Flow cytometric gating strategy for discerning T_H subsets. (A) Shown is a representative healthy donor PBMC analysis, whereby gated populations (from left to right) are indicated defining viable CD3⁺CD4⁺ T cells. (B) As indicated, viable CD3⁺CD4⁺ T cells, defined in panel A, are differentiated into T_CM (CD45RA⁻CCR7⁺), T_EM (CD45RA⁻CCR7⁻), and T_N (CD45RA⁺) populations. T_CM and T_EM populations are further gated on the basis of CXCR3 expression with CXCR3⁺ T_CM and T_EM populations being associated with a pre-T_H1 and T_H1 population, respectively, and CXCR5⁺ T_CM and T_EM populations being associated with non–pre-T_H1 and non-T_H1 populations, respectively. (C) Total CD45RA⁻ memory CD4⁺ T cells are gated based on CXCR5 expression. CXCR5⁺ cells are further divided into CD25 and CD57 subsets: germinal center (GC; CD57⁻CD25⁺), Dual (CD57⁺CD25⁺), light zone (LZ; CD57⁺CD25⁻), and outer zone (OZ; CD57⁻CD25⁻).