TFIs reinvigorate T-cell function. (A) Timeline of continuous vs TFI T-cell stimulation with AMG 562 over 28 days. (B) Percentage of CD8⁺ T cells coexpressing PD-1, Tim-3, and LAG-3; n = 6. The spider plot (right) indicates coexpression on day 28 in continuously stimulated vs rested T cells from 1 representative donor. (C) Cytokine levels determined by CBA in coculture supernatants on day 17; n = 6. (D) Percentage of IFN-γ and TNF-α double-positive CD4⁺ and CD8⁺ T cells after PMA/ionomycin restimulation on day 28 of coculture; n = 3. Representative plots of CD8⁺ T cells from 1 donor are shown. (E) AMG 562-mediated CD2⁺ fold change (n = 3), cytotoxic capacity against hCD19-Ba/F3 cells (n = 6) and granzyme B expression (n = 6) of isolated T cells after 14 or 28 days of coculture. (F) TCR expression of T cells (n = 3) quantified by immunophenotyping during coculture. (G) Timeline of AMG 562+dasatinib-mediated T-cell stimulation in comparison with continuous or TFI stimulation over 17 days. Dasatinib = 100 nM. (H) Percentage of CD8⁺ T cells coexpressing PD-1, Tim-3, and LAG-3; n = 3. (I) T-cell proliferation, cytotoxicity, Granzyme B, and TCR expression; n = 3. Boxplot whiskers indicate minima and maxima, and boxes represent the lower quartile, the median, and the upper quartile. Bar graphs present mean ± SEM values. Statistical analysis: 2-way ANOVA and Sidak’s multiple comparison test (B,C,E-F,H–I); ^nsP > .05; *P < .05; **P < .01; ***P < .001; ****P < .0001. CBA, cytometric bead array; LAG-3, lymphocyte activation gene 3; ns, not significant; PD-1, programmed cell death protein 1; PMA, phorbol myristate acetate; ± SEM, standard error of the mean; TCR, T-cell receptor; TFI(s), treatment-free interval(s).