AbstractIn the gut, various subsets of intraepithelial T cells (IELs) respond to self or non-self-antigens derived from the body, diet, commensal and pathogenic microbiota. Dominant subset of IELs in the small intestine are TCRαβCD8αα⁺ cells, which are derived from immature thymocytes that express self-reactive TCRs. Although most of TCRαβCD8αα⁺ IELs are thymus-derived, their repertoire adapts to microbial flora. Here, using high throughput TCR sequencing we examined how clonal diversity of TCRαβCD8αα⁺ IELs changes upon exposure to commensal-derived antigens. We found that fraction of CD8αα⁺ IELs and CD4⁺ T cells express identical αβTCRs and this overlap raised parallel to a surge in the diversity of microbial flora. We also found that an opportunistic pathogen (Staphylococcus aureus) isolated from mouse small intestine specifically activated CD8αα⁺ IELs and CD4⁺ derived T cell hybridomas suggesting that some of TCRαβCD8αα⁺ clones with microbial specificities have extrathymic origin. We also report that CD8ααCD4⁺ IELs and Foxp3CD4⁺ T cells from the small intestine shared many αβTCRs, regardless whether the later subset was isolated from Foxp3^CNS1 sufficient or Foxp3^CNS1 deficient mice that lacks peripherally-derived Tregs. Overall, our results imply that repertoire of TCRαβCD8αα⁺ in small intestine expends in situ in response to changes in microbial flora.