搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

During persistent murine cytomegalovirus (MCMV) infection,the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells,which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation,MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead,we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported,at least in part,by memory T cells primed early in infection,as well as by recruitment of naive T cells at late times. Thus,these data show that memory inflation is maintained by a continuous replacement of short-lived,functional cells during chronic MCMV infection. View Publication

Targeting of myeloid-dendritic cell receptor DC-SIGN by numerous chronic infectious agents,including Porphyromonas gingivalis,is shown to drive-differentiation of monocytes into dysfunctional mDCs. These mDCs exhibit alterations of their fine-tuned homeostatic function and contribute to dysregulated immune-responses. Here,we utilize P. gingivalis mutant strains to show that pathogen-differentiated mDCs from primary human-monocytes display anti-apoptotic profile,exhibited by elevated phosphorylated-Foxo1,phosphorylated-Akt1,and decreased Bim-expression. This results in an overall inhibition of DC-apoptosis. Direct stimulation of complex component CD40 on DCs leads to activation of Akt1,suggesting CD40 involvement in anti-apoptotic effects observed. Further,these DCs drove dampened CD8(+) T-cell and Th1/Th17 effector-responses while inducing CD25(+)Foxp3(+)CD127(-) Tregs. In vitro Treg induction was mediated by DC expression of indoleamine 2,3-dioxygenase,and was confirmed in IDO-KO mouse model. Pathogen-infected &CMFDA-labeled MoDCs long-lasting survival was confirmed in a huMoDC reconstituted humanized mice. In conclusion,our data implicate PDDCs as an important target for resolution of chronic infection. 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

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here,we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen,while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner,while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells,a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack. View Publication

Wild-type (WT) NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) when given 0.05% NaI in their drinking water,whereas B cell-deficient NOD.H-2h4 mice are SAT resistant. To test the hypothesis that resistance of B cell-deficient mice to SAT was due to the activity of regulatory CD4+CD25+ T (T reg) cells activated if autoantigen was initially presented on non-B cells,CD25+ T reg cells were transiently depleted in vivo using anti-CD25. B cell-deficient NOD.H-2h4 mice given three weekly injections of anti-CD25 developed SAT 8 wk after NaI water. Thyroid lesions were similar to those in WT mice except there were no B cells in thyroid infiltrates. WT and B cell-deficient mice had similar numbers of CD4+CD25+Foxp3+ cells. Mice with transgenic nitrophenyl-specific B cells unable to secrete immunoglobulin were also resistant to SAT,and transient depletion of T reg cells resulted in severe SAT with both T and B cells in thyroid infiltrates. T reg cells that inhibit SAT were eliminated by day 3 thymectomy,indicating they belong to the subset of naturally occurring T reg cells. However,T reg cell depletion did not increase SAT severity in WT mice,suggesting that T reg cells may be nonfunctional when effector T cells are activated; i.e.,by autoantigen-presenting B cells. View Publication

Abnormal activation of B lymphocytes is a feature commonly seen in human immunodeficiency virus type 1 (HIV-1)-infected persons. However,the mechanism(s) responsible for this dysfunction is still poorly understood. Having recently shown that CD40L,the ligand for CD40,is inserted within emerging HIV-1 particles,we hypothesized that the contact between virus-anchored host CD40L and CD40 on the surface of B lymphocytes might result in the activation of this cell type. We report here that CD40L-bearing viruses,but not isogenic virions lacking host-derived CD40L,can induce immunoglobulin G and interleukin-6 production. Furthermore,such viral entities were found to induce B-cell homotypic adhesion. These effects were paralleled at the intracellular level by the nuclear translocation of the ubiquitous transcription factor NF-kappaB. The presence of host-derived CD40L within virions resulted in an increased virus attachment to B cells and a more-efficient B-cell-mediated transfer of HIV-1 to autologous CD4(+) T lymphocytes. All the above processes were independent of the virus-encoded envelope glycoproteins. Altogether,the data gathered from this series of investigations suggest that the incorporation of host-encoded CD40L in HIV-1 is likely to play a role in the B-cell abnormalities that are seen in infected individuals. View Publication

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity,we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo,activated and memory B cells expressed lower levels of CD1d compared with resting,naive,and marginal zone-like B cells. In vitro,CD1d was downregulated by all forms of B cell activation,leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone,whereas activation via the BCR significantly upregulated CD1c,particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes,an effect that was reversed by RARα agonists. However,BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells,in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity. View Publication

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease,expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis,little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture,resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell,in characterizing the effect of this oncogene on human stem cell biology,and in defining its contribution to the development of leukemia. View Publication

PURPOSE: B-cell receptor signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However,blocking B-cell receptor signaling with dasatinib,an inhibitor of SRC kinase,produced variable results in preclinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. EXPERIMENTAL DESIGN: Fresh CLL B cells were treated with dasatinib,and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of B-cell receptor signaling molecules,as well as with molecular and cytogenetic prognostic factors. RESULTS: Among 50 CLL cases,dasatinib treatment reduced cell viability by 2% to 90%,with an average reduction of 47% on day 4 of culture. The drug induced CLL cell death through the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly,phosphorylation of SRC family kinases was inhibited by dasatinib in good,as well as poor,responders. As opposed to SRC family kinases,activities of two downstream molecules,SYK and phospholipase Cgamma2,correlate well with the apoptotic response of CLL cells to dasatinib. CONCLUSIONS: Thus,SYK inhibition predicts cellular response to dasatinib. SYK,together with phospholipase Cgamma2,may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective,our study suggests the existence of alternative mechanisms or pathways that activate SYK,independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment. 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

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells,including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM,we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients,we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However,we discovered,next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC,a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients),as well as CDC (56 patients). Similarly,experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly,all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients. View Publication

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However,we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study,25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not have any effect on the T cell FCXM reactivity. Pretreatment with reducing agents did not affect the T cell FCXM reactivity. 15 of 21 HIV-infected kidney allograft recipients with pronase-treated T cell FCXM reactivity display long-term graft survival (1193±631days). These data indicate that HIV-infected patients have nondeleterious autoantibodies recognizing cryptic epitopes exposed by pronase on T cells. View Publication