Background: Novel and recent discoveries have shown that antigen presentation to gut T cells can be carried out by gut epithelial cells in the colon. Here we show, in the inflamed stomach, that gastric epithelial cells highly express MHC-I and interact with intraepithelial CD8⁺ T cells, and that Absent in Melanoma 2 (Aim2) can regulate the frequency of intraepithelial CD8⁺ T cells. Understanding this mechanism and modulating CD8⁺ T cell education by gastric epithelial MHC-I can be useful in regulating the outcome of gastric pre-neoplastic and neoplastic lesions. Methods: We used a combination of methods including (1) immunofluorescence of intraepithelial E-cadherin⁺ CD8⁺ T cells in tissue sections from human gastric biopsies, (2) single-cell RNA sequencing (scRNA-Seq) of WT vs Aim2^-/- 6-month H. felis-infected stomachs, (3) WT → Aim2^-/- vs Aim2^-/- → WT bone marrow chimeras, (3) CD19^CreAim2^flox/flox vs Aim2^flox/flox mice, and (4) H. pylori-pulsed BMDC:T cell co-culture of WT vs Aim2^-/- splenocytes. Results: Intraepithelial CD8⁺ T cells, which also expressed E-cadherin, were identified, by immunofluorescence, in biopsies from subjects with chronically inflamed gastric mucosae due to chronic H. pylori infection. The cell membranes of these cells were in contact with β₂ microglobulin (MHC-I subunit) expressed by gastric mucous pit and neck cell membranes. In mice, MHC-I was similarly induced in gastric epithelial cells by 6-month H. felis or H. pylori infection, relative to uninfected. Intraepithelial CD8⁺ T cells were identified by scRNA-Seq, and they clustered with gastric epithelial cells rather than the T cell cluster, as they co-expressed a combination of CD8⁺ T cell and epithelial cell markers. scRNA-Seq showed that Aim2 was downregulated in these hybrid intraepithelial CD8⁺ T cells, relative to stromal E-cadherin^- CD8⁺ T cells. To determine which compartment Aim2 exerts its impact on CD8⁺ T cells, we performed bone marrow chimera experiments (e.g., Aim2^-/- → WT vs WT → Aim2^-/-) followed by 6mo H. felis infection, and observed an increase in intraepithelial CD8⁺ T cells in Aim2^-/- → WT but not WT → Aim2^-/-, thus implicating a non-epithelial-intrinsic Aim2 effect. Moreover, CD19^CreAim2^flox/flox followed by 6mo H. felis also failed to recapitulate the Aim2 phenotype, thereby excluding B cell-intrinsic Aim2 involvement. Myeloid Aim2 involvement was also ruled out in a previous report. In contrast, CD8⁺ T cell-intrinsic Aim2 deficiency increased the expression of IFN-γ and tissue-resident memory T cell markers CD69 and CD103 in H. pylori-pulsed BMDC:T cell ex-vivo co-culture experiments. Conclusion: Our study suggests that intraepithelial engagement of gastric CD8⁺ T cells with epithelial cells may lead to downregulation of CD8 T cell Aim2 expression, thereby increasing pathogenic CD8 T cell phenotype.

Nobuhiko Kamada

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REGULATION OF INTRAEPITHELIAL CD8+ T CELL INTERACTION WITH EPITHELIAL MHC-I BY ABSENT IN MELANOMA 2 IN GASTRIC PRE-NEOPLASTIC LESIONS.

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