Fig. 3: NKG2D upregulation enhances T and NK cell cytotoxicity, sensitizes tumors to combined αPD1 and αVEGF therapy.

A Flow cytometry analysis of the number of CD8⁺ T cells, NK cells, and MDSC in Nf2^−/− tumors treated with control IgG (Ctrl, n = 10), αPD1 (n = 9), αVEGF (n = 10), and combination antibodies (Comb, n = 10). *P = 0.01. B ELISA of murine IFNγ and TNFα in Nf2^−/− tumor from different treatment groups (N = 6 tumors/group). P = 0.01. C Flow cytometry analysis of the proportion of CD8⁺ T cells expressing granzyme B (P = 0.0008) and perforin (P = 0.008) and NK cells expressing granzyme B (P = 0.0008) and perforin (P = 0.0007) in Nf2^−/− tumors treated with control IgG (n = 11) or αVEGF antibodies (n = 12). D Schematic and timeline of CD8 T cell and NK cell depletion in Nf2^−/− sciatic nerve model. Mice bearing Nf2^−/− tumors in the sciatic nerve were treated with anti-CD8 (200 μg/mouse) and anti-NK (200 μg/mouse) antibodies, followed by control IgG, αPD1, αVEGF antibody treatments two days later. E Flow cytometry analysis of the percentage of CD8⁺ T cells and NK cells in Nf2^−/− tumors treated with control IgG (Ctrl, n = 6), αPD1 + αVEGF (n = 7), αPD1 + αVEGF + αΝΚ (n = 7, P = 0.006 compared to αPD1 + αVEGF), and αPD1 + αVEGF + αCD8 (n = 5, P = 0.008 compared to αPD1 + αVEGF). F Sciatic nerve Nf2^−/− tumor diameter was measured with a caliper every 3 days following treatment. αPD1 + αVEGF vs. αPD1 + αVEGF + αCD8: P = 0.001 (pink); αPD1 + αVEGF vs. αPD1 + αVEGF + αΝΚ: P = 0.003 (blue). N = 24 mice/group. G In vitro T cell and NK cell cytotoxicity assay. Nf2^−/− tumor-bearing mice were treated with control IgG or αVEGF for 21 days. Tumor-associated CD8⁺ T cells and spleen NK cells were isolated. CD8⁺ T cells were stimulated with IL-2 (50 IU/ml). CD8⁺ T cells and NK cells were co-cultured with Nf2^−/− tumors and YAC-1 NK target cells at an E:T ratio of 10:1. After 8 hours of co-culture, target cell lysis was quantified by measuring the fluorescent intensity in the supernatant. P = 0.0001. N = 6 mice/treatment, in vitro cytotoxicity assay performed in triplicates for each condition. H qRT-PCR analysis of murine Nkg2d, Nkg2a, Rae-1, and H60a mRNA in Nf2^−/− tumor (N = 6 tumors/group). P = 0.0001. I Schematic and timeline of NKG2D blockade in Nf2^−/− sciatic nerve model. Mice bearing Nf2^−/− tumors in the sciatic nerve were treated with anti-NKG2D (αNKG2D, 200 μg/mouse, i.p., for two doses: seven days before implantation and seven days after implantation), followed by control IgG, αPD1, αVEGF antibody treatments. The schematic in panel I was created in BioRender. Xu, L. (2026) http://BioRender.com/yltfaz6. J Sciatic nerve Nf2^−/− tumor diameter was measured with a caliper every 3 days following treatment. αVEGF vs. αVEGF + αNKG2D: P = 0.02 (red); αVEGF + αPD1 vs. αVEGF + αPD1 + αNKG2D: P = 0.04 (blue). N = 24 mice/group. Flow cytometry, ELISA, qPCR, and cytotoxicity studies are presented as mean ± SD, and analyzed using two-sided Student’s t test and the Mann-Whitney U test. All animal studies are presented as mean ± SEM, representative of at least three independent experiments. Differences in tumor growth were analyzed using repeated-measures two-way ANOVA. Source data are provided as a Source Data file.