To quantify the suppressive function of CD8+ Treg cells, naive CD4+ T cells (5 × 104 cells per 100 μL) were mixed with CD8+ Treg cells (2.5 × 104 cells per 100 μL) and activated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, 11132D) at a 1:1 ratio for 15 minutes. CD4+ T cell activation was evaluated by quantifying phosphorylated ZAP70 by flow cytometry. For DAPT treatment, DAPT was added during the CD8+ Treg differentiation process and washed off before mixing the cells with CD4+ T cells. The inhibition efficiency of CD8+ Treg cells was calculated as follows: Inhibition efficiency = (pZAP70%(No Treg) – pZAP70%(Treg)) / pZAP70%(No Treg).

Naive CD4+ T cells were CFSE labeled and incubated with CD8+ Treg cells for 30 minutes. CD8+ Treg cells were then removed and CD4+ T cells were stimulated with T-Activator anti-CD3/anti-CD28 beads for 5 days. Proliferation of CD4+ T cells was determined by flow cytometric measurement of CFSE dilution.

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