搜索结果: 'gmp t cell activator'

Na{\{i}}ve CD8+ T cells can differentiate into effector (Teff) memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff Tmem and Tex populations remain poorly understood. Here we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2 as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator Btg1 in establishing the Tex population. Finally these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections." View Publication

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein,we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore,the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFN$\gamma$ production,also Ca2+- and CaM-dependent,was instead not reduced in HNSCC T cells,which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity,but not IFN$\gamma$ production,and reduced their chemotaxis in the presence of adenosine,thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally,1-EBIO enhanced INF$\gamma$ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore,they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers. View Publication

Glucocorticoids (GCs) play an important role in controlling acute graft-versus-host disease (aGvHD),a frequent complication of allogeneic hematopoietic stem cell transplantation. The anti-inflammatory activity of GCs is mainly ascribed to the modulation of T cells and macrophages,for which reason a genetically induced GC resistance of either of these cell types causes aggravated aGvHD. Since only a few genes are currently known that are differentially regulated under these conditions,we analyzed the expression of 54 candidate genes in the inflamed small intestine of mice suffering from aGvHD when either allogeneic T cells or host myeloid cells were GC resistant using a microfluidic dynamic array platform for high-throughput quantitative PCR. The majority of genes categorized as cytokines (e.g. Il2,Il6),chemokines (e.g. Ccl2,Cxcl1),cell surface receptors (e.g. Fasl,Ctla4) and intracellular molecules (e.g. Dusp1,Arg1) were upregulated in mice transplanted with GC resistant allogeneic T cells. Moreover,the expression of several genes linked to energy metabolism (e.g. Glut1) was altered. Surprisingly,mice harboring GC resistant myeloid cells showed almost no changes in gene expression despite their fatal disease course after aGvHD induction. To identify additional genes in the inflamed small intestine that were affected by a GC resistance of allogeneic T cells,we performed an RNAseq analysis,which uncovered more than 500 differentially expressed transcripts (e.g. Cxcr6,Glut3,Otc,Aoc1,Il1r1,Sphk1) that were enriched for biological processes associated with inflammation and tissue disassembly. The changes in gene expression could be confirmed during full-blown disease but hardly any of them in the preclinical phase using high-throughput quantitative PCR. Further analysis of some of these genes revealed a highly selective expression pattern in T cells,intestinal epithelial cells and macrophages,which correlated with their regulation during disease progression. Collectively,we identified an altered gene expression profile caused by GC resistance of transplanted allogeneic T cells,which could help to define new targets for aGvHD therapy. View Publication

Background: Differences in DNA methylation have been reported in B and T lymphocyte populations,including CD4+ T cells,isolated from rheumatoid arthritis (RA) patients when compared to healthy controls. CD4+ T cells are a heterogeneous cell type with subpopulations displaying distinct DNA methylation patterns. In this study,we investigated DNA methylation using reduced representation bisulfite sequencing in two CD4+ T cell populations (CD4+ memory and na{\{i}}ve cells) in three groups: newly diagnosed disease modifying antirheumatic drugs (DMARD) na{\"{i}}ve RA patients (N = 11) methotrexate (MTX) treated RA patients (N = 18) and healthy controls (N = 9) matched for age gender and smoking status. Results: Analyses of these data revealed significantly more differentially methylated positions (DMPs) in CD4+ memory than in CD4+ na{\""{i}}ve T cells (904 vs. 19 DMPs) in RA patients compared to controls. The majority of DMPs (72{\%}) identified in newly diagnosed and DMARD na{\""{i}}ve RA patients with active disease showed increased DNA methylation (39 DMPs) whereas most DMPs (80{\%}) identified in the MTX treated RA patients in remission displayed decreased DNA methylation (694 DMPs). Interestingly we also found that about one third of the 101 known RA risk loci overlapped (±500 kb) with the DMPs. Notably introns of the UBASH3A gene harbor both the lead RA risk SNP and two DMPs in CD4+ memory T cells. Conclusion: Our results suggest that RA associated DNA methylation differences vary between the two T cell subsets but are also influenced by RA characteristics such as disease activity disease duration and/or MTX treatment.""" View Publication

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular,T cell regeneration is generally delayed,resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled,feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect,indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore,the addition of AA to feeder cocultures resulted in development of DP and SP T cells,whereas without AA,a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy. View Publication

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo,the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE,whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However,the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone,RTE,as well as mature naive and memory CD4(+) T cells,are rendered only minimally sensitive to Fas-mediated cell death. However,in the presence of the two cytokines,Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast,equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus,IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets. View Publication

Human immunodeficiency virus type 1 (HIV-1) utilizes Vpu,Env,and Nef to down-modulate its primary CD4 receptor from the cell surface,and this function seems to be critical for the pathogenesis of AIDS. The physiological relevance of CD4 down-modulation,however,is currently not well understood. In the present study,we analyzed the kinetics of CD4 down-modulation and the susceptibility of HIV-1-infected T cells to superinfection using proviral HIV-1 constructs containing individual and combined defects in vpu,env,and nef and expressing red or green fluorescent proteins. T cells infected with HIV-1 mutants containing functional nef genes expressed low surface levels of CD4 from the first moment that viral gene expression became detectable. In comparison,Vpu and Env had only minor to moderate effects on CD4 during later stages of infection. Consistent with these quantitative differences,Nef inhibited superinfection more efficiently than Vpu and Env. Notably,nef alleles from AIDS patients were more effective in preventing superinfection than those derived from a nonprogressor of HIV-1 infection. Our data suggest that protection against X4-tropic HIV-1 superinfection involves both CD4-independent and CD4-dependent mechanisms of HIV-1 Nef. X4 was effectively down-regulated by simian immunodeficiency virus and HIV-2 but not by HIV-1 Nef proteins. Thus,maximal protection seems to involve an as-yet-unknown mechanism that is independent of CD4 or coreceptor down-modulation. Finally,we demonstrate that superinfected primary T cells show enhanced levels of apoptosis. Accordingly,one reason that HIV-1 inhibits CD4 surface expression and superinfection is to prevent premature cell death in order to expand the period of effective virus production. View Publication

