Project description:We used microarrays to detail the global programme of gene expression by circulating TCRVgamma9+ gamma delta T cells isolated from healthy individuals,tested either as resting cells or cells activated by phosphoantigen BrHPP and IL-2at an early(+6hrs) and a late (+7days) timepoint. We find that with more “NK cell” genes than alphabeta T cells and more “T cell” genes than NK cells, the circulating TCRVgamma9+ gamma delta T cells cells have a hybrid transcriptome. The gene signature of the activated cells recapitulates their physiological functions: Th1 cytokine, chemokine and cytotoxic activities at first and mitotic activity at later time points. The gene expression pattern of activated normal gamma delta T cells is nevertheless clearly distinctive from that of NK/T and peripheral T cell lymphomas of the gamma delta subtype.
Project description:We used microarrays to detail the global programme of gene expression by circulating TCRVgamma9+ gamma delta T cells isolated from healthy individuals,tested either as resting cells or cells activated by phosphoantigen BrHPP and IL-2at an early(+6hrs) and a late (+7days) timepoint. We find that with more M-bM-^@M-^\NK cellM-bM-^@M-^] genes than alphabeta T cells and more M-bM-^@M-^\T cellM-bM-^@M-^] genes than NK cells, the circulating TCRVgamma9+ gamma delta T cells cells have a hybrid transcriptome. The gene signature of the activated cells recapitulates their physiological functions: Th1 cytokine, chemokine and cytotoxic activities at first and mitotic activity at later time points. The gene expression pattern of activated normal gamma delta T cells is nevertheless clearly distinctive from that of NK/T and peripheral T cell lymphomas of the gamma delta subtype. Human TCRVg9positive gamma delta T cells were isolated from PBMC by cell sorting (>98% purity) and activated for RNA extraction and hybridization on Affymetrix microarrays. Samples comprise cells before activation (control time 0), early after activation with BrHPP/IL2 (+6 hours) and at a later timepoint of the activated in vitro culture with BrHPP/IL2 (day 7).
Project description:Epithelial cells provide an initial line of defense against damage and pathogens in barrier tissues such as the skin; however this balance is disrupted in obesity and metabolic disease. Skin gamma delta T cells recognize epithelial damage and release cytokines and growth factors that facilitate wound repair. To determine the impact of obesity and metabolic disease on skin gamma delta T cells, we isolated skin gamma delta T cells from 10-week old C57BLKS/J lean db/+ and obese db/db animals for further study. Due to a deficiency in the leptin receptor (db), homozygous db/db animals do not process satiety signals, continually eat and develop severe obesity and metabolic disease. Skin gamma delta T cells isolated from these animals were compared for changes in mRNA expression using microarray. We have determined that obesity and metabolic disease negatively impacts homeostasis and functionality of skin gamma delta T cells, rendering host defense mechanisms vulnerable to injury and infection. The goal of this experiment was to compare skin gamma delta T cells in a control mouse to skin gamma delta T cells isolated from an obese mouse to see what homeostatic changes occur in obesity and metabolic disease. gamma delta T cells were isolated from two 10-week old lean db/+ control and two 10-week old obese db/db animals for comparison. We wanted to determine which growth factors and signaling pathways were altered in skin gamma delta T cells residing in the obese environment.
Project description:Epithelial cells provide an initial line of defense against damage and pathogens in barrier tissues such as the skin; however this balance is disrupted in obesity and metabolic disease. Skin gamma delta T cells recognize epithelial damage and release cytokines and growth factors that facilitate wound repair. To determine the impact of obesity and metabolic disease on skin gamma delta T cells, we isolated skin gamma delta T cells from 10-week old C57BLKS/J lean db/+ and obese db/db animals for further study. Due to a deficiency in the leptin receptor (db), homozygous db/db animals do not process satiety signals, continually eat and develop severe obesity and metabolic disease. Skin gamma delta T cells isolated from these animals were compared for changes in mRNA expression using microarray. We have determined that obesity and metabolic disease negatively impacts homeostasis and functionality of skin gamma delta T cells, rendering host defense mechanisms vulnerable to injury and infection.
