NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses

The objective of this study was to explore the role of NOD2 and TLR2 in cross-presentation in human dendritic cells undertaking an unbiased screen. We have used a quantitative phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by a computational analysis to identify the proteins as differentially abundant in response to NOD2 and TLR2 sensing. Validation of the phosphoproteomic analysis was performed by the detection of proteins in phosphoenriched lysates and detected by western blot. Techniques for the modulation of gene expression (shRNA and siRNA) were used to confirm the results of observational studies. Immunoprecipitation, in-gel or in-solution digestions and HLA-associated peptide purification were all performed in primary DCs isolated from healthy donors and analyzed on an ultra-high performance liquid chromatography system. Cellular analysis of cross-presentation experiments was performed using CD8+ T cells from OT-1 C57BL/6 TCR-transgenic mice or human HLA-A2 NY-ESO-11571−65 CD8+ T cell clones and analyzed by flow cytometry. The sample size is outlined in the figure legends. Generation of Human Monocyte-Derived Dendritic Cells and Cell Lines Human monocytes were purified from peripheral blood mononuclear cells (PBMCs) from healthy donors by positive immunoselection with anti-CD14-conjugated MACS beads (Miltenyi Biotec). Monocytes were also purified from PBMCs from either HLA-A2 WT NOD2 donors or HLA-A2 ho...
Source: Frontiers in Immunology - Category: Allergy & Immunology Source Type: research