BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients 
with non-small-cell lung cancer (NSCLC), but robust biomarkers that predict 
response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding 
of the diversity of the NSCLC tumor immune microenvironment remains limited. 
METHODS. We performed comprehensive flow cytometric immunoprofiling on both 
tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical 
and histopathologic characteristics, next-generation sequencing, mRNA 
expression, and PD-L1 immunohistochemistry (IHC). RESULTS. Cytometric profiling 
identified an immunologically "hot" cluster with abundant CD8+ T cells 
expressing high levels of PD-1 and TIM-3 and an immunologically "cold" cluster 
with lower relative abundance of CD8+ T cells and expression of inhibitory 
markers. The "hot" cluster was highly enriched for expression of genes 
associated with T cell trafficking and cytotoxic function and high PD-L1 
expression by IHC. There was no correlation between immunophenotype and KRAS or 
EGFR mutation, or patient smoking history, but we did observe an enrichment of 
squamous subtype and tumors with higher mutation burden in the "hot" cluster. 
Additionally, approximately 20% of cases had high B cell infiltrates with a 
subset producing IL-10. CONCLUSIONS. Our results support the use of immune-based 
metrics to study response and resistance to immunotherapy in lung cancer. 
FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles 
Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer 
Cancer Foundation, International Association for the Study of Lung Cancer, 
National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research 
Foundation.