Figure 2.

ABL501 weakens T_reg-mediated suppression through promoting effector T cell responses

(A–F) FACS sorted and ex vivo expanded CD25⁺CD127⁻CD4⁺ T_reg cells were co-cultured with conventional CD4⁺ or CD8⁺ T cells isolated from the same human donor in the presence of A375 melanoma cells expressing membrane-bound anti-CD3 scFv (A375-OKT3). (A) Schematic illustration of the ex vivo human T_reg cell suppression assay is shown. (B) FACS analysis of Foxp3 expression in ex vivo expanded T_reg cells is shown. (C and D) FACS analysis showing CTV dilution (C) or ELISA of IL-2 secretion (D) by CD4⁺ T or CD8⁺ T cells in the presence or absence of T_reg cells (effector T: T_reg = 4:1) is shown. (E and F) FACS analysis shows CTV dilution (E) or ELISA of IL-2 secretion (F) by CD4⁺ T or CD8⁺ T cells with the indicated antibodies at a final concentration of 66.8 nM in the presence of T_reg cells. (G) Isolated and ex vivo expanded T_reg cells were co-cultured with A375-OKT3 cells in the presence of the indicated antibodies at a final concentration of 66.8 nM. Proliferation index of T_reg cells was calculated by FlowJo software based on CTV dilution. (H) Isolated conventional CD4⁺ or CD8⁺ T cells were co-cultured with A375-OKT3 cells in the presence of the indicated antibodies at a final concentration of 66.8 nM. ELISA of IL-2 secretion by CD4⁺ T or CD8⁺ T cells is shown. Data were pooled from three to five independent experiments with two replications. Statistical significance was determined by unpaired t tests with two-tailed analysis in (D) or one-way ANOVA with Holm-Sidak multiple comparisons in (E)–(H). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and ns.