Nathanael Cox

Supplementary Table 1 shows the list of antibodies used in this study. Supplementary Table 2 shows the input data and variables used for computational modeling. Supplementary Table 3 shows parameter estimates and confidence intervals for modeled cell populations. Supplementary Table 4 shows patient demographics, tumor characteristics, and treatment regimens.

Supplementary Figure S1 shows PD-1 blockade therapy combined with NK cell depletion improves tumor control and increases CD8⁺ T cell infiltration. Supplementary Figure S2 shows the combination of NK cell depletion and ICB is most effective when NK cells are depleted at an early stage. Supplementary Figure S3 shows NKRT accumulation is tumor-site specific. Supplementary Figure S4 shows phenotypic changes in CD4⁺ T cells upon NK cell depletion. Supplementary Figure S5 shows NKRTs are antigen-specific and exhibit enhanced tumor-controlling efficacy when combined with ICB. Supplementary Figure S6 shows NKRT formation depends on the timing of NK cell depletion. Supplementary Figure S7 shows NK cell depletion delays CD8⁺ T cell exhaustion in day 15 MC38 tumors. Supplementary Figure S8 shows NK cell depletion delays CD8⁺ T cell exhaustion in day 20 MC38 tumors. Supplementary Figure S9 shows NK cell depletion improves tumor control in the ACT setting. Supplementary Figure S10 shows distinct gene expression profiles in CD8⁺ T cells following NK cell depletion. Supplementary Figure S11 shows volcano plots of differentially expressed genes under specific conditions. Supplementary Figure S12 shows elevated p-STAT3 and p-STAT5 levels in CD8⁺ T cells from NK cell-depleted tumors. Supplementary Figure S13 shows cytokines play a critical role in NKRT formation and anti-tumor activity. Supplementary Figure S14 shows computational model fits of tumor growth curves and CD8⁺ T cell dynamics. Supplementary Figure S15 shows phenotypic variation in tumor-infiltrating NK cells across tumor models. Supplementary Figure S16 shows expression patterns of tissue-resident markers in CD8⁺ T cells and NK cell proportions in responders versus non-responders to immunotherapy.

Immune checkpoint blockers (ICB) targeting the PD-1/PD-L1 axis represent established therapies for many cancers. However, resistance occurs in most patients due to complex immune-suppressive mechanisms in the tumor microenvironment. NK cells can play effector roles in tumor control, but their impact on T-cell dysfunction and ICB efficacy remains controversial. Through genetic and antibody-mediated NK cell depletion, we found that a subset of tumor-associated NK cells plays a negative role in ICB sensitivity; they further impede CD8+ T-cell differentiation toward a CD69+ BCL2+ EOMES+ GZMB+ TIM3− GITR− phenotype. Mechanistically, the retinoic acid receptor α–dependent differentiation program in CD8+ T cells is hindered by tumor-infiltrating NK cells via competition for IFNα and IL-2. Finally, we observed that lower frequencies of NK cells correlate with better clinical responses to ICBs in patients with cancer. These findings suggest potential avenues for enhancing CD8+ T cell–centered immunotherapy by targeting regulatory NK cells. Significance: Although NK cells are traditionally viewed as antitumor effectors, our study uncovers their unexpected suppressive role in CD8+ T cell–based immunotherapy. By competing for cytokines, they disrupt retinoic acid receptor α–driven CD8+ T-cell differentiation and limit ICB efficacy. Clinically, reduced NK cell presence is associated with an enhanced immunotherapy response. See related article by Pozniak et al. p. XX