Spatial analysis reveals combinative role for natural killer and CD8 T cells in antitumor immunity despite profound MHC class I loss in non-small cell lung cancer

Background. MHC class I (MHC-I) loss is frequent in NSCLC and renders tumor cells resistant to T cell lysis. NK cells kill MHC-I deficient tumor cells, and although previous studies indicate their presence in NSCLC tumor margins, they were also functionally impaired. The goal of this study was to evaluate whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. Methods. We used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFNγ-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis. Results. Spatial quantitation of tumor cell MHC-I expression revealed intra- and inter-tumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high numbers of CD56+ cells in patient tumors were associated with disease-free and overall survival (p<0.05). The overall survival association strengthened considerably with high counts of both CD56+ and CD8+ cells (p<1x10^-4). mIF imaging and multivariate discriminant analysis revealed an enrichment of both CD3+CD8+ T cells and CD3- CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single cell neighborhood profiles to delineate the cellular environments of IFNγ+/- NK cells and T cells. We discovered that both IFNγ+ NK and CD8 T cells were more frequently associated with other IFNγ+ lymphocytes in comparison to IFNγ- NK cells and CD8 T cells (p<1x10^-30). Moreover, IFNγ+ lymphocytes were most often found clustered near MHC-I+ tumor cells. Conclusions. We demonstrate that tumor-infiltrating NK cells and CD8 T cells jointly affect vital control of NSCLC tumor progression. Robust association of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent co-localization of IFNγ+ NK cells with other IFNγ+ lymphocytes in near-neighbor analysis indicates lymphocyte activation is coordinately regulated in NSCLC. ### Competing Interest Statement CLS: Research support to the University of Virginia from Celldex (funding, drug), Merck (funding, drug), Theraclion (device staff support); Funding to the University of Virginia from Polynoma for PI role on the MAVIS Clinical Trial; Funding to the University of Virginia for roles on Scientific Advisory Boards for Immatics and CureVac. CLS also receives licensing fee payments through the UVA Licensing and Ventures Group for patents for peptides used in cancer vaccines. RDG: Research support to the University of Virginia from Pfizer, Amgen, Chugai, Merck, AstraZeneca, Janssen, Daiichi Sankyo, Alliance Foundation, Takeda, ECOG/ACRIN, Jounce Therapeutics, Bristol Myers Squibb, SWOG, Helsinn, Dizal Pharmaceuticals, and Mirati. RDG received payment for service on Scientific Advisory Boards including AstraZeneca, Takeda, Gilead, Janssen, Mirati, Daiichi Sankyo, Sanofi, Oncocyte, Jazz Pharmaceuticals, Blueprint Medi-cines, and Merus.