Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer

Abstract

Background Major histocompatibility complex class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated 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 intratumoral and intertumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (HR=0.58, p=0.064) and overall survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1×10−3). mIF imaging and multivariate discriminant analysis revealed 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<1×10–30). Moreover, IFNγ+ lymphocytes were most often found clustered near MHC-I+ tumor cells.Conclusions Tumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Coassociation of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent colocalization of IFNγ+ NK cells with other IFNγ+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated.