Figure 5

LRRC32⁺ CD4⁺CD25^hiFoxP3 T _regs appear to be more potent suppressors than LRRC32^- CD4⁺CD25^hiFoxP3 and exhibit decreased CD62L upon activation. a) Expression of LRRC32 and LAP in CD4+ T cells rested overnight (top panel) or stimulated with plate bound anti-CD3 and soluble anti-CD28 (bottom panel). T_regs were selected from the top 5% CD25-expressing and FoxP3⁺ populations, as previously described. Confirmation of activation by expression of the surface markers CD40L and CD69 are also shown (top of each panel). b) The expression patterns of various T_reg and activation surface markers (CD62L, CD69, GITR, CTLA4, CD45RO, and HLA-DR) in FoxP3⁺ and LRRC32⁺-gated populations of CD25^hi cells were studied using flow cytometry. Stimulated CD4⁺FoxP3⁺CD25^hi T_regs (top panel) & unstimulated CD4⁺FoxP3⁺CD25^hiT_regs (bottom panel). c) Composite summary of phenotypic analysis of unstimulated LRRC32^-CD4⁺CD25^hiFoxP3⁺ T_regs and stimulated LRRC32⁺ and LRRC32^- CD4⁺CD25^hiFoxP3⁺ T_regs. Black bars = unstimulated LRRC32^- T_regs. Dark grey bars = stimulated LRRC32^- T_regs. Light grey bars = stimulated LRRC32⁺ T_regs. Data are expressed as the mean ± SEM from 3 individuals. Heteroscedastic variances and an independent t-test comparing stimulated LRRC32⁺ and LRRC32^- subsets were used for calculations of the p values which are reported along the x-axis, below each surface marker (*). d) CD25^hi cells were sorted and activated overnight using anti-CD3-coated plates and soluble anti-CD28 (1 microgram/ml). Cells were then resorted based upon LRRC32 expression. The suppressive capacities of these LRRC32⁺ and LRRC32^- T_regs were subsequently tested in a mixed lymphocyte reaction utilizing syngeneic effectors (T_eff, 20,000/well) and allogenic antigen presenting cells (50,000/well). T_reg:T_eff ratios are depicted above. Data summarize 3 independent experiments. Results are expressed as the mean ± SEM. p = 0.0001 and R²= 0.7244. Absolute proliferation values for the 3 experiments were as follow: T_effs alone: average of 31094 cpm to average of 47483 cpm (at least 6 replicates per assay), background: average of 24 cpm to 35 cpm (at least 6 replicates per assay); T_reg:T_eff ratio of 1:1: 89 cpm to 346 cpm. When titrating T_regs vs. T_effs, 3 replicates were performed at each titration for the LRRC32⁺ and LRRC32^- T_regs except for in one assay set in which there was limited number of LRRC32⁺ T_regs. In this case, only one replicate was performed at the 1:1 and 1:2 titrations, and two replicates were performed for the other titrations (0:1, 1:4, 1:8, and 1:16). We performed 3 replicates for each titration utilizing the LRRC32^- T_regs.