Figure 8
Colonic migratory Tregs in dMLN display superior immunosuppressive phenotype in colitis. Proximal colons of DSS-treated and control KikGR/Foxp3hCD2/hCD52 mice were photoconverted on day 12 (Colitis) and flow cytometric analysis or cell sorting were performed 24 h later. CD103, ICOS, LAG3, PD-1, and CTLA-4 expression in total Tregs in control mice, CD25++ and CD25+/– Tregs in DSS-treated mice in dMLN (a), and CTLA-4 expression in CD25++ and CD25+/– photoconverted and non-photoconverted Tregs (b). These flow cytometry data are representative of at least four independent experiments. (c) The proportions of IL-10-producing cells out of photoconverted and non-photoconverted Tconvs and Tregs in dMLN stimulated with PMA/ionomycin and analyzed by flow cytometry; values in the plots indicate the percentage of the parent population. These data are representative of at least two independent experiments. Data in bar graphs are represented means±s.e.m. (n=4). Statistical comparisons were performed using Mann–Whitney’s U-test (*P<0.05). (d) CellTrace Violet-labeled CD4+CD25- cells were co-cultured for 72 h with sorted photoconverted and non-photoconverted Tregs from dMLN of DSS-treated mice on day 12 (in the ratio of 1 Treg to 4 CD4+CD25− cells) with or without stimulation with anti-CD3ɛ and anti-CD28 antibodies. Fluorescent intensity of CellTrace Violet-labeled CD4+CD25- cells was measured by flow cytometry. These data are representative of five independent experiments; values in the histogram plots indicate the percentage of the parent population. The graph shows percentage inhibition relative to “No added Treg” group. Each symbol represents an individual replicate. Data in bar graph are represented means±s.e.m. (n=5). Statistical comparisons were performed using Mann–Whitney’s U-test (**P<0.01, *P<0.05).
