Fig 2: ICOSLG upregulation in SEM::EGR3 cells leads to the rapid development of Tregs upon co-culture with primary T-cells(A) Flow cytometry gating strategy of the co-cultured cells. Cells with low granularity (low SSC) were gated out of all cells. CD3+ cells were selected to subsequently separate CD4+ and CD8+ cells. Viability after co-culture of these populations was assed using Annexin V and 7-AAD staining to ensure that the majority of T-cells were alive throughout co-culture. CD25+FOXP3+ cells (Tregs) were quantified out of the CD4+ population.(B) Percentages of CD4+CD25+FOXP3+ Tregs among CD4+ T-cells after co-culturing T-cells from six healthy donors (HD1-HD6) with SEM::mock or SEM::EGR3. SEM::EGR3 co-culture led to 7.96%-23.94% more Tregs than SEM::mock co-culture (geometric mean: 1.14 ± 0.009991, p = 0.0023 in a two-tailed ratio paired t test).(C) Replication of the co-culture experiment of HD1-HD3 under the addition of 20 µg/mL mouse IgG1 isotype control or neutralizing monoclonal mouse a-human ICOSLG antibody demonstrated that EGR3-mediated Tregs induction could be impaired through ICOSLG antibody blockade.(D and E) ELISAs of the co-culture supernatants depicted that ICOSLG antibody blockade has no effect on the IL-2 level (D) but strongly decreased the IL-10 level (SEM::mock: p = 0.0424; SEM::EGR3: p = 0.0002) (E) of the media. Significance was tested with multiple t tests.