Figure 4: Myeloma progression is associated with a reduced ratio of IFNγ⁺ T_PHEX and T_PEX to myeloma cells.

Recipient C57Bl/6 × PTPRCA (CD45.1/CD45.2) mice were transplanted with HULK donor (IFNγ-YFP × IL-10-GFP × FoxP3-RFP; CD45.2) grafts. Recipients were either never injected with tumor (MM-free) or had controlled (MM-controlled) or progressive myeloma (MM-relapsed) at 6–7 weeks post-transplant. BM was harvested for analysis of CD8 T cells using flow cytometry. (A) Representative contour plots and frequency of PD-1+TIM-3⁻ and PD-1⁺TIM-3⁺ cells (MM-free n = 8; MM-controlled n = 10; MM-relapsed n = 19). Kruskal-Wallis with Dunn’s Test. (B) Representative contour plot and frequency of T_PEX (Ly108^hi PD-1⁺) cells. (MM-free n = 5; MM-controlled n = 9; MM-relapsed n = 24). Kruskal-Wallis with Dunn’s Test. (C) Representative contour plots and frequency of IFNγ⁺IL-10⁻ and IFNγ⁺IL-10⁺ cells (MM-free n = 8; MM-controlled n = 10; MM-relapsed n = 19). One-way ANOVA with Tukey’s test. (D) Representative contour plots and frequency of Pfp⁺GzmB⁻ and Pfp⁺ GzmB⁺ cells and (E) histograms and MFI of perforin in all Pfp⁺ cells (MM-free n = 5; MM-controlled n = 4; MM-relapsed n = 11). One-way ANOVA with Tukey’s test. (F) Sternum was harvested at MM relapse for VECTRA multispectral imaging of myeloma lesions in BM. White circles highlight populations of interest including TOX⁺ and TCF1⁺ CD8 T cells. (G) Quantification and (H) correlation of TIM-3⁺PD-1⁺ CD8 T (left) and MM (right) cell numbers in controlled and relapsed recipients. (I) ratio of T cells to myeloma for TIM-3⁺PD-1⁺ cells and T_PEX cells (MM-controlled n = 10; MM-relapsed n = 24). Mann-Whitney t test and Pearson r correlation. (J) Fold change in myeloma cell and TIM-3⁺ T cell number in MM-relapsed mice relative to MM-controlled mice. Mann-Whitney t test. (K) Sort purified PD-1⁺TIM-3⁺CX3CR1⁻ (IFNγ⁺ T_PHEX), CX3CR1⁺, or PD-1⁻ T cells were cultured with myeloma cells for 17 hours followed by Annexin V and 7AAD staining. % Killing (7AAD⁺) was calculated using a viability baseline from myeloma only wells. Error bars are from 3 biological replicates (PD-1⁻ from 2 replicates) with 5 mice pooled per biological replicate. Data is mean ± SEM. Each symbol represents an individual mouse. ** p<0.01, *** p<0.001, **** p<0.0001.