Exhausted CD8⁺T cells can be defined by lack of DNAM-1 expression in mice with relapsed myeloma after SCT. MM-bearing or naive (MM-free; green) recipients were transplanted as previously described with BM+T grafts from B6 donors. MM-bearing mice were categorized as MM relapsed (purple) or MM controlled (orange) at 6 or 8 weeks after SCT. Mice were sacrificed, BM and spleen were harvested, and CD8⁺ T cells were analyzed using flow cytometry. FACS plots and histograms are representative. (A-C) Phenotyping performed on CD8⁺ T cells from BM of MM-relapsed mice. (A) CD44 and CD62L populations in PD-1⁻ or TIGIT⁺ or PD-1⁺ or LAG-3⁺ or TIM-3⁺ subsets. Frequency of T_EFF/EM (CD44⁺CD62L⁻) cells within each positive subset was compared with PD-1⁻ cells (n = 5-11 combined from 2 to 4 experiments). (B) Frequency of TIGIT and PD-1 coexpression (n = 13-18 combined from 5 experiments). (C) Frequency of TIM-3 and LAG-3 coexpression on PD-1⁺ CD8⁺ T cells from BM of MM-relapsed mice (n = 11 combined from 4 experiments). (D) Frequency of CD101⁺CD38⁺ and CD101⁻CD38⁻ CD8⁺ T cells (n = 8-11 from 2 experiments) and (E) t-distributed stochastic neighbor embedding (tSNE) analysis and frequency of exhausted and responsive CD8⁺ T-cell phenotypes in BM of MM-relapsed, MM-controlled, and MM-free mice (n = 4-8; representative of 2 independent experiments). (F) Frequency of PD-1 and TIGIT expression on DNAM-1⁺ (teal) and DNAM-1⁻ (magenta) subsets (n = 16 combined from 6 experiments). (G) Frequency of Eomes^hiT-bet^dim/− CD8⁺ T cells in DNAM-1⁺ and DNAM-1⁻ subsets from MM-relapsed mice (n = 10 combined from 2 experiments). (H) Frequency of Eomes expression within DNAM-1⁺ and DNAM-1⁻ subsets from BM of MM-relapsed mice (n = 10 combined from 2 experiments). (I) Recipients were transplanted with FoxP3-RFP × IL-10-GFP reporter donors. Frequency of IL-10 producing CD8⁺ T-cells within DNAM-1⁺ and DNAM-1⁻ subsets in BM of MM-relapsed mice (n = 8 combined from 3 experiments). Data represent mean ± SEM. Mann-Whitney U test. **P < .01, ***P < .001.