Abstract 896: Investigating tissue-resident memory T cell differentiation of CAR T cells reveals an unexpected role for CD69
Authors: Cheng Menglin , Bergsbaken Tessa 
Published: April 2023

Abstract
Adoptive cell therapy using chimeric antigen receptor-expressing (CAR) CD8+ T cells has revolutionized the treatment of liquid tumors; however, CAR T cells have had more limited success against solid tumors. The tumor microenvironment can impair T cell function within solid tumors through a variety of mechanisms, including nutrient deprivation, hypoxia, and recruitment of immuno-suppressive cells. CD8+ tissue-resident memory T cells (Trm), which are characterized by surface expression of CD69 and CD103 and a unique transcription factor circuit, are found within solid tumors, important for tumor surveillance, and responsive to checkpoint inhibitor blockade. However, it is unclear whether CAR T cells adopt Trm features in the tumor microenvironment and how the nature of the CAR costimulatory domain could impact Trm differentiation and efficacy against solid tumors. To investigate Trm differentiation in solid tumors, we utilized murine the MC38 syngeneic graft tumor model and found three populations of tumor-infiltrating CD8+ T lymphocytes: CD69-CD103-, CD69+CD103- and CD69+CD103+. CD69+ populations downregulated circulating T cells genes and expressed Trm signature genes often found in non-tumor Trm cells. To examine if tumor infiltrating CAR T cells follow a similar differentiation pattern, human carcinoembryonic antigen (hCEA)-specific CAR T cells with either a CD28 or 4-1BB costimulatory domain were transferred into hosts bearing MC38-hCEA tumors. hCEA-CD28 CAR T cells accumulated in the tumor, while hCEA-41BB CAR T cells and T cells without a CAR were present at much lower numbers. hCEA-CD28 CAR T cells displayed delayed Trm differentiation with an increased proportion of CD69-CD103- cells when compared to hCEA-41BB CAR T cells or T cells without a CAR. While hCEA-CD28 CAR signaling drove T cell accumulation, there was significant heterogeneity in the degree of T cell expansion, and the size of CD69-CD103- population correlated strongly with the number of hCEA-CD28 CAR T cells within the tumor. CD69-CD103- hCEA-CD28 CAR T cells were more proliferative and expressed lower levels of surface markers associated with dysfunction, including PD-1 and LAG3. These observations suggested CD69 could be a negative regulator of T cell proliferation and function. Indeed, deletion of CD69 in hCEA-CD28 CAR T cells further enhanced proliferation, which resulted in greater numbers of CAR T cells in tumor. In summary, hCEA-CD28 CAR signaling prolonged T cell proliferation in solid tumors by delaying Trm differentiation and the upregulation of CD69, whose expression impairs T cell proliferation.
Citation Format: Menglin Cheng, Tessa Bergsbaken. Investigating tissue-resident memory T cell differentiation of CAR T cells reveals an unexpected role for CD69 [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 896.