Intestinal CD4-CD8αβ-TCRαβ+ T cells function as tolerogenic antigen presenting cells in mice

To distinguish harmless from harmful antigens (Ag), anatomic, cellular and molecular mechanisms operate in the intestinal tract to acquire, process, present and interpret luminal Ags. The intestinal mucosa is populated by many T cell types in the intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) populations, including double negative CD4-CD8αβ-TCRαβ+ T (double-negative T/DNT) cells. Here we show that murine DNT cells in the small intestine reach across the epithelial barrier to capture luminal Ags. A sizeable fraction of DNT cells in Peyer’s patches and mesenteric lymph nodes express MHC-II, but little or no classical co-stimulatory molecules. Indeed, DNT cells acquire, process and present antigenic proteins to tolerize Ag-specific CD4+ T cells. Conditional genetic ablation of MHC-II in T cells disable this tolerogenic function and rendered mutant mice hypersusceptible to intestinal inflammation. Intriguingly, intestinal T cells in patients with Crohn’s disease express lower levels of HLA-DR than those from controls. Our findings thus suggest that MHC-II+ DNT cells may regulate intestinal immune homeostasis and their dysfunction may contribute to the pathogenesis of inflammatory bowel disease. PP cells were collected from pool of 3-5 C57BL/6 mice (10-week-old, male and female), stained with anti-CD8β-PE, anti-CD4-APC, anti-TCRβ-APC-Cy7, and anti-γδTCR-PE-Cy7, and then sorted by the MofFlo-XDP as the following fractions: γδTCR+TCRβ- (γδ), γδTCR-TCRβ+CD4-CD8β- (DN), γδTCR-TCRβ+CD4+CD8β (CD4), and γδTCR-TCRβ+CD4-CD8β+ (CD8). More than 98% purity of each fraction was confirmed by analysing post-sorted cells with the FACS Canto II. Total RNA was collected from each fraction with a RNeasy Mini Kit (QIAGEN# 74106). RNA collection was repeated 3 times. Microarray analysis including PCA plots and gene expression analysis was outsourced to KAMAKURA TECHNO-SCIENCE INC. Samples were analysed as triplicated with a 3D gene chip (Mouse oligo 24k).