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. 2025 Mar 21;5(3):477–496. doi: 10.1158/2767-9764.CRC-24-0478

Figure 3.

Figure 3

ULBP6 may be the most potent NKG2D ligand. A, Equilibrium dissociation constant KD for human NKG2DLs binding to NKG2D as measured by a Biacore surface plasmon resonance assay. Data represent mean ± SD of three technical replicates per condition. ULBP4 is not shown, as no binding to NKG2D was detected. B, IFNγ concentration of the supernatants of IL-2/IL-15–primed PBMCs cocultured with COV644 cells and 0.05 to 200 nmol/L recombinant sULBP6-02 or sMICA for 24 hours, depicted as a concentration-dependent response. No IFNγ was detected in COV644 cell supernatants without PBMC. Data represent mean ± SEM of three technical replicates per condition from one of three biological replicates. C, IFNγ concentration of the supernatants of IL-2/IL-15–primed PBMCs cocultured with 200 nmol/L plate-bound ULBP4 or MICA ± 250 nmol/L recombinant sULBP6-02 for 24 hours. Data represent mean ± SD of three technical replicates per condition from one of four biological replicates and were analyzed using one-way ANOVA for statistical significance. D, Cell surface NKG2D expression on human NK (CD45+ CD3– CD56+) cells or CD8+ T (CD45+ CD3+ CD56– CD8+) cells analyzed from IL-2/IL-15–primed PBMCs cultured with 50 nmol/L recombinant sULBP6-02 or PBS. Data represent mean ± SD of three technical replicates per condition from one of two biological replicates. Welch’s t test was used for statistical analysis. E, Quantification of COV644-GFP cell growth, as measured by GFP area per well by an Incucyte live cell analysis system, in the presence of IL-2/IL-15–primed PBMCs and 100 nmol/L recombinant sULBP6-02. Quantification is represented continuously over a 5-day time course. Data represent mean ± SD of three technical replicates per condition from one of four biological replicates. The last time point was analyzed using an unpaired t test for statistical significance. F, μMT−/− mice were inoculated with syngeneic MC38 EV or MC38 ULBP6-02 cells by subcutaneous injection (n = 10 mice per group). Nineteen days after inoculation, tumors were resected and processed for flow cytometric analysis. Bar graphs show the percentage of total tumor-infiltrating CD45+, NK (CD45+ Thy1.2– NK1.1+), NKT (CD45+ Thy1.2+ NK1.1+), and CD8+ T (CD45+ Thy1.2+ NK1.1– CD8+) cells and the percentage of CD25+ NK and NKT cells. Data represent mean ± SEM from one of two independent experiments and were analyzed using Welch’s t test for statistical significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. ns, not significant.