Reciprocal responsiveness to IL-12 and IFN-α/β correlates to development of T_EM and T_CM cells. (A) Day 3, neutralized or IL-12 plus IFN-α–activated cells were assessed for surface expression of IL-12Rβ2 or IFNAR2 or (B) expression of CCR7, IL-12Rβ2, and IFNAR2. (C) Day 5, PBSE-labeled cells were assessed for IL-12Rβ2 and IFNAR2 expression as a function of division (top), and relative mean fluorescence intensity was quantified (bottom). (D) Day 5, PBSE-labeled cells were gated on division 0 (magenta), division 1-3 (teal), or division 4⁺ (orange), and chemokine receptor (middle panels) and cytokine receptor (right panels) expression was measured. (E,F) Day 5 cells were polarized with IL-12 + IFN-α, reactivated with cytokines for 30 minutes, and assessed for intracellular STAT or phospho-STAT protein expression. (E) Total STAT, STAT4 (left panels), or phospho-STAT1, phospho-STAT4 (right panels) expression in live CD8⁺ gated cells. The 2° + 3° antibody alone (gray), unstimulated (black), IFN-α (teal), or IL-12 treated (orange). (F) Dot plot overlays of phospho-STAT1 (top panel, right) or phospho-STAT4 (bottom panel, left) expression as a function of CFSE dilution. Unstimulated (black), IFN-α treated (teal), or IL-12 treated (orange). Quantification of mean fluorescence intensity as a function of CFSE dilution (right panels); phopho-STAT1 (top), phospho-STAT4 (bottom).