技术资料

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

BACKGROUND: Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD),little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming,or whether allogeneic Treg are safe to use clinically. METHODS: To address these questions,we used an in vitro human skin explant GVHD model,which mimics the physiopathology of GVHD. First,donor"-derived CD8 T cells were stimulated with human leukocyte antigen-unmatched "recipient" dendritic cells (priming phase)� View Publication

Natural killer T (NKT) cells are innate-like lymphocytes that recognize lipid antigens and have been shown to enhance B-cell activation and antibody production. B cells typically recruit T-cell help by presenting internalized antigens recognized by their surface antigen receptor. Here,we demonstrate a highly efficient means whereby human B cells present lipid antigens to NKT cells,capturing the antigen using apolipoprotein E (apoE) and the low-density lipoprotein receptor (LDL-R). ApoE dramatically enhances B-cell presentation of alpha-galactosylceramide (alphaGalCer),an exogenous CD1d presented antigen,inducing activation of NKT cells and the subsequent activation of B cells. B cells express the LDL-R on activation,and the activation of NKT cells by B cells is completely LDL-R dependent,as shown by blocking experiments and the complete lack of presentation when using apoE2,an isoform of apoE incapable of LDL-R binding. The dependence on apoE and the LDL-R is much more pronounced in B cells than we had previously seen in dendritic cells,which can apparently use alternate pathways of lipid antigen uptake. Thus,B cells use an apolipoprotein-mediated pathway of lipid antigen presentation,which constitutes a form of innate help for B cells by NKT cells. View Publication

Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds,we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia,myeloma,and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore,oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition,oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models,thereby establishing its ability to target leukemia stem cells. Mechanistically,CPX bound intracellular iron,and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance,CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus,in summary,CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore,CPX could be rapidly repurposed for the treatment of malignancies,including leukemia and myeloma. 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

Although natural Abs (NAbs) are present from birth,little is known about what drives their selection and whether they have housekeeping functions. The prototypic T15-NAb,first identified because of its protective role in infection,is representative of a special type of NAb response that specifically recognizes and forms complexes with apoptotic cells and which promotes cell-corpse engulfment by phagocytes. We now show that this T15-NAb IgM-mediated clearance process is dependent on the recruitment of C1q and mannose-binding lectin,which have known immune modulatory activities that also provide eat me" signals for enhancing phagocytosis. Further investigation revealed that the addition of T15-NAb significantly suppressed in vitro LPS-induced TNF-alpha and IL-6 secretion by the macrophage-like cell line� 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

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

In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication

The transcription factor T-bet (Tbx21) is critical for Th1 polarization of CD4(+) T cells. Genetic deletion of Tbx21 can cause either exacerbation or attenuation of different autoimmune diseases in animal models. In the nonobese diabetic (NOD) mouse,genetic deletion of the Ifng or the Il12b (IL-12p40) genes,which are both critical Th1 cytokines,does not reduce the incidence of autoimmune diabetes. These results suggest that autoimmune diabetes in the NOD may not be a Th1-driven disease. However,we report that Tbx21 deficiency in the NOD mouse completely blocks insulitis and diabetes due to defects both in the initiation of the anti-islet immune response and in the function of CD4(+) effector T cells. We find defective priming of naive islet-reactive T cells by the innate immune system in Tbx21(-/-) animals. By contrast to naive cells,activated islet-reactive BDC2.5 TCR-transgenic T cells do not require Tbx21 in recipient animals for efficient adoptive transfer of diabetes. However,when these BDC2.5 TCR-transgenic effector cells lack Tbx21,they are less effective at entering the pancreas and promoting diabetes than Tbx21(+/+) cells. Tbx21(-/-) regulatory T cells function normally in vitro and diabetes can be restored in Tbx21(-/-) mice by reducing regulatory T cell numbers. Thus,the absence of diabetes in the NOD.Tbx21(-/-) is due to intrinsic defects in both T cells and cells of the innate immune system paired with the relative preservation of regulatory T cell function. View Publication

Cytoplasmic APOBEC3G has been reported to block wild-type human immunodeficiency virus type 1 (HIV-1) infection in some primary cells. It is not known whether cytoplasmic APOBEC3G has residual activity in activated T cells,even though virion-packaged APOBEC3G does restrict HIV-1 in activated T cells. Because we found that APOBEC3G expression is greater in activated CD4(+) T-helper type 1 (Th1) lymphocytes than in T-helper type 2 (Th2) lymphocytes,we hypothesized that residual target cell restriction of incoming Vif-positive virions that lack APOBEC3G,if present,would be greater in Th1 than Th2 lymphocytes. Infection of activated Th1 cells with APOBEC3-negative virions did result in decreased amounts of early and late reverse transcription products and integrated virus relative to infection of activated Th2 cells. Two-long terminal repeat (2-LTR) circles,which are formed in the nucleus when reverse transcripts do not integrate,were increased after APOBEC3-negative virus infection of activated Th1 cells relative to infection of activated Th2 cells. In contrast,2-LTR circle forms were decreased after infection of APOBEC3G-negative cells with APOBEC3G-containing virions relative to APOBEC3G-negative virions and with Th1 cell-produced virions relative to Th2 cell-produced virions. Increasing APOBEC3G in Th2 cells and decreasing APOBEC3G in Th1 cells modulated the target cell phenotypes,indicating causation by APOBEC3G. The comparison between activated Th1 and Th2 cells indicates that cytoplasmic APOBEC3G in activated Th1 cells partially restricts reverse transcription and integration of incoming Vif-positive,APOBEC3G-negative HIV-1. The differing effects of cytoplasmic and virion-packaged APOBEC3G on 2-LTR circle formation indicate a difference in their antiviral mechanisms. 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