Myeloma-experienced CD8 T cells generated during SCM with Treg depletion mediate potent myeloma-specific immunity after ASCT. MM-bearing FoxP3-DTR donor mice were treated with DT or vehicle control during mobilization with G-CSF and AMD3100. T cells were harvested from spleens containing mobilized cells unless otherwise specified. (A-C) Phenotype and function of antigen-experienced memory (CD44⁺CD49d⁺) and virtual memory (T_VM, CD44⁺CD49d^–) CD8 T cells in spleens from mice treated with DT (Treg-depleted) or vehicle control (non-Treg-depleted) during mobilization (n = 5 per group from 2 independent experiments). (D-G) MM-bearing recipient mice were lethally irradiated and transplanted with mobilized splenocytes including 5 × 10⁶ T cells from Treg-depleted or non-Treg-depleted donors (D-E; n = 10 per group) or 10 × 10⁶ BM cells and 2 × 10⁶ CD4 T cells from nonmobilized B6 donors and 2 × 10⁶ CD8 T cells from mobilized Treg-depleted or non-Treg-depleted MM-bearing donors (F,G; n = 15 per group from 2 independent experiments). Recipient mice were monitored for survival and tumor burden using M-band (G/A ratio) level. Dotted line indicates a statistically determined threshold for myeloma relapse. (H) M-band and survival after tumor rechallenge in long-term survivors (>100 days after ASCT) who were transplanted with CD8 T cells from Treg-depleted donors. Naïve B6 mice were injected with the same tumor as a nonimmune control. (I) Mice from (H) were then rechallenged with a different clone of Vk∗MYC (Vk12598) and their survival was monitored. Data represents mean ± SEM. Mann-Whitney test for 2 sample comparison and log-rank test for survival data. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.