Abstract 2706: Comprehensive immune profiling of primary tumor-infiltrating lymphocytes isolated from primary non-small cell lung cancer specimens
Authors: Koguchi Yoshinobu , Miller William , Piening Brian D. , Rajamanickam Venkatesh , Bernard Brady , Sun Zhaoyu , Wu Yaping , Kaufmann Johanna K. , Redmond William L. 
Published: August 2019

Abstract
In the past years, multiple inhibitors of the immune checkpoint receptor (ICR) PD-1 or its ligand PD-L1 have been approved in the U.S. in a selected, yet growing number of indications including non-small cell lung cancer (NSCLC). In patients with advanced NSCLC, checkpoint inhibition significantly prolongs overall survival and, due to its overall responsiveness, additional novel immunotherapeutic combinations are being investigated. Rational design of future combination treatment with immunomodulatory agents like PD-1 inhibitors will greatly benefit from a detailed understanding of the immune contexture and the functional state of tumor infiltrating lymphocytes (TILs) within NSCLC. To this end, we characterized the immune infiltrate in 13 primary NSCLC resections using multiple orthogonal techniques: (1) surface expression of multiple ICRs on various T cell subsets as well as the functional markers granzyme B and Ki-67 were assessed by flow cytometry; (2) the spatial localization of immune infiltrates was analyzed by 7-color multiplex immunohistochemistry (mIHC); (3) gene expression profiling enabled in depth molecular characterization of TILs and indicated their functional status. Flow cytometry revealed differential contributions of ICR expression in various T cell compartments. Of note, around 30% (median=29.1%; 95% CI, 20.3-38.5%) of CD8+ T cells co-expressed CD39 and CD103, which represents a recently described population of tumor-reactive CD8+ T cells. Furthermore, approximately 90% of these double-positive CD8+ T cells expressed PD-1 (median=91.3%; 95% CI, 78.3-96.4%) and about 50% were granzyme B-positive (median=51.8%; 95% CI, 32.5-63.95%), indicative of functionally active effector status. Interestingly, more than 90% of PD-1+ CD8+ T cells co-expressed at least one more ICR (TIM-3 and/or LAG-3) (median=90.6%; 95% CI, 70.3-95%), suggesting ongoing development of T cell exhaustion in response to chronic T cell activation. In contrast, the CD4+ T cell compartment was dominated by PD-1 single-positive cells and contained a median of 15.4% (95% CI, 11.7-20%) regulatory T cells.The overall composition of the immune infiltrates in these patients were corroborated by mIHC, revealing strong immune infiltration within the tumor stroma. Furthermore, immune cell deconvolution of bulk RNA-seq data from FFPE tissues revealed a high concordance with mIHC. In conclusion, we established a pipeline for high content immune profiling of human TIL enabling correlative analyses of proteomic and transcriptomic data. The high proportion of markers indicative of TIL with anti-tumor effector potential was in line with the responsiveness of NSCLC to immunotherapy. Integration of additional gene expression data, mutational burden, and T cell clonality will deepen our understanding of the NSCLC tumor microenvironment.
Citation Format: Yoshinobu Koguchi, William Miller, Brian D. Piening, Venkatesh Rajamanickam, Brady Bernard, Zhaoyu Sun, Yaping Wu, Johanna K. Kaufmann, William L. Redmond. Comprehensive immune profiling of primary tumor-infiltrating lymphocytes isolated from primary non-small cell lung cancer specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2706.