Project description:gd T cells have an important yet incompletely defined role in inflammation associated with a variety of infectious and autoimmune conditions. To better understand the precise roles of gd T cells relative to ab T cells in a specific infection, we utilized Salmonella Enterica Serovar Typhimurium (S. typhimurium) infection in cattle as it is a leading cause of disease in cattle and closely approximates S. typhimurium-induced enterocolitis in humans. To best represent phenotype and gene expression changes occurring in the gut mucosa early in S. typhimurium infection, gd and ab T cells were collected directly from the mesenteric lymphatic ducts and analyzed by FACS or immediately sorted and processed for microarray analysis. Gene expression profiles were compared at intervals during infection within T cell subsets. The majority of gene expression changes in both subsets occurred 48 hours after infection. In response to S. typhimurium infection there was an increase in expression of several genes in gd T cells which were indicative of activation, proliferation and innate function, whereas in ab T cells gene expression changes suggested a lack of S. typhimurium-specific response. This work represents the first focus on gene expression trends in tissue-derived T lymphocytes in an in vivo model that is highly relevant to human S. typhimurium-induced enterocolitis. Experiment Overall Design: For one mock infection (calf 156) and two experimental S. typhimurium infections (calves 112 and 162), gd and ab lymphatic T cells were stained with GD3.8 directly conjugated to FITC, washed, and sorted on a Vantage SE cell sorter (BD Immunocytometry Systems) as previously described. Sorted gd and ab T cells were collected directly into TRIzol reagent (Invitrogen; calf 112) and lysed or suspended in Buffer RLT (Qiagen; calves 156 and 162) and lysed using Qiashredder columns, then frozen at -80oC. RNA was extracted following the manufacturerâs protocol for Trizol (Invitrogen) extraction, or RNeasy (Qiagen) column purification, assessed on a Bioanalyzer 2100 (Agilent Technologies), and amplified either using Affymetrix Two-cycle (calf 112) target labeling protocol with 100 ng total RNA or the One-cycle protocol (calves 156 and 162) with approximately 1.6 micrograms of total RNA as described in the GeneChipÂ® Expression Analysis Technical Manual (June 2004). Hybridizations to GenechipÂ® Bovine Genome Arrays (Affymetrix) were performed with 15 micrograms biotin labeled cRNA. Washing and staining was performed in the GeneChipÂ® Fluidics Station 450 using the Midi_euk2v3 protocol. Chip scans were performed on the Affymetrix GeneChipÂ® Scanner 3000. GeneChipÂ® Operating Software (GCOS v.1.1, Affymetrix) was used for data collection. Experiment Overall Design: Table I represents annotated genes of potential interest that changed 2 fold or greater in expression between 0 and 48 hours post-Salmonella infection (calves 112 and 162) or mock-infection (calf 156) in T cell subsets.

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