Fig 1: Copanlisib strongly inhibits Treg development, while having minor effects on CD8+ T cells in vitro. Naïve CD4+ T cells from two healthy donors were differentiated in vitro to Tregs. The expression of FOXP3+CD25+ was analyzed by flow cytometry after 6 days of treatment with copanlisib (pan-PI3K inhibitor), idelalisib (PI3Kδ inhibitor), alpelisib (PI3Kα inhibitor), TGX-221 (PI3Kβ inhibitor), or eganelisib (PI3Kγ inhibitor) as single agents (A) or in combinations (B) and shown as % positive cells, normalized to the DMSO control. After 6 days of treatment with copanlisib or DMSO, the viability (C) and proliferation (D) of cells undergoing in vitro Treg differentiation was assessed by flow cytometry. Data are presented as mean ± SD (n = 2). The Secretion of IL-10 in Treg differentiation culture of naïve CD4+ T cells was measured after 5 days of treatment with DMSO or copanlisib (E). Data are presented as mean + SD (n = 3). CD8+ T cells were cultured in vitro and treated with copanlisib or DMSO. After 6 days, the viability (F) and the expression of T cell activation markers CD25, 4-1BB, and PD-1 (G) was assessed by flow cytometry. After 7 days, the proliferation was assessed by flow cytometry (H). Data are presented as mean ± SD (n = 2). CD8+ T cells were cultured with or without Tregs and treated with copanlisib (10 nM) or DMSO. CD8+ T cell proliferation was measured by flow cytometry after 6 days (I). Data are presented as mean ± SD (n = 3). p values were calculated by one-way ANOVA and Šídák's multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)