Project description:The two major human gd T cell subsets, Vd1 and Vd2, display differences in tissue tropism and agonist responses, but we have little insight into global differences that may exist at the gene expression level. This is due to the small numbers of these cells that can be obtained from healthy donors, which limit comprehensive, comparative gene expression analyses. We established a culture method that expands Vd1 and Vd2 cells from the same PBL preparation to levels sufficient for sorting and microarray analysis. Although the subsets were expanded identically (anti-TCR mAb, plus IL-15), 392 and 614 genes were identified, which were differentially expressed in the two subsets, from two donors, respectively. Approximately 4,500 genes changed in both subsets following PMA/ionomycin treatment; about 50% of these genes were subset-specific. Both subsets responded to a crude LPS preparation, but only 6% of the responsive genes were the same. The differentially expressed genes were consistent with Vd2 cells being more inflammatory and Vd1 cells having more of a regulatory phenotype. Both subsets expressed transcripts encoding an array of innate and NK cell receptors, supporting the relationship of gd?T cells to the innate immune system. Our results show that circulating Vd1 and Vd2 subsets in humans have considerable, inherent differences in gene expression following treatment with non-TCR agonists, supporting unique functional roles for these cells in vivo.
Project description:Gene expression analysis comparison of ex vivo isolated GD T cell subpopulations Under non-pathological conditions, human gd T cells represent a small fraction of CD3+ T cells in peripheral blood (1-10%). They constitute a unique subset of T lymphocytes that recognize stress ligands or non-peptide antigens through MHC-independent presentation. Major human gd T cell subsets, Vd1 and Vd2, expand in response to microbial infection or malignancy, but possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that differences at the gene, phenotypic, and functional level would provide evidence that gd T cell subpopulations belong to distinct lineages. Comparisons between each subset and the identification of the molecular determinants that underpin their differences has been hampered by experimental challenges in obtaining sufficient numbers of purified cells. By utilizing a stringent FACS-based isolation method, we compared highly purified human Vd1 and Vd2 cells in terms of phenotype, gene expression profile, and functional responses. We found distinct genetic and phenotypic signatures that define functional differences in gd T cell populations. Differences in TCR components, repertoire, and responses to calcium-dependent pathways suggest that Vd1 and Vd2 T cells are different lineages. These findings will facilitate further investigation into the ligand specificity and unique role of Vd1 and Vd2 cells in early immune responses.
Project description:The two major human gd T cell subsets, Vd1 and Vd2, display differences in tissue tropism and agonist responses, but we have little insight into global differences that may exist at the gene expression level. This is due to the small numbers of these cells that can be obtained from healthy donors, which limit comprehensive, comparative gene expression analyses. We established a culture method that expands Vd1 and Vd2 cells from the same PBL preparation to levels sufficient for sorting and microarray analysis. Although the subsets were expanded identically (anti-TCR mAb, plus IL-15), 392 and 614 genes were identified, which were differentially expressed in the two subsets, from two donors, respectively. Approximately 4,500 genes changed in both subsets following PMA/ionomycin treatment; about 50% of these genes were subset-specific. Both subsets responded to a crude LPS preparation, but only 6% of the responsive genes were the same. The differentially expressed genes were consistent with Vd2 cells being more inflammatory and Vd1 cells having more of a regulatory phenotype. Both subsets expressed transcripts encoding an array of innate and NK cell receptors, supporting the relationship of gd?T cells to the innate immune system. Our results show that circulating Vd1 and Vd2 subsets in humans have considerable, inherent differences in gene expression following treatment with non-TCR agonists, supporting unique functional roles for these cells in vivo. Keywords: cell type comparison
Project description:gd T cell infiltration into tumours usually correlates with improved patient outcome, but both tumour-promoting and tumoricidal effects of γδ T cells have been documented. Human γδ T cells can be divided into functionally distinct subsets based on T cell receptor Vd usage. Still, the contribution of these different subsets to tumour immunity remains elusive. Here, we provide a detailed gd T cell profiling in colon tumours, comprising mRNA quantification using the Nanostring platform, in combination with mass and flow cytometry and TCR sequencing. δ chain usage in both the macroscopically unaffected colon mucosa and tumours varied considerably between patients, with substantial fractions of Vδ1, Vδ2, and non-Vd1Vd2 cells. Nanostring analyses of flow cytometry sorted Vd1, Vd2 and non-Vd1Vd2 cells showed a large variation in gd T cell subsets between individual tumours, and we suggest that individual gd T cell composition in colon tumours may contribute to the balance between favourable and adverse immune responses, and thereby also patient outcome.
Project description:We hypothesised that sustained exposure of naïve CD4+ T cells to IL-6 might imprint them with a distinct transcriptional programme, whose molecular and functional consequences following subsequent TCR ligation was relevant to disease development. Preexposure of healthy naïve CD4+ T-cells to pathophysiological levels of the cytokine caused induction of STAT3 target genes known to discriminate RA patients from disease controls in the clinic. After TCR stimulation IL-6 pre-exposed cells exhibited enhanced proliferative capacity, activation and a propensity towards Th1 differentiation, compared to non-exposed cells.
Project description:Vg9Vd2 gd T cells, nonVg9Vd2 gd T cells and ab T cells were sorted from human fetal peripheral blood (<30 weeks of gestation) and RNA was isolated from 10,000-100,000 sorted T cells. RNA was amplified using the Ovation PicoSL WTA System (NuGen), labeled with biotin using the Encore BiotinIL Module (NuGen) and applied on Illumina HT12 bead arrays.
Project description:Interferon alpha (IFNa) is a pro-inflammatory cytokine that is rapidly upregulated as part of an innate immune response. Gamma delta intraepithelial lymphocytes (gd IEL) mount a rapid antimicrobial response to luminal microorganisms and previous report indicate that the T cell receptor (TCR) of these cells is constantly triggered at steady-state. We assessed the contribution of tonic TCR activation in response to acute, systemic IFNa administration among murine gd IELs. Our data demonstrate that inhibiting basal TCR signaling has a minimal effect on the transcriptional profile of gd IELs and acute IFNa exposure induces antiviral gene programs independent of TCR signaling.
Project description:Naive T cells experience accumulation of TCR signaling in response to self-antigens in the steady state. However, how these signals influence the responsiveness of naive CD8+ T cells to subsequent agonist TCR stimulation remains incompletely understood. We investigated how naive CD8+ T cells that experienced relatively low or high levels of TCR signaling in response to self-antigens respond to stimulation with foreign antigens. A transcriptional reporter of Nr4a1 (Nur77-GFP) revealed substantial heterogeneity of the amount of TCR signaling naive CD8+ T cells accumulate in the steady state. To further understand the impact of differential TCR signaling on CD8 T cells we performed RNA-seq on naive Nur77-GFPhi and lo populations.