Project Summary An intact epithelial barrier is key for maintaining intestinal homeostasis and preventing inflammation. Barrier dysfunction has been associated with inflammatory bowel disease and increased susceptibility to enteric infection. Previous work from the Kuhn lab has demonstrated that IL-6 produced by TCRαβ+ CD4+ colon intraepithelial lymphocytes (cIELs) are required to maintain epithelial barrier integrity and thus intestinal homeostasis. In contrast with phenotypically distinct small intestine IELs, which have been widely characterized, very little is known about cIEL function and development beyond the importance of IL-6 production. This project aims to define the mechanism that recruits cIELs to and retains them in the epithelium. Preliminary data indicate that upon exposure to luminal bacteria, myeloid cells produce sphingosine-1-phosphate (S1P) in a MyD88- dependent fashion. Conditional knockout of MyD88 in myeloid cells and S1P signaling blockade significantly reduce the number of TCRαβ+ CD4+ cIELs in the epithelium. However, further experiments are required to confirm this mechanism and understand the characteristics of recruited cIELs. Intestinal epithelial cells (IECs) express MHC class II (MHCII) and act as non-professional antigen presenting cells capable of influencing T cell function independent of local dendritic cells, and conditional knockout of MHCII on IECs changes cIEL effector- memory ratios. This proposal hypothesizes that intestinal bacteria are recognized by myeloid cells to recruit commensal reactive cIELs through S1P signaling to the epithelium, where interactions with epithelial MHCII lead to tolerance. Aim 1 seeks to demonstrate the mechanism by which myeloid cells recruit commensal reactive cIELs to the colon epithelium. The requirement for cIEL S1P signaling and myeloid cells will be shown by inhibiting each variable using the S1P receptor 1 inhibitor W146 and CD11c-DTR mice, respectively. cIELs will be stained for markers of differentiation, antigen experience, and activation. Commensal-reactive cIELs will also be identified by staining with MHCII-restricted tetramers containing the commensal antigenic peptide Cbir1, and their TCR Vβ chains will be identified using a flow screening panel. Aim 2 will investigate the mechanism by which cIELs are recruited to the colon epithelium by utilizing a conditional knockout mouse line in which IECs are deficient in MHCII (MHCIIΔIEC). Single cell assay for transposase-accessible chromatin (ATAC) sequencing and bulk T cell receptor (TCR) sequencing will be performed on cIELs from MHCIIΔIEC and MHCIIWT mice. These experiments will identify how epithelial MHCII influences cIEL gene regulation and TCR repertoire diversity, respectively. Lastly, MHCIIΔIEC and MHCIIWT mice will be infected with the murine enteric pathogen Citrobacter rodentium. This experiment will demonstrate the importance of epithelial MHCII in influencing cIELs in the immune response.