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. 2026 Oct 16;17:1910235. doi: 10.3389/fimmu.2026.1910235

Figure 4.

Panel A shows flow cytometry contour plots comparing IFNγ and Granzyme B expression between 0 micromolar and 1 micromolar Nav treatment, with a bar graph quantifying higher percentages of IFNγ and Granzyme B double-positive cells at 1 micromolar. Panel B shows similar plots and quantification with lower but significant increases in IFNγ and Granzyme B at 1 micromolar Nav. Panel C displays microscope images at 0 and 160 hours illustrating reduced cell confluence after 1 micromolar Nav treatment. Panel D is a line graph of tumor confluence percentages over time, showing greater reduction with higher Nav concentrations and PBMC ratios. Panel E presents four bar graphs comparing mean fluorescence intensity of Tox, IFNγ, PD-1, and Tim-3 in human PBMCs, highlighting statistically significant increases with 1 micromolar Nav. Panel F shows flow cytometry plots of PBMCs for Tox, Tim-3, PD-1, and TOX expression under both conditions.

MDM2 inhibition constrains functional exhaustion and improves anti-tumor immunity of MAGE-A4 directed CD8 T cells. Isolated human naïve CD8+ T cells from healthy donors were activated with CD3/CD28/IL-2 for 5 days with or without 1µM of Navtemadlin. Tumor-specific MAGE-A4 TCR was transduced to CD8+ T cells on day 2. On day 6, TCR-engineered T cells were co-cultured with MAGE-A4 positive melanoma cells (M407) without additional use of Navtemadlin. Different ratios of effectors (MAGE-A4 TCR+ CD8+ T cells)-to-target (M407 melanoma) were tested, including 1:1, 1:5, and 1:10. Cytotoxic functions of CD8+ T cells were measured and quantified before (A) and after (B) co-culture with M407 melanoma (n ≥ 2 biological replicates). (C) Representative images of the co-culture before and after the tumor-killing assay show increased clearing of tumor cells (red dots) by TCR-engineered T cells that were previously treated with 1 µM of Navtemadlin. (D) Previously NAV-treated CD8+ T cells showed increased tumor clearing across different effector-to-target (E:T) ratios (n = 3 biological replicates). (E) TOX, TIGIT, PD-1 and Tim-3 MFI values were measured after the co-culture when gating on CD8+ and MAGE-A4 TCR+ cells at 1:1 E:T ratio. (F) Representative flow plots showing PD-1, TIM-3, and TOX expression gated on live CD8+ TCR+ cells. Unpaired T-test was performed, and statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent mean and error bars indicate SEM.