Figure 6.

Poly-functional CD4⁺ T cells display both T_EM and T_CM phenotypes. PBMC isolated from five subjects following ZOSTAVAX vaccination were thawed and rested overnight before VZV antigen stimulation. PBMC were stimulated with various VZV antigens for 5.5 h at 37°C/5% CO₂, with brefeldin A and monensin added 30 min after the initial incubation. Cells were stained with Vividye, surface stained with fluorescent primary antibodies against CCR7, CD45RA, and CD45RO. After washing off excess antibodies, PBMC were fixed, permeabilized, and stained with fluorescent primary antibodies to identify T cells producing IFNγ, IL-2, TNFα, and CD154 as described in Figure 3. Boolean analysis was performed to identify poly-functional CD4⁺ T cells with 4+ functions. (A) CCR7, CD45RA, and CD45RO expressions of 4+ (CD154⁺IFNγ⁺IL-2⁺TNFα⁺) antigen-specific CD4⁺ T cells (red dots) overlayed on CD3⁺ T cells (gray contours). Numbers in dot plots represent percentage mean ± SEM for the five subjects. (B) The GMFI of IFNγ, IL-2, TNFα, and CD154 was assessed in both T_CM (CCR7⁺CD45RA⁻CD45RO⁺) and T_EM (CCR7⁻CD45RA⁻CD45RO⁺) 4+ antigen-specific CD4⁺ T cells.