Fig. 1.

Screening of epigenetic targets that regulate allogeneic T-cell responses. a Peripheral blood CD3⁺ T cells were stimulated with artificial antigen-presenting cells that express a membrane-bound form of anti-CD3 mAb (clone OKT3), CD80, and CD83 (aAPC/mOKT3) and individually treated with epigenetic chemical probes for 6 days. The probe-treated T cells were labeled with CFSE and cocultured with T-cell-depleted peripheral blood mononuclear cells (PBMC) from a different donor to evaluate allogeneic T-cell responses or cultured in the presence of IL-2 and IL-15. b, c T-cell division (b) and expression of CD69 (c) in the CFSE-labeled CD4⁺ and CD8⁺ T cells were analyzed on day 4 (n = 3 technical replicates, ordinary one-way ANOVA with Tukey’s multiple comparisons test). *P < 0.05, **P < 0.01 compared with the DMSO control. Dashed lines denote the average values of the DMSO control samples. d, e CD3⁺ T cells treated with 0.5 μM SGC0946 or DMSO were labeled with CFSE and cocultured with allogeneic PBMC. The frequency of CFSE-diluted cells (d) and CD69⁺ cells (e) was analyzed on day 4 (n = 4 cultures, unpaired two-sided t-test). Experiments were repeated three times with different samples, and similar results were obtained. f CD3⁺ T cells were transduced with control shRNA or shRNA against DOT1L and cocultured with allogeneic PBMC. The expression of CD69 in the shRNA-transduced T-cell population was analyzed on day 4 (n = 5 cultures, ordinary one-way ANOVA with Tukey’s multiple comparisons test compared with the control). Horizontal lines represent the means ± s.d