Abstract PO1-25-06: Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells
Authors: Jeong Byung-Kwan , Kim Young-Ae , Park Jungwook , Ham Baknoon , Kim Jiheyong , Lim Chae Lyul , Lee Hee Jin 
Published: May 2024

Abstract
Background
In recent years, the advancement of adoptive cell transfer-based immunotherapies has significantly contributed to the evolution of cancer treatment. The crucial step in achieving successful treatment lies in the identification of T cells that exhibit reactivity towards tumor-specific antigens. However, the heterogeneous composition of tumor antigen-specific T cells poses a challenge as there are no standardized biomarkers available for their identification. To overcome this limitation, we investigated alternative approaches, employing single cell RNA and VDJ sequencing to identify antigen-specific T cells.
Methods
First, peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors were stained with carboxyfluorescein succinimidyl ester (CFSE) to facilitate the identification of proliferating antigen-specific T cells. Then the PBMCs were exposed to 1 ug/ml of CMV pp65 peptide. After a 2-day incubation period with the CMV peptide, interleukin-2 (IL-2) was administered to stimulate T cell expansion. At various time points (0, 1, 2, 3, 5, and 7 days) following the CMV peptide treatment, CD8+ T cells were isolated through fluorescence-activated cell sorting (FACS) analysis. On day 7, the CD8+ T cells were further segregated into CFSE- and CFSE+ cell populations. The single-cell transcriptomes and T cell receptor (TCR) repertoire of the CD8+ T cells were subsequently examined using the Chromium Single Cell Reagent Kit.
Results
The TCR repertoire analysis of CD8+ CFSE- T cells revealed the dominance of 10 major clones, accounting for 93.9% of the total cells. These clones were confirmed to be CMV-specific through various in vitro assays, including NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assay. In contrast, CD8+ CFSE+ T cells did not exhibit significant overlap with the major TCR clones found in CFSE- CD8+ T cells, comprising only 0.02% of the total cells. Gene set enrichment analysis demonstrated that the upregulated genes in CFSE- CD8+ T cells, when compared to CFSE+ T cells, were related to cell cycle proliferation and T cell cytotoxicity. Furthermore, these genes exhibited a matching upregulation pattern with CFSE- T cells from another batch of the same donor, as well as from two other healthy donors. In addition, there was a moderate increase in the expression of the upregulated genes in CFSE- CD8+ T cells at day 3 compared to day 0-2. In this end, we selected a set of nine genes that were consistently upregulated in CFSE- CD8+ T to serve as markers for antigen-specific T cells, employing a logistic regression model. This gene set showed high sensitivity (97.7%) and specificity (95.6%) in identifying T cell clones that respond to CMV antigen at day 3.
Conclusions
We have discovered potential markers for antigen-specific CD8+ T cells. These results can offer the promising opportunity for earlier isolation of T cells that exhibit reactivity towards tumor-specific antigens. This breakthrough has significant implications, as it can potentially contribute to cost savings in adoptive cell transfer-based immunotherapies.
Citation Format: Byung-Kwan Jeong, Young-Ae Kim, Jungwook Park, Baknoon Ham, Jiheyong Kim, Chae Lyul Lim, Hee Jin Lee. Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-06.