PMA/ionomycin-stimulated Treg and T_consubpopulations. PBMCs from 16 patients and 5 HDs were stimulated for 4 hours with PMA, a calcium ionophore (ionomycin), and protein transport inhibitor (brefeldin A) and stained with a panel of antibodies based on 29 surface markers, transcription factors, and cytokines (supplemental Table 1). Treg subpopulations A and B were identified within Tregs after stimulation with Treg profiles that distinguished responders from nonresponders to IST (supplemental Figure 3). (A) Following 4 hours stimulation with PMA and ionomycin in the presence of brefeldin, PBMCs were stained for surface and intracellular markers (supplemental Table 1, panel 2) followed by mass cytometry and viSNE on CD4⁺ T cells. Cytokine-secreting CD4⁺ T cells, including IFN-γ, IL-2–, IL-17–, and IL-4–secreting cells, localized distinctly with minimal overlap and outside “Treg’s area.” Both Treg A and Treg B populations show higher expression of IL-10 compared with “non-Tregs” and TNF-α–secreting Tregs, but there was no significant difference between Tregs A and B in terms of IL-10 expression. (B) Unlike the rest of cytokine-secreting CD4⁺ T cells, TNF-α–secreting cells were spread over several areas, including the Treg A subpopulation (red arrow). (C) SPADE analysis of Tregs A and B following 4 hours’ stimulation with PMA/ionomycin and intracellular staining (supplemental Table 1, panel 2). Although the TNF-α–secreting cells within the Treg A subpopulation reduces after IST in responder AA patients, there is no similar reduction in nonresponder patients (patient AA-11 is an IST nonresponder and AA-6 is a responder patient). (D-E) SPADE clustering based on CD45RA, CD45RO, CD27, and CD62L. The T_con subpopulations were defined as naïve (CD45RA⁺ CD45RO^- CD27^hi), memory (CD45RA⁻ CD45RO⁺ CD27^lo), central memory (CM; CD45RA⁻ CD45RO⁺ CD27^lo CD62L^hi), effector memory (EM; CD45RA⁻ CD45RO⁺ CD27^lo CD62L^lo), effector (CD45RA⁻ CD45RO⁺ CD27^lo), and terminal effectors (Tee; CD45RA⁺ CD45RO⁻ CD27^lo). T_con with effector phenotype expresses higher CD161 in nonresponder patients at the time of diagnosis compared with responder AA patients (patient AA-11 is an IST nonresponder and AA-6 is a responder patient). The frequencies of these subpopulations were not significantly different between IST responder and IST nonresponder patients at the time of diagnosis. The naïve T_con from IST nonresponder patients were expressing slightly higher CCR4 compared with responder patients (supplemental Table 2).