CD8⁺T-cell exhaustion correlates with tumor burden in mice with relapsed myeloma after SCT. MM-bearing or naive (MM-free; green) B6 recipients were lethally irradiated (total body irradiation [TBI], 1000 cGy) and transplanted with B6 donor cells (10 × 10⁶ BM; 5 × 10⁶ T cells). MM-bearing mice were categorized as MM relapsed (above threshold; purple) or MM controlled (below threshold; orange) at 8 weeks after SCT. Mice were sacrificed, BM was harvested, and CD8⁺ T cells were analyzed using flow cytometry. (A) Illustration of experimental design. (B) Representative histograms and frequency of expression of DNAM-1, TIGIT, PD-1, LAG-3, and TIM-3 (n = 16-21 for MM relapsed; n = 19 for DNAM-1, TIGIT, and PD-1 for MM controlled and MM-free; n = 3-12 for LAG-3 and TIM-3 for MM controlled and MM-free; data are combined from 2 to 7 experiments [LAG-3 MM-free from 1 experiment]). (C) Spearman r correlation of DNAM-1, PD-1, and TIGIT expression on CD8⁺ T cells with MM cell (CD138⁺CD19⁻) number in the BM of MM-bearing mice (n = 36 combined from 7 experiments). (D) Ratio of CD45.1 splenocytes to CFSE-labeled MM (CTL index) was determined in BM of MM-controlled and MM-free mice at 18 hours after transfer of 25 × 10⁶ purity adjusted cells at 3 weeks after SCT (n = 3-4). FACS plots show MM frequency within CFSE⁺ cells. (E) Histograms and Spearman r correlation of CD107a⁺ (n = 7 from 1 experiment), IFN-γ⁺ (n = 14 from 3 experiments), and Ki67⁺ (n = 7 from 1 experiment) CD8⁺ T cells in BM with MM cell number. Control represents isotype control staining for CD107a and IFNγ, and staining of myeloma-free mice for Ki67. Data represent mean ± SEM. *P < .05, **P < .01, ***P < .001 (Mann-Whitney U test).