65 Sex-Dependent NK-Cell Immunosurveillance and Control of Ccrcc Liver Metastasis

Abstract Background Sex disparities in renal cell carcinoma (RCC) are well documented, with males exhibiting higher incidence, mortality, and more aggressive tumor features than females. While these differences have been partly attributed to risk factors such as smoking, hypertension, and obesity, emerging evidence implicates sex-specific differences in anti-tumor immunity. One proposed mechanism underlying female protection is escape from X-chromosome inactivation (XCI), which permits biallelic expression of select X-linked tumor suppressor genes (EXITS). Among these, the epigenetic regulator Kdm6a (UTX) has been implicated in modulating natural killer (NK) cell function in tumor-free mice. Notably, males exhibit increased NK cell abundance, but reduced effector function compared to females. Historically, most studies of sex bias in RCC have focused on adaptive immunity, particularly CD8+ T-cell dysfunction and exhaustion within the tumor microenvironment in males. In contrast, the contribution of innate immune mechanisms, particularly NK cell–mediated tumor surveillance, to sex-based differences in RCC progression remains poorly characterized. Methods Two ccRCC models, representing distinct clinical subtypes, were used to investigate sex-specific differences: an immune checkpoint blockade (ICB)-resistant model, Vhl⁻/⁻; p53⁻/⁻; Rb1⁻/⁻; Myc-overexpression (VpRM), and an ICB-responsive model, Vhl-mutant; Cdkn2a/2b⁻/⁻; Bap1⁻/⁻ (VCCB). Metastatic derivatives of these models were used to assess sex-dependent metastatic tropism and immune surveillance. Immune cell depletion studies targeting CD4+ T-cells, CD8+ T-cells, B-cells, NK-cells, and group 1 innate lymphoid cells were performed to define immune populations’ contributing to sex bias. Genetically engineered mouse models (GEMMs) with selective immune cell deficiencies were used to validate these findings and bone marrow chimeras to evaluate host-intrinsic determinants of sex-biased tumor progression. Results We found that sex bias in RCC models is immune dependent. In immune-deficient settings, male mice in both models exhibited smaller tumors than females; however, in immune-competent settings, female mice developed smaller tumors, recapitulating human disease. In the metastatic setting, tropism was model dependent: the immune-inflamed VCCB model showed preferential lung colonization, whereas the VpRM model preferentially seeded the liver. Notably, in the VpRM model, female mice exhibited near-complete protection from liver metastases, suggesting a highly protective or immune-surveilled female liver microenvironment. Flow cytometry revealed increased NK cells and effector ILC1s (NK1.1+, NKp46+, Eomes⁻, CD49a+, CD127⁻) in female livers. These effector ILC1s displayed higher expression of cytotoxic granzyme B and C. NK cells in females also showed increased granzyme B expression and a more mature, terminally differentiated CD11b+ phenotype, consistent with enhanced effector function. While depletion of CD4+ T-cells, CD8+ T-cells, B-cells, and ILC1s did not alter liver metastatic incidence, depletion of NK cells strikingly exacerbated liver mets in females. In Rag1⁻/⁻ mice, characterized by the absence of mature T-cells and B-cells and the enrichment of circulating and splenic NK cells, both sexes showed near-complete protection from liver metastases; this phenotype was reversed with NK cell depletion, confirming a central role for NK cells in hepatic metastasis control. Bone marrow chimera experiments where CD45.1 male and female bone marrow were transplanted into male and female CD45.2 hosts, further demonstrated that female protection is conferred by both hematopoietic and non-hematopoietic compartments, indicating that either host or NK-intrinsic female factors are sufficient to enhance immune surveillance. Conclusions Our findings identify a sex-dependent axis of innate immune surveillance, where female-intrinsic factors prime NK populations for elevated tumor immune surveillance. This highlights innate immunity as a critical, underappreciated driver of sex-based clinical outcomes in RCC. DOD CDMRP Funding yes