Extended Data Fig. 5: TCF-1 overexpression promotes memory T cells formation in ECSIT deficiency CD8⁺ T cells in vitro.

(a, b) Ecsit^fl/fl OT-1 (WT) and Ecsit^fl/fl Rosa26^CreERT2 OT-1 (iKO) splenocytes were activated with OVA and IL-2 for 2 days, followed by sorting of CD8⁺ T cells, then CD8⁺ T cells were transduced with vector (EV) or TCF-1 (TCF-1 OE) by the retroviral system and cultured in IL-15 for 3 days to induce IL-15-mediated differentiation of memory T cells in the presence of 4⁻OHT. (a) Flow cytometry gating strategy of TCF-1 overexpression CD8⁺ T cells. (b) Immunoblot analysis of ECSIT expression in WT + EV, WT + TCF-1 OE, iKO+EV and iKO+TCF-1 OE cells. (c) Schematic of the experiments. Ecsit^fl/+ CD45.1.2 OT-1(WT) and Ecsit^fl/fl Rosa26^CreERT2 CD45.2 OT-1(iKO) mice splenocytes were activated with OVA and IL-2 for 2 days, followed by sorting of CD8⁺T cells, then CD8⁺T cells were transduced with vector (EV) or TCF-1 (TCF-1 OE) by the retroviral system and cultured in IL-15 for 3 days to induce IL-15-mediated differentiation of memory T cells in the presence of 4-OHT. WT and iKO memory CD8⁺T (GFP⁺) were mixed at a 1:1 ratio and transferred into CD45.1 recipient mice, and then the recipient mice were infected with VSV-OVA 1 day later. OT-1 cells were analysed on day 5 after infection in the spleen. (d, e) Representative flow cytometry plots (left) and the analysis of percentage (right) of OT-1 cells (GFP⁺ Kb-OVA⁺ CD44⁺ CD8⁺ T) (d) and IFN-γ⁺ cells (e) gated on GFP⁺ Kb-OVA⁺ CD44⁺ CD8⁺ T cells in the spleen. Data are pooled from two independent experiments and n = 4 for (d-e). Error bars show mean ± sem. ***P ≤ 0.001, ns, not significant. Two-tailed unpaired student’s t-test for (d-e).

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