(A) Schematic for in vitro stimulation of GP33-specific P14 CD8⁺ T cells isolated from spleens of naive mice. P14 cells were activated with anti-CD3 and anti-CD28 day 0–2, followed by resting (“Acute-2d”), or by 2 rounds of GP33 peptide stimulation day 2–7 followed by resting until day 19 (“Acute-7d”). (B) Representative FACS plots showing expression of PD-1 and CD44 or Tim3 on P14 cells from day 0 to day 2 and summary bar graphs showing frequency of PD-1⁺CD44⁺ or PD-1⁺Tim3⁺ P14 cells on days 0 and 2. (C) Longitudinal tracking of % PD-1⁺CD44⁺ P14 cells for either Acute-2d (bottom line, green) or Acute-7d (top line, blue) or (D) % PD-1⁺Tim3⁺ P14 cells from day 0–19. (E) Expression levels (shown by geometric mean fluorescence intensity, gMFI) of CD62L from Acute-2d or Acute-7d P14 cells on days 7 and 19. (F) Bar graphs showing % of CD62L⁺Ccr7⁺, total CD62L⁺, and total Ccr7⁺ P14 cells on day 19. (G) Schematic for adoptive transfer of 100K Acute-2d or Acute-7d in vitro–stimulated P14 cells in congenically distinct C57BL/6 mice on day 19, followed by acute LCMV infection and analysis day 6–7 postinfection. (H) Fold expansion of P14 cells relative to endogenous GP33-specific CD8⁺ T cells on day 6–7 postinfection. (I) Representative FACS plots and % Ifnγ⁺CD107a⁺ P14 cells after ex vivo GP33 peptide restimulation of splenocytes. For B–F n = 4 biological replicates, representative of 2 to 3 independent experiments. For H and I, data were pooled from 2 independent experiments, each with n = 3–4 biological replicates per group. *P < 0.05, ***P < 0.001, ****P < 0.0001. Comparisons were determined by 2-way ANOVA (C and D) or Mann–Whitney U test (unpaired, 2-sided) (B, E, F, H, and I). Error bars indicate mean ± SEM.