Supplementary Figure 8: NKILA expression in activated T cells is finely tuned by calcium-influx-mediated histone acetylation.

CD8⁺ T cells were primed by the autologous DCs pulsed with MDA-MB-231 lysates and retrieved from the co-cultures on indicated days. (a) A typical CpG island is present at the promoter region of NKILA. Each vertical bar represents a single CpG site. The transcription start site (TSS) is indicated by a curved arrow. BGS analysis confirmed the methylation status of NKILA promoter in T cells primed for 0 day, 1 day and 6 days (n = 3, P>0.05). 8–10 clones were selected for sequencing in one test. Black, deep gray, light gray and white circles represent complete methylation (70.1~100.0%), partial methylation (30.1~70%), little methylation (0.1~30.0%) and unmethylation (0%) on each site, respectively. (b) NKILA expression in the primed CTLs pre-treated with inhibitors for DNA methyltransferase 5’-Aza or DAC was evaluated by qRT-PCR (mean ± SEM, n = 3; P>0.05 compared with DMSO). (c) ChIP analysis for the localization of H3K9me3 and H3K27me3 at NKILA promoter in the primed CTLs (DC group) (mean ± SEM, n = 3, P>0.05 compared with CTLs co-cultured with naive DCs (control group)). (d) NKILA expression in the primed CTLs pre-treated with pan HDAC inhibitors TSA and SAHA, selective Class I HDAC inhibitors MS-275 and FK228, selective Class II HDAC inhibitors TMP269 and Class III HDAC inhibitors droxinostat was evaluated by qRT-PCR (mean ± SEM, n = 3, **P < 0.01, ***P < 0.001 compared with DMSO-treated primed T cells). (e) ChIP analysis for the localization of H4Ac and STAT1 at NKILA promoter in the primed CTLs pre-treated with DMSO, TSA and MS-275 (mean ± SEM, n = 3; *P<0.05; ***P<0.001). (f) ChIP analysis for the localization of HDAC8 and HDAC4, 5 and 7 at NKILA promoter in the primed CTLs (DC group) (mean ± SEM, n = 3; P>0.05 compared with CTLs co-cultured with naive DCs (control group)). (g) Representative images of calmodulin (CaM) nuclear translocation and binding to HDAC1 in the CTLs primed for 1 day, assayed by CaM and HDAC1 double staining and immunofluorescent confocal microscopy (n = 3). Scale bar, 10 μm. (h) Interactions of HDACs (HDAC1, HDAC2, HDAC3) with nuclear-translocated CaM in the nucleus of CTLs upon co-culturing with DCs pulsed with MDA-MB-231 lysates for 1 day were determined by immunoprecipitation (n = 3). (i) The scheme of the mechanism underlying NKILA expression and contribution to the sensitivity to AICD of different T cell subsets. P-values, unpaired two-tailed Student’s t-test (a-f).