Fig. 4.

Flow cytometric analysis, immunosuppressive function, and FOXP3 Treg-Specific Demethylated Region (TSDR) methylation status of CD4+CD127lowCD25+ Treg subsets within CD4+ peripheral blood lymphocytes. Quantification for the dependent variables included (a) CD45RA-RO+ Treg, (b) CD31+ Treg, (c) CTLA+ Treg, (d) CD49+ Treg, and (e) ItgB7+ Treg subset frequencies within peripheral blood over time. Difference in means (± SD) for each dependent variable grouped by time of treatment were determined by one-way ANOVA and Dunnett's post hoc test where p ≤ 0.05 compared to baseline (*). (f) Percent demethylation (± SD) within the TSDR of the FOXP3 intron from isolated Tregs before and at 2 and 6 months after initiation of sargramostim treatment. Differences in means (± SD) were determined by one-way ANOVA where p ≤ 0.05 compared to baseline (*). (g) Quantification of Treg-mediated suppression (± SD) of Tresp (CD4+CD25-) proliferation at various Tresp:Treg ratios. Treg-mediated suppression is calculated as % Inhibition = 1 – (% Proliferating Tresp:Treg ÷ % Proliferating Stimulated Tresp Alone) and is reported as percent inhibition. Linear regression analysis indicates r² ≥ 0.81, p < 0.0001 for all lines and significant elevation (p < 0.0001) from baseline (blue) compared to each month of treatment. (h) Correlation analysis for percent TSDR demethylation and corresponding Treg-mediated inhibition (Treg activity, AUC) at baseline (blue), 2-month (red), and 6-month (green) during sargramostim treatment, indicating a direct correlation of TSDR demethylation and Treg activity with r = 0.3212, p = 0.0004.