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
Remziye E. Wessel, Nardin Ageeb, Joseph M. Obeid, Ileana S. Mauldin, Kate A. Goundry, Gabriel F. Hanson, Mahdin Hossain, Chad P. Lehman, Ryan D. Gentzler, Nolan A. Wages, Craig L. Slingluff, Timothy N. J. Bullock, Sepideh Dolatshahi, Michael G. Brown
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.