456 Functional and transcriptomic evaluation of pleural mesothelioma tumor expanded infiltrating lymphocytes and their TCR repertoire

<h3>Background</h3> Pleural mesothelioma (PM) is a highly aggressive cancer associated with asbestos exposure and fiber-associated inflammation. Immunotherapy is now an approved standard-of-care for PM; however, response rates remain low. Defining the tumor-infiltrating lymphocytes (TIL) functional landscape can provide an avenue to identify targetable mechanisms to rewire immune suppression. We hypothesized that while PM is considered a cold tumor with a suppressive profile, specific TIL subsets may be harnessed to drive an anti-tumor response. <h3>Methods</h3> TIL were expanded utilizing the ‘TIL 3.0’ method¹ from PM surgical resected specimens (n=46). TIL phenotype was assessed by flow cytometry and CyTOF. Functionality testing by CD107a and IFNg production was performed using a redirected lysis assay with P815 pulsed with anti-CD3 (OKT3) at a 1:1 ratio. Cytokine production was assessed using CyTOF following stimulation with PMA and ionomycin. TIL from metastatic melanoma were included for control purposes. Expanded TIL transcriptome was evaluated using 10x Genomics 5’ single cell (sc)RNA- and scTCR-seq profiling (n=20). Sc data was processed using 10x Genomics cell ranger pipeline. Downstream analysis was performed in R (Seurat and scRepertoir packages). Wilcoxon Sum Ranking test was applied for the determination of the statistical significance of genes. One-way ANOVA with Tukey’s multiple comparisons test was used to compare TIL function at assessed ratios. <h3>Results</h3> Pre-REP TIL expansion success defined as &gt;40 million TIL within 3 weeks was 86.96%. Principal TIL populations were CD8+ and double negative (median 86.73% and 8.84%) enriched for TCRgd (median 40.05%), CD8+ TIL expressed LAG3, ICOS and PD-1. CD8+ TILs were effector memory (EM; 97.75%) T cells, 39.43% EM3 T cells and 46.81% EM4, determined through CD27 and CD28 expression. PM TIL were comparable to melanoma TIL in their ability to degranulate (median Mel-32.72% and PM-28.61%, respectively) and in their production of IFNg (median Mel-22.24% and PM-12.90%) in a redirected lysis assay. However, polyfunctionality was reduced as 49.61% of CD8+ TIL made more than 1 cytokine. scTCR-seq data confirmed presence of hyper-expanded CD8+ clones [100&lt;X≤500] in 15 TIL lines with granzymes, perforin, <i>TIGIT, LAG3,</i> and <i>TOX</i> but a paucity of <i>PDCD1</i> and<i> TCF7</i> expression. <i>IFNg</i> and <i>TNF</i> expression in hyper-expanded cells is clonally specific indicating polyfunctional heterogeneity in TIL. <h3>Conclusions</h3> PM expanded TIL showed distinctive dysfunctional and memory profiles. The dysfunctional state across hyper-expanded clones was independent of <i>PDCD1</i> while retaining cytotoxic machinery preserving their ability to degranulate and produce cytokines while reducing their polyfunctionality upon activation. <h3>Acknowledgements</h3> This study was supported in part by the Dillon &amp; L Bourg Mesothelioma Fund, the Re &amp; RM Kennedy Lung Cancer Fund, the Fleming Endowed Fund, the MDA Rare Tumor Initiative, the NIH CCSG Award (CA016672 (Institutional Tissue Bank (ITB), Research Histology Core Lab (RHCL), MDACC Advanced Technology Genomics Core (ATGC), and the TMP-IL MDA Moonshots Platform. We would like to thank the MDA Thoracic and Cardiovascular Surgery nurses and staff for their support of this study, Carlos Ramos for his efforts early in this study and the patients who consented to be included in the research protocol. <h3>Reference</h3> Tavera RJ, <i>et al</i>., Utilizing T-cell activation signals 1, 2, and 3 for tumor-infiltrating lymphocytes (TIL) expansion: the advantage over the sole use of interleukin-2 in cutaneous and uveal melanoma. <i>J Immunother</i> 2018;<b>41</b>(9):399–405. <h3>Ethics Approval</h3> The study was performed under the MDA IRB approved LAB08-0380 protocol following patient informed consent.