NS398 inhibition of PGS₂ enzymatic activity affects T-cell expression of CD25 during PHA activation. PBMCs were cultured in the presence of PHA (5 μg/mL) with or without NS398 (5 μM) for 24 hours. The expression of CD25 on CD3⁺ T cells was determined by dual-color flow cytometry. “All subjects tested” includes ICA⁺ diabetics, ICA⁺ moderate-risk subjects, ICA⁺ high-risk subjects, and those classified as AI. Baseline values for the percentage of CD25⁺/CD3⁺ cells in freshly isolated PBMCs were not significantly different between the groups assayed (controls: 4.61 ± SD 4.6, n = 12; other HLA subjects: 6.2 ± SD 6.7, n = 10; and DR04/DQ0302: 4.5 ± SD 3.9, n = 14). The data are presented as a fold increase, calculated as the percent of CD3⁺ T cells expressing CD25 when activated with PHA in the presence of NS398, relative to CD25 expression on CD3⁺ T cells activated with PHA alone. For individuals with multiple sample runs, the fold increase of each assay run was averaged to give a mean fold increase shown here. Statistical analysis demonstrated a significant difference between controls and all subjects (P = 0.04; Student’s t test). When HLA data were available, the CD25 data were also analyzed by this parameter. Enhancement of T-cell CD25 expression was most prevalent in subjects expressing both DR04 and DQβ0302 (P = 0.012; 1-way ANOVA), when compared with other subjects expressing HLA DR alleles 16, 03, 01, 07, 13a, and 08 and DQβ alleles 05, 0201, 0303, 0604, and 04.