Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Immunobiology
First Advisor
Pober, Jordan
Abstract
Celiac disease (CeD) is a common autoimmune disorder triggered by HLA-DQ2/DQ8-restricted CD4 T cells responding to gluten-derived peptides that have been deamidated by tissue transglutaminase 2 (TG2). These activated CD4 T cells then provide help to B cells that produce IgA antibodies to deamidated gluten peptides (DPG) and TG2. Histologically, the epithelial lining of the small intestinal villi is destroyed by infiltrating cytotoxic T cells (CTLs) that replace the normal resident T cell populations, resulting in villous flattening and absorptive dysfunction. The link between the response of CD4+ T cells to DPGs and the generation, recruitment, and activation of intraepithelial CTLs is unknown. In this thesis, I will apply morphological techniques to analyze the interactions of CTLs with villous epithelial cells which is less well characterized than the CD4+ T cell response that develops in the lamina propria. Specifically, I performed both multiparameter immunofluorescence and fluorescent in situ hybridization on FFPE celiac and control duodenal biopsies along with single cell RNA sequencing of isolated villous enterocytes performed by my collaborators from fresh celiac biopsies. Intraepithelial CD3+ T cells numbers per enterocyte were increased in CeD over controls and were a mixture of T cell receptor (TCR) ab and gd cells, consistent with prior reports. Also similar with prior reports, TCRab CTL numbers changed more dramatically with disease activity than TCRgd cell numbers. To investigate the mechanisms of recruitment, we assessed candidate chemokine receptor expression on T cells in tissues and chemokine ligands expressed by the epithelial cells using single cell RNA sequencing. Chemokine ligands CCL3, CCL4, and CCL25 were found to be increased the most on surface enterocytes in CeD which was corroborated by positive CCR5 and CCR9 protein staining on the majority of IELs. We also evaluated for CXCR3 expression and found it was downregulated on IELs in celiac biopsies by immunofluorescence and showed a minimal increase in ligand transcript expression on enterocytes. IELs in CeD also expressed increased levels of IFNg mRNA, CD45RO, granzyme B, and nuclear Ki67 antigens, indicative of activated CTL. Although increased in the surface epithelium of celiac patients, IFNg mRNA positive T cells in the lamina propria did not differ between CeD and control biopsies. Surface enterocytes in CeD but not controls also displayed an IFNg response signature comprised of nuclear phosphoSTAT1, MHC class II (HLA-DR) and HLA-E, an MHC Class ib. molecule. Class Ia proteins (e.g. HLA-B) were expressed at baseline and did not appear to be increased. In short term organ culture studies of normal duodenum, IFNg alone was not able to induce cell death but could increase expression of HLA-E. The molecules involved in CTL recognition of epithelial cells is controversial. Since the epithelium contains mostly CD8 T cells, recognition of MHC class II molecules is unlikely. Classical CTL activation could occur with TCRab and TCRgd CTLs recognizing gluten peptides presented by MHC class Ia molecules. Both types of TCRs utilize ZAP70 adaptor protein to signal which is expressed by these intraepithelial T cells but the lack of MHC-I restriction and minimal evidence of these cells in literature suggests this mechanism may be unlikely. Alternatively, HLA-E can be recognized independently of TCR signaling by NKG2C which signals through DAP12. A third possibility is that HLA-E could present gluten peptides to TCRs. We found that intraepithelial CTLs in celiac biopsies show minimal expression of NKG2C and its adaptor protein DAP12, favoring the hypothesis that killing is mediated by TCR recognition of antigen. These findings propose that antigen experienced CTLs are recruited to the surface epithelium where they are activated and release IFNg, altering enterocytes to render them susceptible to killing, potentially through HLA-E recognition.
Recommended Citation
Johnson, Justin, "Signaling Between Intraepithelial T Cells And Villous Enterocytes In Celiac Disease" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1414.
https://elischolar.library.yale.edu/gsas_dissertations/1414
