Figure 1.

Representative T-cell gating plots and subsets. Following doublet exclusion PBMCs were gated for CD3⁺CD4⁺ T cells (not shown), before being separated into four core subsets (a): Treg, Tfr, Tfh and CXCR5⁻FoxP3⁻ (designated ‘DN’ for brevity)). Treg (b) and Tfr (e) were comprised of ‘resting’ FrI (CD45RA⁺FoxP3^lo), ‘activated’ FrII (CD45RA⁻FoxP3^hi) and ‘cytokine-producing’ FrIII (CD45RA⁻FoxP3^lo) fractions. Treg (c) and Tfr (f) contained both Helios⁺ and Helios⁻ subsets. The majority of Treg (d) and Tfr (g), contrasted here (grey) against CD4⁺FoxP3⁻ T cells (black), fit the conventional CD25⁺CD127^lo Treg cell description. Conventional and follicular regulatory fractions and helper populations were analysed for their composition of Th1-like (CCR6⁻CXCR3⁺), Th2-like* (CCR6⁻CXCR3⁻), Th17-like (CCR6⁺CXCR3⁻) and Th17.1-like (CCR6⁺CXCR3⁺) subsets (h). Th17-like (i) and Th17.1-like (j) subsets were further examined for CD146 and CD161 expression. CD45RA⁻CCR7⁻ effector memory (EM) cells were gated within conventional and follicular helper subsets (k). *Analysis of Th2-like cells was confined to CCR6⁻CXCR3⁻ EM cells.