Figure 2. CD4⁺ CD28⁺ T_EM and CD4⁺ CD57⁺ PD1⁻ subsets exhibit high CD69 upregulation and CD4⁺ CD28⁺ T_EM exhibit high CD38 and HLA-DR upregulation following stimulation.

Human PBMCs from healthy donors were stimulated ex vivo with anti-CD3/CD28 beads for 3 d followed by brief restimulation in the presence or absence of PMA/Iono. PBMCs were analyzed directly ex vivo or following in vitro stimulated as described above by flow cytometry. (A) Representative flow plots of frequencies of CD69⁺ cells within risky memory T cell subsets pre-(top) and post-(bottom) stimulation. (B) Summary data of fold changes of frequencies of CD69⁺ cells within risky memory T cell subsets upon stimulation. (C) Representative flow plots of frequencies of CD38⁺ and HLA-DR⁺ cells within risky memory T cell subsets post-stimulation. (D) Summary data of fold changes of frequencies of CD38⁺ and HLA-DR⁺ cells within risky memory T cell subsets upon stimulation. (E) Representative viSNE plots of the median fluorescence intensity (MFI) of CD57, PD-1, CD28, CCR7 and CD45RA within the CD4⁺ T cell compartment. Red indicates high expression of a given marker, and blue represents low intensity. The red-rimmed population represents CD4⁺ CD28⁺ T_EM subset, and the purple-rimmed population indicates CD4⁺ CD57⁺ PD1⁻ subset. (F) Summary data of frequencies of CD57⁺, PD-1⁺ and CD28⁺ cells within risky memory T cell subsets (*, p < 0.05; n = 6 per experiment; data are representative of three independent experiments. Risky memory T cell subsets were defined by the following gating strategy: CD4⁺ CD28⁺ T_EM, CD3⁺ CD4⁺ CCR7⁻ CD45RA⁻ CD28⁺; CD8⁺ CD28^null, CD3⁺ CD8⁺ CD28⁻; CD4⁺ CD57⁺ PD1⁻, CD3⁺ CD4⁺ CD57⁺ PD1⁻; CD4⁺ CD28⁺ T_EMRA, CD3⁺ CD4⁺ CCR7⁻ CD45RA⁺ CD28⁺; CD8⁺ CD28⁺ T_EM, CD3⁺ CD8⁺ CCR7⁻ CD45RA⁻ CD28⁺; CD8⁺ CD28⁺ T_EMRA, CD3⁺ CD8⁺ CCR7⁻ CD45RA⁺ CD28⁺). G, Frequencies of CD57⁺ PD-1⁻ cells within the CD4⁺ CD28⁺ T_EM subset. H, Frequencies of CD28⁺ T_EM cells within the CD57⁺ PD-1⁻ subset.