High dimensional analyses of CD4+ T cells in human lymph nodes
ABSTRACT: 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: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: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:This study set out to examine CD4 T cell differentiation in a mouse model of diabetes based on transgenic expression of ovalbumin under the control of the rat insulin promoter and co-expression of the DO11.10 transgene (DO11 x rip-mOVA mice). The transcriptome of T cells isolated from the pancreatic lymph nodes (lymph nodes draining the site of self antigen expression) was compared with that of T cells isolated from inguinal lymph nodes (non-draining lymph nodes). T cells were sorted based on expression of CD4, DO11.10 TCR (KJ-126), CD25 and CD69. Primary cells from 6 week old DO11 x rip-mOVA mice were isolated ex-vivo from the pancreatic lymph nodes or inguinal lymph nodes. Cells were sorted by flow cytometry using antibodies to CD4, DO11.10 TCR (KJ-126+), CD25 and CD69. 3-6 replicates were collected per experimental group with each replicate deriving from 14 mice. RNA was isolated using the RNeasy micro kit (Qiagen).
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: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:The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T-cells reside in the fetal intestine. We combined functional assays with mass cytometry, single-cell RNA-sequencing, and high-throughput TCR-sequencing to characterize the fetal intestinal CD4+ T-cell compartment. We identified 22 CD4+ T-cell clusters, including naive-like, regulatory-like, and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like cells expressed high levels of Ki-67, indicating cell division, and CD5, a surrogate marker of TCR-avidity, and produced IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR-repertoire analysis demonstrated clonal expansions, distinct repertoire characteristics, and interconnections between subpopulations of memory-like CD4+ T-cells. Imaging-mass cytometry further showed that memory-like CD4+ T-cells colocalized with antigen-presenting cells. Collectively, these results provided evidence for the generation of memory-like CD4+ T-cells in the human fetal intestine, consistent with exposure to foreign antigens. Overall design: Single, live, CD3+CD8a–TCRγδ–CD4+/– T-cells from one human fetal intestines were processed on the Chromium 3' single cell platform (10x Genomics) and sequenced on an Illumina HiSeq 4000.
Project description:Exhaustion markers are expressed by T lymphocytes in Follicular Lymphoma (FL). Through these, TIM-3 has been recently identified as a poor pronostic factor when expressed by FL CD4+ T cells. Before this study, there was no molecular characterization of unstimulated CD8+ T cells, expressing - or not - TIM-3. We used microarrays to compare the global gene expression profile between CD8+TIM-3+ T cells and CD8+TIM-3- T cells freshly sorted from follicular lymphoma biopsies. Abstract from the associated publication: Up-regulation of T-cell immunoglobulin-3 (TIM-3) has been associated with negative regulation of the immune response in chronic infection and cancer, including lymphoma. Here, we investigated the possible correlation between TIM-3 expression by ex vivo cytotoxic T cells (CTL) from follicular lymphoma (FL) biopsies and their functional unresponsiveness that could limit the favorable impact of CTL on disease progression. We report a high percentage of CD8+TIM-3+ T cells in lymph nodes of FL patients. When compared to their CD8+TIM-3- counterparts, CD8+TIM-3+ T cells exhibited defective cytokine production following TCR engagement. Furthermore CD8+TIM-3+ T cells display ex vivo markers of lytic granule release and remain unresponsive to further TCR-induced activation of the lytic machinery. Although confocal microscopy showed that TIM-3 expression on CD8+ T cells correlated with minor alterations of immunological synapse, a selective reduction of ERK signaling in CD8+TIM-3+ T cells was observed by phospho-flow analysis. Finally, short relapse free survival despite rituximab(R)-chemotherapy was observed in patients with high content of TIM-3+ cells and a poor infiltrate of granzyme B+ T cells in FL lymph nodes. Together, our data indicate that, besides selective TCR early signaling defects, TIM-3 expression correlates with unresponsiveness of ex vivo CD8+ T cells in FL. They show that scores based on the combination of exhaustion and cytolytic markers in FL microenvironment might be instrumental to identify patients at early risk of relapses following R-chemotherapy. Overall design: CD3+CD8+TIM-3+ and CD3+CD8+TIM-3- T cells from FL lymph nodes (n=3 patients) were sorted by flow cytometry and then the 6 samples were selected for RNA extraction and hybridization on GeneChip® Human Gene 2.0 ST microarrays.