Fig 6. Allosteric MALT1 inhibitors affect de novo induction of human Tregs in vitro but not the function of in vitro expanded blood Tregs.

(A) Naïve human Tregs expanded in vitro for 14 days in the presence of rapamycin and then treated for 2 days with DMSO (n = 9), Compound 2 (n = 5), mepazine (n = 5), z-VRPR-fmk (n = 5) or rapamycin (n = 9). Expanded and treated Tregs were then co-cultured with CTV labeled CD4+ T cells and stimulated with anti-CD3 at the indicated ratios for 3 days. Suppressive activity was assessed as the reduction in CD4+ responder T cell proliferation normalised to responder cell proliferation in the absence of Tregs. Data is presented as mean ± SEM. (B) Number of CD4⁺CD25⁺FoxP3⁺ human Tregs after 14-days expansion in vitro in the presence of Compound 2, mepazine or z-VRPR-fmk or rapamycin (n = 6). Data are presented as Box and whiskers of number of live Tregs. (C) Percentage of de novo induced Tregs from naïve human CD4⁺ T cells stimulated for 7 days with anti-CD3 antibody plus anti-CD28 antibody in the absence or the presence of TGFβ + IL-2 + rapamycin in the presence of Compound 2, mepazine or z-VRPR-fmk (n = 5). Media indicates the basal induction of FoxP3 in activated CD4+ T cells in the absence of TGFβ + IL-2 + rapamycin. Coloured dots indicate data from individual donors across different stimulations. Data are presented as Box and whiskers plot. For all experiments, Compound 2 was used at 10 μM, mepazine at 5 μM, z-VRPR-fmk at 100 μM and rapamycin at 100 nM. One-way ANOVA with Dunnett’s for multiple comparison test was used for statistical analyses.**:p<0.01.