Intestinal surveillance by intraepithelial lymphocytes

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Project Summary Intestinal intraepithelial lymphocytes (IELs) are T cells that form one of the key branches of the mucosal immune system, providing a first line of immune defense against pathogens and possibly against epithelial cancers due to their location at the critical interface between the intestinal lumen and the core of the body. Consistently, dysregulation of IELs leads to loss of mucosal barrier integrity, susceptibility to enteric infections and inflammatory bowel diseases (IBD) and cancer. In recent years some of the mechanisms controlling the development and function of IEL populations against enteric pathogens have been elucidated, including work developed during the first funding cycle of this proposal. In addition to their role in immune surveillance against enteric infections, recent data suggest a γδ T cell-associated gene signature as the most favorable prognostic factor across cancer types, including colorectal cancer (CRC). CRC is the second most deadly cancer in the United States, affecting over 140,000 people each year, killing approximately 50,000 in the US. Up to 20% of IBD patients develop CRC, although the majority of CRCs develop in patients without underlying inflammation. In both the common forms of CRC and IBD-induced CRC tumor-elicited inflammation triggers EC damage resulting in microbial invasion, which sustains inflammation that in turn drives cancer progression. Therefore, IEL surveillance of the mucosal barrier may play dual roles in CRC: (i) prevention of CRC progression and early dissemination by immune cell-mediated killing or additional anti-tumor responses; (ii) promotion of CRC progression and metastasis through inflammatory cytokines or immune-regulatory molecules. Based on existing literature in murine and human CRC, our recent work, and extensive preliminary data presented here, we hypothesize that γδ IEL epithelial surveillance is crucial for the regulation of tumor formation. We show that at steady state, the majority of intestinal γδ IELs express Vγ7 or Vγ1 TCRs and IEL hallmarks including a cytotoxic machinery. However, in both colitis-associated (AOM+DSS) and mutation-associated (CDX2-APC) models, CRC progression was associated with relative reduction of Vγ7 or Vγ1+ and accumulation of γδ IELs expressing Vγ6 or Vγ4 TCRs, which produce IL-17 and express PD-1. In Aim 1, we will address whether tissue-resident Vγ7+ or Vγ1+ γδ IEL subsets play a role in immune surveillance of the epithelium, preventing tumor formation. In Aim 2, we will characterize γδ IEL subsets that accumulate during CRC progression and may facilitate tumor growth. Studies proposed here will characterize γδ IEL behavior during early and late stages of CRC development using a combination of innovative imaging approaches. We will also track interacting ECs and surrounding IELs during CRC using a novel mouse model to identify cellular partners and single cell transcriptomics. Inducible intersectional genetics will be used to target differentiation or function of γδ IELs, while γδ IEL subsets enriched in different stages of CRC will be targeted using novel strains lacking specific V-gamma; these murine lines will be subjected to gnotobiotic and infection models using complementary CRC approaches.