T Cell Receptors for Antigen on Intraepitheial Cytolytic T Lymphocytes in Celiac Disease Engage Enterocyte HLA-E and HLA-B.

We compared duodenal biopsies showing active celiac disease (CeD) to normal controls using single cell RNA sequencing, cyclic immunofluorescence, RNAScope and proximity ligation assays. There is increased infiltration of villous but not crypt epithelium T cells bearing either αβ or γδ T cell receptors (TCRs) in CeD. Both T cell subsets are activated cytotoxic T lymphocytes (CTLs) and surprisingly are the predominant mucosal source of IFNγ. In response to this IFNγ,villous but not crypt enterocytes show an IFNγ signature, including nuclear phospho-STAT1 protein, class II HLA molecules and IFNγ-inducible chemokines known to recruit CTLs (e.g., CCL3, CCL4, CXCL10, and CXCL11) and receptors for these chemokines are expressed on the infiltrating CTLs. Villous enterocytes also display increased HLA-E and HLA-B mRNAs and proteins. Bioinformatic analyses (NICHES) and proximity ligation assays show frequent binding of both αβ and γδ TCRs with enterocyte HLA-E or HLA-B, but not HLA-DR. In contrast, NKG2C, proposed as an alternative trigger of CTL activation, is infrequently-expressed and shows few interactions with HLA-E. Our data suggest that activated intraepithelial CTLs produce IFNγ which recruits additional CTLs and increases antigen-dependent killing of villous epithelium using either conventional or HLA-E antigen presentation. Signifance: Analyses of diagnostic biopsies from celiac disease patients reveals critical roles for IFNγ actions on enterocytes and a surprising role of HLA-E in antigen presntation. Activated cytotoxic T lymphocytes (CTLs) that are the primary source of mucosal IFNγ, leading to IFNγ signaling in villous enterocytes, including expression of phospho-STAT1 protein, class II HLA molecules, and chemokines that attract CTLs. Bioinformatic ligand receoptor analyses and proximity ligation assays showed frequent interactions between intraepithelial CTLs and enterocyte HLA-E or HLA-B, indicating that IFNγ production by the CTLs likely promotes further CTL recruitment and antigen-dependent killing of the villous epithelium.