),viability,CD34 and granulocytes by flow cytometry,along with colony-forming activity in methylcellulose. RESULTS The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+),colony-forming units (CFU) and granulocytes. Specifically,relative to the FHCRC standard formulation,cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005),a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition,it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins,and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however,these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections,along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO. View Publication

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-gamma secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family,such as NKp46,by ligands expressed on tumour cells. However,it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here,we investigated the early events occurring during T cell-tumour cell interactions,and their impact on NK cell functions. We observed that on co-culture with some melanomas,activated CD4(+) T cells promoted degranulation,and NKG2D- and NKp46-dependent IFN-gamma secretion by NK cells,probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4(+),CD8(+) and resting T cells,which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new,so far,unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity. View Publication

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium,following acute myocardial infarction,may be,in part,due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep,keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells,endothelial progenitor cells,T lymphocytes,B lymphocytes and NK cells using cell surface markers CD34(+),CD133(+)VEGFR2(+),CD45(+)CD3(+),CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+),respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells,granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep. View Publication

The antigen recognition interface formed by T helper precursors (Thps) and antigen-presenting cells (APCs),called the immunological synapse (IS),includes receptors and signaling molecules necessary for Thp activation and differentiation. We have recently shown that recruitment of the interferon-gamma receptor (IFNGR) into the IS correlates with the capacity of Thps to differentiate into Th1 effector cells,an event regulated by signaling through the functionally opposing receptor to interleukin-4 (IL4R). Here,we show that,similar to IFN-gamma ligation,TCR stimuli induce the translocation of signal transducer and activator of transcription 1 (STAT1) to IFNGR1-rich regions of the membrane. Unexpectedly,STAT1 is preferentially expressed,is constitutively serine (727) phosphorylated in Thp,and is recruited to the IS and the nucleus upon TCR signaling. IL4R engagement controls this process by interfering with both STAT1 recruitment and nuclear translocation. We also show that in cells with deficient Th1 or constitutive Th2 differentiation,the IL4R is recruited to the IS. This observation suggest that the IL4R is retained outside the IS,similar to the exclusion of IFNGR from the IS during IL4R signaling. This study provides new mechanistic cues for the regulation of lineage commitment by mutual immobilization of functionally antagonistic membrane receptors. 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