Supplementary Figure 6: Tumor-specific T cells from patients with breast and lung cancer have high amounts of NKILA and are sensitive to AICD.

(a) The expression of BCL2, BCL2L1, IER3 and GADD45B, normalized to GAPDH, in different T cell subsets isolated from peripheral blood (red) and tumor tissues (green) of breast cancer patients was evaluated by qRT-PCR (mean ± SEM, n = 24, ** P<0.01, *** P<0.001 compared with tumor-specific CTLs (CD8⁺MUC1-tetramer⁺ cells)). (b) IFN-γ releasing T cells from the peripheral blood of breast cancer patients were quantified by IFN-γ ELISPOT (n = 3) and isolated by IFN-γ capture assay (n = 48). (c, d) NKILA expression in IFNγ-releasing CD8⁺ (upper panel) and CD4⁺ (lower panel) T cells isolated from the tumor tissues (c) and peripheral blood (d) of breast (n = 48) and lung cancer patients (n = 32) was evaluated by qRT-PCR. ***, P<0.001 compared with non-IFNγ-releasing T cells. (e) Apoptosis of IFNγ-releasing CD8⁺ and CD4⁺ T cells isolated from the peripheral blood of breast (n = 5) and lung cancer patients (n = 5) induced by FasL was determined by Annexin V/PI staining and the bars correspond to FasL-induced apoptotic rates (mean ± SEM). ***, P<0.001. (f-h) The isolated tumor-infiltrating T_H1 were transduced with shNK1, shNK2, or shvec (f) and the isolated tumor-infitrating T_H2 (g) and T_reg (h) were transduced with NKILA or vec, followed by co-culturing with autologous tumor cells with or without anti-FasL antibody. Apoptosis of T cells was determined by Annexin V/PI staining and the bars correspond to percentages of Annexin V⁺ cells (mean ± SEM, n = 3, *P<0.05, **P<0.01, ***P<0.001 compared with the untreated T cell subsets (-); ^#P<0.05, ^##P<0.01, ^###P<0.001 compared with the T cell subsets co-cultured with primary tumor cells). P-values, unpaired two-tailed Student’s t-test (a, e-h) and paired two-tailed Student’s t-test (c, d).