Project description:T cells bearing gamma delta T cell antigen receptors (TCRs) function in lymphoid stress surveillance. However, the contribution of gamma delta TCRs to such responses is unclear. Here we found that the TCR of a human V gamma4Vdelta5 clone directly bound endothelial protein C receptor (EPCR), which allowed gamma delta T cells to recognize both endothelial cells targeted by cytomegalovirus and epithelial tumors. EPCR is a major histocompatibility complex–like molecule that binds lipids analogously to the antigen-presenting molecule CD1d. However, the V gamma4Vdelta5 TCR bound EPCR independently of lipids, in an antibody-like way. Moreover, the recognition of target cells by gamma delta T cells required a multimolecular stress signature composed of EPCR and costimulatory ligand(s). Our results demonstrate how a gamma delta TCR mediates recognition of broadly stressed human cells by engaging a stress-regulated self antigen.
Project description:Gamma delta T cells were sorted from the lungs of 4 female FVB/n mice (labeled wild-type [WT] 1-4) and 4 female mammary tumour-bearing K14-Cre;Brca1F/F;Trp53F/F (KB1P) mice (labeled Tumour 1-4).The sorted cells were loaded onto the Chromium Single Cell 30 Chip Kit v2 (10xGenomics) to generate libraries for scRNAseq, following the manufacturer’s instructions. The sequencing-ready library was cleaned up with SPRIselect beads (Beckman Coulter, High Wycombe, UK). Quality control of the library was performed prior to sequencing (Qubit, Bioanalyzer, qPCR). Illumina sequencing was performed using NovaSeq S1 by Edinburgh Genomics (University of Edinburgh).
Project description:T cells bearing gamma delta T cell antigen receptors (TCRs) function in lymphoid stress surveillance. However, the contribution of gamma delta TCRs to such responses is unclear. Here we found that the TCR of a human V gamma4Vdelta5 clone directly bound endothelial protein C receptor (EPCR), which allowed gamma delta T cells to recognize both endothelial cells targeted by cytomegalovirus and epithelial tumors. EPCR is a major histocompatibility complex–like molecule that binds lipids analogously to the antigen-presenting molecule CD1d. However, the V gamma4Vdelta5 TCR bound EPCR independently of lipids, in an antibody-like way. Moreover, the recognition of target cells by gamma delta T cells required a multimolecular stress signature composed of EPCR and costimulatory ligand(s). Our results demonstrate how a gamma delta TCR mediates recognition of broadly stressed human cells by engaging a stress-regulated self antigen. 2E9 is an antibody that blocks gd-TCR recognition of human cells that are the targets of the clone LES. 2E9 also stains cells that are targeted by LES. Therefore, to identify the TCR ligand expressed by 2E9-positive cells, Gene Expression was compared in two cell lines that stain with 2E9 (K562 and U937) versus two cell lines that do not (Hutu80 and Huh7).
Project description:gamma delta intraepithelial lymphocytes were isolated from the colons of DSS-treated and untreated mice. Total RNAs were isolated and compared by Affymetrix DNA microarray. Duplicate comparisons were made between DSS-treated and untreated gamma delta intraepithelial lymphocytes.
Project description:The contribution of chronic antigen stimulation to the occurrence of lymphoproliferative disorder (LPD) with the gamma-delta T-cell lineage is unclear, despite the fact that Epstein-Barr virus (EBV) positive T-cell LPD is derived from antigen-stimulated cytotoxic T-cells. Given the possible association of antigen stimulation with the development of cytotoxic T-cell LPD, we compared gene expression patterns in Epstein-Barr virus (EBV)-positive gamma-delta T-cell lines derived from patients with nasal T-cell lymphoma and chronic active EBV infection and those in gamma-delta T-cells from healthy volunteers. Three EBV-positive gamma-delta T-cells lines, SNT cells (SNT-8, SNT-13 and SNT-15), were used in this study. SNT-8 was established from patients with nasal T-cell lymphoma and SNT-13, -15 were established from patients with chronic active EBV infection (Zhang Y, et al., Br J Cancer 94:599-608, 2006). All the SNT cells exhibits common rearrangement of Vgamma9-JgammaP and Jdelta3 genes. The gamma-delta T-cells obtained from healthy volunteers were expanded ex vivo by 1 microM of zoledronate (ZOL) plus IL-2 for 14 days incubation.