Treg mediated suppression is lower in polyclonally activated CD4⁺ Teff cells in PTB.

Teff and Treg populations were isolated either by magnetic beads or flow cytometry cell sorting. Data shown in A–C is from experiments carried out using Teff and Treg cells isolated by magnetic beads and that shown in D–E is from experiments carried out using Teff and Treg cells isolated by flow cytometry sorting. CFSE labelled Teff cells were co-cultured with Treg cells at a 1:0.5 or 1:1 (C) or 1:1 (E) ratio. Cells were activated with mitogenic anti-CD3/anti-CD28 beads at a bead:Teff cell ratio of 2:1 (A–C) or 1:1 (D–E). Proliferation was measured by CFSE dilution after 4 days of culture. Representative FACS plots of Teff cell CFSE dilution in presence or absence of Treg cells from different clinical categories are shown in (A). Percentage suppression was calculated as described in Materials and Methods and is compared between different categories (C, E). Proliferation of Teff cells in absence of Tregs was compared among all clinical categories used and is shown in B and D. Data shown in B and D is mean + SEM and in C and E is median frequency/range. All data shown is from multiple donors in each category: IGRA-ve N = 12 (B, C) and 8 (D, E); IGRA+ve N = 4 (B, C) and 7 (D, E); PTB N = 10 (B, C) and 17 (D, E); ATT 6 months N = 5 (D, E). P value was determined by non-parametric One-Way ANOVA Kruskal–Wallis test and Dunn’s multiple comparisons test. ***p < 0.001, **p < 0.01, *p < 0.05.