LAUSR

The frequency of CD8+ tumor infiltrating lymphocytes (TILs) has been positively associated with overall survival in several cancers. The interaction of T cell receptors (TCRs) on T cells with Major Histocompatibility Complex (MHC)-peptide complexes on cancer cells elicits the cytotoxic activity of CD8+ T cells. Here we set out to understand how the strength of this interaction, or T cell avidity, shapes the phenotype of CD8+ TILs and how that dictates anti-tumor immunity. We developed a novel tetramer decay assay to isolate T cells based on their TCR avidities. We then used this method to isolate from tumors of Meth A tumor-bearing mice (28 days after tumor challenge), Low (K off < 15 min), Medium (75 min > K off > 45 min), and High (K off > 105 min) avidity CD8+ TILs responding to the tumor rejection neoepitope, PDPRMUT of this fibrosarcoma (Ebrahimi-Nik et al. JCI Insight 2019). Unbiased clustering of single cell RNA sequencing, TCR sequencing, and CITE-seq of these PDPRMUT -specific CD8+ TILs revealed five clusters: Stem-Like (LY108+Tcf7+), Effector-Exhausted (Eff-Exh PD-1Low,CX3CR1+) Terminally-Exhausted-1 (Term-Exh1 PD-1+TIM3High), Terminally-Exhausted-2 (Term-Exh2 PD-1HighToxHigh), and NK-Like (PD-1Low, KlrHigh). The low avidity CD8+ TILs have more than twice the proportion of Eff-Exh (60.0%) when compared to high avidity CD8+ TILs (28.39%). The high avidity CD8+ TILs have more Stem-Like (12.37% vs 6.31%), Term-Exh1 (27.38% vs 21.88%), and Term-Exh2 (24.03% vs 5.33%) cells than low avidity CD8+ TILs. Furthermore, trajectory inference of these clusters, by scVelo, predicts that Stem-Like cells differentiate into Eff-Exh cells that can differentiate into either Term-Exh1 or Term-Exh2. Our data demonstrates that low avidity Eff-Exh cells preferentially differentiate into Term-Exh1, while high avidity Eff-Exh cells differentiate into both Term-Exh1 and Term-Exh2. To validate these results, we isolated CD8+ TILs responding to PDPRMUT with low or high avidity from tumors of 21 Meth A bearing mice 28 days after tumor challenge and analyzed their exhaustion phenotype by flow cytometry. There were significantly more TIM3+PD-1+ cells (Paired t-test; P < 0.0001) and TOX+PD-1+ cells (Paired t-test; P < 0.05) in the high avidity CD8+ TILs than the low avidity CD8+ TILs. We conclude that CD8+ TILs with high avidity are more exhausted and less effector like than low avidity CD8+ TILs. This finding has major implications for T cell-based cancer immunotherapy. Citation Format: Summit Singhaviranon, Joseph P. Dempsey, Adam T. Hagymasi, Pramod K. Srivastava. Low avidity neoepitope-specific CD8+ tumor infiltrating lymphocytes are less exhausted and more effective than their high avidity counterparts in a BALB/c murine fibrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 608.