LRRC32+ CD4+CD25hiFoxP3 T
appear to be more potent suppressors than LRRC32- CD4+CD25hiFoxP3 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). Tregs 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 Treg and activation surface markers (CD62L, CD69, GITR, CTLA4, CD45RO, and HLA-DR) in FoxP3+ and LRRC32+-gated populations of CD25hi cells were studied using flow cytometry. Stimulated CD4+FoxP3+CD25hi Tregs (top panel) & unstimulated CD4+FoxP3+CD25hiTregs (bottom panel). c) Composite summary of phenotypic analysis of unstimulated LRRC32-CD4+CD25hiFoxP3+ Tregs and stimulated LRRC32+ and LRRC32- CD4+CD25hiFoxP3+ Tregs. Black bars = unstimulated LRRC32- Tregs. Dark grey bars = stimulated LRRC32- Tregs. Light grey bars = stimulated LRRC32+ Tregs. 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) CD25hi 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- Tregs were subsequently tested in a mixed lymphocyte reaction utilizing syngeneic effectors (Teff, 20,000/well) and allogenic antigen presenting cells (50,000/well). Treg:Teff 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: Teffs 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); Treg:Teff ratio of 1:1: 89 cpm to 346 cpm. When titrating Tregs vs. Teffs, 3 replicates were performed at each titration for the LRRC32+ and LRRC32- Tregs except for in one assay set in which there was limited number of LRRC32+ Tregs. 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- Tregs.