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The distinct response of gd T cells to the Nod2 agonist muramyl dipeptide

ABSTRACT: We have begun to approach gd T cells more as prospective innate cells than as conventional T cells. Recent results indicated that purified gd T cells are primed directly in response to pathogen associated molecular patterns (PAMPs) to better respond to secondary signals and increase expression of chemokine and activation-related genes. In microarray and real time PCR analyses of RNA derived from bovine and human gd T cells, transcripts encoding Nod2 were repeatedly amplified. Nod2 is the intracellular receptor for muramyl dipeptide (MDP), a subunit of PGN, functions in regulating innate activities, and was thought to be expressed primarily in APCs. Given our repeated detection of Nod2 transcripts in gd T cells, the specific direct response of gd T cells to MDP was analyzed by microarray, real time PCR, proteome array and in a functional priming assay. The results indicate a subtle activation in response to MDP akin to priming, and suggest a unique mechanism for differential gene expression. Keywords: Comparison of genes expressed after stimulation of bovine gd T cells with either PBS or MDP

ORGANISM(S): Bos taurus

PROVIDER: GSE6918 | GEO | 2009/02/10

SECONDARY ACCESSION(S): PRJNA98133

REPOSITORIES: GEO

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Project description:We have begun to approach gd T cells more as prospective innate cells than as conventional T cells. Recent results indicated that purified gd T cells are primed directly in response to pathogen associated molecular patterns (PAMPs) to better respond to secondary signals and increase expression of chemokine and activation-related genes. In microarray and real time PCR analyses of RNA derived from bovine and human gd T cells, transcripts encoding Nod2 were repeatedly amplified. Nod2 is the intracellular receptor for muramyl dipeptide (MDP), a subunit of PGN, functions in regulating innate activities, and was thought to be expressed primarily in APCs. Given our repeated detection of Nod2 transcripts in gd T cells, the specific direct response of gd T cells to MDP was analyzed by microarray, real time PCR, proteome array and in a functional priming assay. The results indicate a subtle activation in response to MDP akin to priming, and suggest a unique mechanism for differential gene expression. Experiment Overall Design: PBLs from calf 129 were stained with GD3.8 directly conjugated to FITC, washed, and sorted on a Vantage SE cell sorter (BD Immunocytometry Systems) as previously described. PBLs from calves 150 and 151 were stained with GD3.8, and anti-Mouse IgG magnetic beads and sorted with MACS magnetic bead system (Miltenyi Biotec) as previously described to a purity of >98%. Sorted cells were rested overnight, stimulated for 4 hours then lysed in Buffer RLT and genomic DNA sheared using Qiashredder columns then frozen at -80oC. RNA was extracted following the manufacturerâs protocol for RNeasy (Qiagen) column purification, assessed on a Bioanalyzer 2100 (Agilent Technologies), and amplified using the Affymetrix One-cycle protocol with approximately 1.7micrograms 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 mg 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) 19;20 was used for data collection.

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The distinct response of gd T cells to the Nod2 agonist muramyl dipeptide

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