Continuous stimulation with AMG 562 induces T-cell exhaustion. (A) Timeline of continuous T-cell stimulation with AMG 562 and functional testing over 28 days. (B) Percentage of CD8⁺ T cells coexpressing PD-1, Tim-3, and LAG-3 and the MFI ratio of TOX during continuous AMG 562 stimulation; n = 6. (C) Cytokine and granzyme B levels in coculture supernatants determined by CBA; n = 3–9. Significant differences compared with day 3 are indicated. (D) Percentage of IFN-γ and TNF-α double-positive CD4⁺ and CD8⁺ T cells after PMA/ionomycin restimulation. Representative examples of CD8⁺ T cells from 1 donor are shown; n = 3. (E-F) AMG 562-mediated CD2⁺ fold change (n = 3) and cytotoxicity against hCD19-Ba/F3 cells (E) or OCI-Ly1 cells (F) after 3 days (AMG 562/cBiTE = 5 ng/mL, E:T = 1:1); n = 6. Data are mean ± SEM values. Statistical analysis: Kruskal–Wallis and Dunn’s multiple comparison test (B,C,E,F); *P < .05; **P < .01; *** P < .001; ****P < .0001. CBA, cytometric bead array; E:T, effector/target ratio; LAG-3, lymphocyte activation gene 3; MFI, median fluorescence intensity; PD-1, programmed cell death protein 1; PMA, phorbol myristate acetate;± SEM, standard error of the mean.