Abstract 5390: microRNA signature of T cell exhaustion
Authors: Greenlees Lydia M. , Kuziora Michael , Sebastian Yinong , Creasy Todd , Lee Young S. , Pilataxi Fernanda , Holoweckyj Nick , Cheng Li , Higgs Brandon W. , Ranade Koustubh , Streicher Katie 
Published: August 2018

Abstract
Background: T cell exhaustion is driven by persistence of antigen and inflammation, common features of cancer. The success of a checkpoint inhibitor blockade may depend upon reactivation of pre-existing tumor-specific CD8+ T cells in the tumor microenvironment. Using an in vitro model and tumor infiltrating lymphocytes (TILs) isolated from multiple tumors, we explored microRNAs (miRNA, miR) involved in T cell dysfunction in order to identify pathways that may be important for altering this phenotype following immunotherapy.
Methods: We used an established in vitro model of T cell exhaustion: healthy donor CD8+ T cells were stimulated with anti-CD3/CD28 for six days. We used RNASeq and quantitative PCR to evaluate genomic (mRNA and microRNA (miR)) changes associated with T cell function and paired differentially expressed microRNAs with predicted target genes. Exhaustion of CD8+ TILs isolated from melanoma (n = 2), NSCLC (n = 3), renal (n = 3), bladder (n = 10), and colorectal tumors (n = 2) was measured by flow cytometry analysis of PD-1/TIM-3. miRNA and mRNA relationships identified by the in vitro model were evaluated in a subset of TILs from NSCLC, renal, and bladder tumors (n = 6).
Results: Results showed expected phenotypic and functional changes across 6 donors stimulated chronically with anti-CD3/CD28: 2-4-fold increased PD-1 and TIM-3 surface expression with a 3-5-fold loss of intracellular IFNg production (p &lt; 0.05). Comparing exhausted T cells with unstimulated T cells revealed ~1300 differentially expressed genes and ~100 differentially expressed miRs (p &lt; 0.05). A set of differentially expressed genes/miRs from the in vitro exhaustion model was then confirmed in TILs from NSCLC, bladder, and renal tumors. Differentially expressed miRs included microRNAs-155 and 181a, previously implicated in regulating IFNg signaling and T cell proliferation, respectively. Focusing on miRs that were altered between activation and exhaustion revealed a novel miR exhaustion signature, which was increased ≥4-fold relative to activated T cells. Evaluating this miR exhaustion signature in TCGA demonstrated a 16-fold range in expression across indications, with melanoma, head and neck, and NSCLC, among the highest median expression, while renal, HCC and rectal cancers were among the lowest. In HCC, GITR and OX40 pathway activation signatures were increased &gt;2-fold in tumors with high miR signature, while in renal cancer, B cell and CD40 pathway signatures were enriched. In melanoma, high miR signature showed a trend toward increased myeloid-derived suppressor cell (MDSC) signature expression (&gt;1.5-fold change), while the opposite trend was observed in head and neck cancer.
Conclusions: We identified a novel miRNA exhaustion signature associated with immune-related pathways in multiple tumor types. This signature may help generate hypotheses to guide prioritization of specific IO combination treatments.
Citation Format: Lydia M. Greenlees, Michael Kuziora, Yinong Sebastian, Todd Creasy, Young S. Lee, Fernanda Pilataxi, Nick Holoweckyj, Li Cheng, Brandon W. Higgs, Koustubh Ranade, Katie Streicher. microRNA signature of T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5390.