Project description:The TCR repertoire of CD4+ T cells in human lymph nodes is a case-based analysis of the TCR repertoire of CD4+ T cells in human lymph nodes. The study aims to determine how HIV infection alters the repertoire complexity of follicular helper T cells (TFH) in relationship to other T cell subsets in the lymphoid compartment. This study performs TCR repertoire sequencing using Molecular Identifier Clustering-based Immune Repertoire Sequencing (MIDCIRS). We found TFH cells become clonally expanded and convergently selected in LNs from patients with chronic HIV infection.
Project description:The High dimensional analyses of CD4+ T cells in human lymph nodes is a case-based analysis of the TCR repertoire, transcriptome, and epigenetic signatures of various CD4+ T cells in human lymph nodes. The study aims to determine clonal overlap between phenotypically distinct CD4+ T cells and the similarities between these populations on transcriptional and epigenetic levels. This study performs TCR repertoire sequencing using Molecular Identifier Clustering-based Immune Repertoire Sequencing (MIDCIRS). We found clonal overlap between a population of CXCR5- T cells and Tfh cells. We also show epigenetic and transcriptional similarities between CXCR5-PD-1+ T cells and Tfh cells.
Project description:To investigate the influence of CNS3, a cis-regulatory element in the Foxp3 locus, on the selection of T cell antigen receptor (TCR) repertoire of regulator CD4+ T cells (Treg), we crossed Foxp3ΔCNS3-gfp or control Foxp3gfp mice to DO11.10 TCRβ transgene and Tcra-/+ background. We isolated Treg and conventional CD4+T cells from thymus, spleen and lymph nodes of Foxp3ΔCNS3-gfp DO11.10 TCRβ Tcra-/+ or Foxp3gfp DO11.10 TCRβ Tcra-/+ male littermates, and sequenced the TCRα chains. Analysis of the diversity of Complementary Determining Region 3 (CDR3) of TCRα showed a distinct clustering of CNS3-deficient Treg cells from the CNS3-sufficient ones. Overall design: DO11.10 TCRβ transgene inhibits the recombination of endogenous Tcrb loci thus restricting TCR repertoire to TCRα chains expressed by T cells. Further limitation of the TCR repertoire was achieved by the presence of one functional Tcra gene. With restricted TCR repertoire, mRNA of TCRα was extracted from Treg and conventional CD4+ T cells for library preparation and high throughput sequencing.
Project description:We were interested in investigating the heterogeneity of CD4+ Th2 cells during infection response. In order to do so we injected Nippostrongylus brasiliensis (Nb) in recipient mice as this is known to elicit a Type 2 response. Mice were subcutaneously injected with Nb larvae and single CD4+ cells were isolated from mediastinal lymph nodes, mesenteric lymph nodes, and lungs 5 days after infection.
Project description:To activate primary T cells, lymph nodes were removed and disaggregated. Cells were cultured in RPMI 1640 containing L-glutamine, 10% FBS, 50 μM β-mercaptoethanol and penicillin/streptomycin. Cells were stimulated with 1 μg/ml of the CD3 monoclonal antibody (2C11) and 2 μg/ml anti-CD28 (37.51) in the presence of cytokines IL12 (10 ng/ml) and 20 ng/ml IL2 (20 ng/ml). Cells were stimulated for 24 hours. Live TCR activated CD4+ cells were sorted for CD4+ and CD45.1+ expression and DAP1 exclusion prior to collection for proteomics processing. Mice were “wild-type” (ie non-TCR transgenic) from OT-2 CD45.1 expressing C57bl6 background maintained in-house line.
Project description:We have previously shown that Tregs infiltrating follicular lymphoma lymph nodes (FLN) are quantitatively and qualitatively different than those infiltrating normal and reactive nodes (NLN, RLN, respectively). To gain insight into how such Treg populations differ, we performed RNA sequence (RNAseq) analyses on flow sorted Tregs from all three sources. We identify several molecules that could contribute to the observed increased suppressive capacity of FLN tregs, including upregulation of CTLA-4, IL-10, and GITR, all confirmed by protein expression. In addition, we identify, and confirm functionally, a novel mechanism by which Tregs target to and accumulate within a human tumor microenvironment, through the down regulation of S1PR1, SELL (L-selectin) and CCR7, potentially resulting in greater lymph node retention. In addition we identify and confirm functionally the upregulation of CXCR5, CXCL13 and IL-16 demonstrating the unique ability of the follicular derived Tregs to localize and accumulate within not only the malignant lymph node, but also localize and accumulate within the malignant B cell follicle itself. Such findings offer significant new insights into how FLN Tregs may contribute to the biology of follicular lymphoma and identify several novel therapeutic targets. Overall design: Cross sectional comparison of transcription profiles of human regulatory T cells from normal lymph nodes (n=10), follicular lymphoma lymph nodes (n=12) and reactive lymph nodes (n=5).
Project description:We isolated CD4+ T cells from draining lymph nodes 7 days post EAE from DA rats treated with vitamin D supplemented, vitamin D deprived or regular diet Overall design: EAE was induced with MOG in age matched DA rats. After isolation of CD4+ T cells with FACS, RNA was isolated and subjected to microarray analysis. We were interested in the effect of vitaimin D on the transcriptome of CD4+ T cells. Thus we did microarray analysis on 3 different groups: DA rats treated with vitamin D supplemented, vitamin D deprived or regular diet
Project description:In order to characterize pathogen specific T cell responses against Salmonella volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). we used mass cytometry, to identify effector CD4+ T cells circulating during infection. We identified a population of CCR7-CD38+ cells accumulating during infection, and via unbiased single cell cloning and expansion we demonstrated that these CCR7-CD38+ cells are enriched in Salmonella specific T cells. In this experiment we performed TCR repertoire analysis of CCR7-CD38+ and CCR7-CD38- cells to determine the clonality of CCR7-CD38+ cells, the overlap between the repertoire of CCR7-CD38+ cells and of non-activated effector CCR7-CD38- cells, and to identify within CCR7-CD38+ and CCR7-CD38- cells the presence of the CDR3b TCR sequence of the pathogen specific T cell clones isolated from CCR7-CD38+ cells Overall design: Analysis of the TCR repertoire of CCR7-CD38+ and CCR7-CD38+ CD4 T cells isolated from frozen PBMC after diagnosis of enteric fever in one volunteer challenged with Salmonella Typhi and one challenged with Salmonella Paratyphi A
Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Analysis of CD4+ TIL by comparing their expression profiles to those of their conterparts from patient axillary lymph nodes and peripheral blood and healthy donor blood CD4+ T cells were isolated from primary tumors, axillary lymph nodes and peripheral blood of 10 patients with invasive breast carcinomas and blood of 4 healthy donors and analyzed on Affymetrix U133 Plus 2.0 arrays