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Summary

Bidirectional communication between the intestinal epithelial barrier and the immune cells in the underlying lamina propria is critical to human health. This study investigates the impact of human T cell subtypes on intestinal epithelial cell phenotype and survival. Blood-derived CD4+ or CD8+ T cells were isolated from healthy human participants using negative magnetic separation and cocultured with healthy human colonic epithelial stem cell spheroids suspended in Matrigel. A subset of T cells was activated by anti-CD3/CD28 cross-linking for 24 h prior to co-culture. Only activated CD4+ T cells (ACD4) caused significant epithelial cell damage and death as measured by histological scoring of brightfield microscopic images and zombie violet+ epithelial cells detected by flow cytometry.

The mechanism of cell damage and death was determined to be via soluble mediators, in that conditioned media from ACD4 but not activated CD8+ T cells (ACD8) significantly induced spheroid cell damage and death. A Luminex cytokine multiplex assay revealed that high levels of IFN-γ, IL17,and TNF-α were produced by ACD4 as compared with ACD8. The addition of neutralizing IFN-γ antibody or neutralizing IL-17 antibody to cocultures completely protected spheroids from death induced by ACD4. However, the addition of only IFN-γ, or IL-17 protein did not induce spheroid cell death. This finding conflicts with currently published literature that indicates IFN-γ protein is able to induce spheroid cell death for both humans and mice. In addition, ACD8 also secrete IFN-γ, although to a lesser extent than ACD4, yet do not initiate spheroid cell death. These results indicate that there is a unique mixture of immune mediator released by ACD4s that mediate spheroid damage and death.

To further investigate the mechanism of cell death and further elucidate the mix of soluble mediators contributing, the Pu·MA microfluidics System was used. T cells and spheroids were mixed in 5% Matrigel or various concentrations of TNF-α, IFN-γ, and IL-17 alone and in combination were added to spheroids and incubated for 72 hours. Flow chips stained with Annexin V indicated that activated CD4 T cells or a triple combination of TNF-α, IFN-γ, and IL-17 initiate apoptosis in epithelial spheroids.