Peripheral CD103(+)Foxp3(+) regulatory T cells (Tregs) can develop both from conventional naive T cells upon cognate Ag delivery under tolerogenic conditions and from thymic-derived,expanded/differentiated natural Tregs. We here show that CD47 expression,a marker of self on hematopoietic cells,selectively regulated CD103(+)Foxp3(+) Treg homeostasis at the steady state. First,the proportion of effector/memory-like (CD44(high)CD62L(low)) CD103(+)Foxp3(+) Tregs rapidly augmented with age in CD47-deficient mice (CD47(-/-)) as compared with age-matched control littermates. Yet,the percentage of quiescent (CD44(low)CD62L(high)) CD103(-)Foxp3(+) Tregs remained stable. Second,the increased proliferation rate (BrdU incorporation) observed within the CD47(-/-)Foxp3(+) Treg subpopulation was restricted to those Tregs expressing CD103. Third,CD47(-/-) Tregs maintained a normal suppressive function in vitro and in vivo and their increased proportion in old mice led to a decline of Ag-specific T cell responses. Thus,sustained CD47 expression throughout life is critical to avoid an excessive expansion of CD103(+) Tregs that may overwhelmingly inhibit Ag-specific T cell responses. View Publication

Retinoic acid (RA),a vitamin A metabolite,modulates mucosal T helper cell responses. Here we examined the role of RA in regulating IL-22 production by γδ T cells and innate lymphoid cells in intestinal inflammation. RA significantly enhanced IL-22 production by γδ T cells stimulated in vitro with IL-1β or IL-18 and IL-23. In vivo RA attenuated colon inflammation induced by dextran sodium sulfate treatment or Citrobacter rodentium infection. This was associated with a significant increase in IL-22 secretion by γδ T cells and innate lymphoid cells. In addition,RA treatment enhanced production of the IL-22-responsive antimicrobial peptides Reg3β and Reg3γ in the colon. The attenuating effects of RA on colitis were reversed by treatment with an anti-IL-22 neutralizing antibody,demonstrating that RA mediates protection by enhancing IL-22 production. To define the molecular events involved,we used chromatin immunoprecipitation assays and found that RA promoted binding of RA receptor to the IL-22 promoter in γδ T cells. Our findings provide novel insights into the molecular events controlling IL-22 transcription and suggest that one key outcome of RA signaling may be to shape early intestinal immune responses by promoting IL-22 synthesis by γδ T cells and innate lymphoid cells. View Publication

OBJECTIVE Systemic sclerosis (SSc) is a complex autoimmune disease with a strong genetic component. However,most of the genes associated with the disease are still unknown because associated variants affect mostly noncoding intergenic elements of the genome. We used functional genomics to translate the genetic findings into a better understanding of the disease. METHODS Promoter capture Hi-C and RNA-sequencing experiments were performed in CD4+ T cells and CD14+ monocytes from 10 SSc patients and 5 healthy controls to link SSc-associated variants with their target genes,followed by differential expression and differential interaction analyses between cell types. RESULTS We linked SSc-associated loci to 39 new potential target genes and confirmed 7 previously known SSc-associated genes. We highlight novel causal genes,such as CXCR5,as the most probable candidate gene for the DDX6 locus. Some previously known SSc-associated genes,such as IRF8,STAT4,and CD247,showed cell type-specific interactions. We also identified 15 potential drug targets already in use in other similar immune-mediated diseases that could be repurposed for SSc treatment. Furthermore,we observed that interactions were directly correlated with the expression of important genes implicated in cell type-specific pathways and found evidence that chromatin conformation is associated with genotype. CONCLUSION Our study revealed potential causal genes for SSc-associated loci,some of them acting in a cell type-specific manner,suggesting novel biologic mechanisms that might mediate SSc pathogenesis. View Publication

Background:A primary barrier to curing HIV is the HIV reservoir. The leading infectious cause of death worldwide for people living with HIV is tuberculosis (TB),but we do not know how TB impacts the HIV reservoir.Methods:Participants in identification and validation cohorts were selected from previously enrolled studies at Groupe Haïtien d'Étude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port au Prince,Haiti. Intact and non-intact proviral DNA were quantified using droplet digital PCR of peripheral blood mononuclear cell (PBMC)-derived CD4+ T cells. Kruskal-Wallis tests were used to compare medians with tobit regression for censoring.Results:In the identification cohort,we found that people living with HIV with a history of active pulmonary TB (n=19) had higher levels of intact provirus than people living with HIV without a history of active TB (n=47) (median 762; IQR,183-1173 vs 117; IQR,24-279 intact provirus per million CD4,respectively; P=0.0001). This difference also was seen in the validation cohort (n=31),(median 102; IQR,0-737 vs 0; IQR,0-24.5 intact provirus per million CD4,P=0.03) for TB vs no-TB history groups,respectively. The frequencies of CD4+ T cells with any detectable proviral fragment was directly proportional to the levels of interleukin-1 beta (r=0.524,P= 0.0025) and interleukin-2 (r=0.622,P=0.0002).Conclusions:People living with HIV with a history of active pulmonary TB have more HIV pro-virus in their circulating CD4+ T cells,even years after TB cure. We need to characterize which CD4+ T cells are harboring intact provirus to consider the impact of T cell-targeting HIV cure interventions for people living in TB-endemic areas. 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