In vivo and in vitro Treg suppression assays

For in vivo suppression studies, 3×10⁵ Miltenyi (negative selection) enriched CD4⁺ and FACS sorted (>98% purity) CD45RB^high YFP⁻ cells from LNs and spleens of Foxp3^YFP-Cre/Cre mice were i.v. injected into the tail vein of Rag1 KO mice with or without 1×10⁵ Miltenyi (negative selection) enriched CD4⁺ and FCAS sorted (>98% purity) YFP^bright Treg cells from LNs and spleens of Foxp3^YFP-Cre/+ x CARMA1^{f/f (or f/+ or +/+)} x Rosa26^YFP mice.
For in vitro suppression studies, 1×10⁴ FACS sorted (>98% purity) CD4⁺ YFP⁻ conventional T cells from LNs and spleens of Foxp3^YFP-Cre/Cre mice were labeled with 5 μM CellTrace Violet and stimulated with 250 ng/ml of αCD3 mAb (145–2c11, Biolegend) in presence of 2.5 × 10⁴ T-cell depleted splenocytes and different concentrations (from 1:1 to 1:16) of Miltenyi (negative selection) enriched CD4⁺ and FACS sorted (>98% purity) YFP^bright Treg cells from LNs and spleens of Foxp3^YFP-Cre/+ x CARMA1^{f/f (or f/+ or +/+)} x Rosa26^{STOP f/f-YFP} mice. CD4⁺ YFP⁻ conventional T cell proliferation was read out after 72h, as previously described.^{41 (link)} Briefly, percentage of suppression was scaled from 0 (proliferation of conventional T cell in absence of Treg) to 100 (complete absence of proliferation).