Supplementary Figure 1: Distribution and cytokine profile of EZH2⁺ T cells.

(a) EZH2⁺ T cells in different tissues. Snap-frozen tissues were stained for CD3 (red) and EZH2 (green). Magnification 40X, representative stainings for N = 15 (colitic colon) or N = 10 (tonsil and spleen) patients tested.

(b) Gating strategy to define polyfunctional T cells. Based on FSC, SSC and T cell staining, single CD3⁺CD4⁺ cells and CD3⁺CD8⁺ cells were gated for further analysis. To define cytokine profile for CD8⁺ T cells, CD8⁺ T cells were analyzed on the basis of TNF-α expression (gates R1 and R2). IFN-γ and granzyme B expression were analyzed within TNF-α-negative cells. This allows identification of the triple-negative population (R13), single positive TNF^–IFN-γ⁺GranB^– (R11), TNF^–IFN-γ⁺GranB^– cells (R14), and double positive and TNF^–IFN-γ⁺GranB⁺ population (R12). Analysis of TNF-positive (R2) cells allows assessment to the percentage of triple-positive (polyfunctional) TNF⁺IFN-γ⁺GranB⁺ population (R22), double positive subsets TNF⁺IFN-γ⁺GranB^– (R21), TNF⁺IFN-γ^–GranB⁺ (R24), and single positive TNF⁺IFN-γ^-GranB^– (R23). Therefore, the percentages of cells from gates R11, R14 and R23 demonstrate the frequency of single positive cells, while the total percentages of cells from R12, R21, and R24 are double-positive cells. Polyfunctional CD4⁺ T cells were similarly analyzed for TNF-α, IFN-γ and IL-2 expression.

(c) Effect of cisplatin on CD8⁺ T cell apoptosis. CD8⁺ T cells were activated with anti-CD3 and anti-CD28. On day 4 cisplatin was added into the culture and apoptosis was measured on day 5 with Annexin V and 7-AAD staining. Representative data for one of 5 different donors is shown.

(d) Effect of cisplatin treatment on EZH2 expression in CD8⁺ T cells. Freshly isolated peripheral blood CD8⁺ T cells were stimulated with anti-CD3/anti-CD28 antibodies. EZH2 expression was measured in the cells on day 4 (pre-treated cells) and compared with the cells treated with cisplatin on day 4 and harvested 24 hours later. Data presented as mean ± SEM, N = 6 donors for pre-treated cells and N = 4 donors for cisplatin-treated cells. *P < 0.05.