Abstract
Background The chimeric antigen receptor T cell targeting CD19 (CD19.CAR-T) has demonstrated encouraging therapeutic efficacy in treating B-cell malignancies. However, current CD19.CAR-T therapies still face major hurdles, one of which is limited T cell persistence, leading to treatment resistance and relapse. The transcription factor TCF-1 has been reported to play a critical role in the maintenance of T cell stemness and survival[1]. Emerging evidence from a recent study indicates that TCF-1-enriched T cell subsets outperform TCF-1-low subsets in CAR-T cell therapy[2]. It is noteworthy TCF-1 expression shows significant heterogeneity in peripheral blood-derived T cells[3]. Nevertheless, whether genetic modulation to enforce TCF-1 expression could further improve CAR-T cell efficacy remains to be elucidated.
Methods A codon-optimized TCF-1 sequence was fused to a third-generation CD19-CAR construct containing 4-1BB and CD28 costimulatory domains via T2A peptide. Human peripheral blood mononuclear cells (PBMCs) were transduced with retroviral supernatants to generate either conventional CAR-T cells or TCF-1-overexpressing CAR-TCF1T cells. CAR expression and T cell phenotypes were assessed by flow cytometry. The serial co-culture tumor rechallenge assay was performed by co-culturing CAR-T cells with tumor cells at a 1:2 ratio, with fresh tumor cells added every 48 hours. NSG mice were intravenously injected with luciferase-expressing Nalm-6 cells and T cells, and tumor engraftment was monitored by bioluminescence imaging. Total RNA was isolated from CAR-T cells and subjected to bulk-RNA sequencing analysis. For reactive oxygen species (ROS) detection, cells were loaded with DCFH-DA followed by measurement of fluorescence intensity.
Results CAR-TCF1T cells were successfully generated and demonstrated comparable CAR expression to CAR-T cells (CAR-T: 82.1% ± 3.5%; CAR-TCF1T: 78.3% ± 5.2%; n = 4). Phenotypic analysis revealed that CAR-TCF1T cells maintained a less differentiated state, with a higher frequency of Tn/scm (naive T cells or stem cell memory-like T cells) and Tcm (central memory T cells), and fewer Tem (effector memory T cells) and Te (effector T cell) cells. In the tumor rechallenge assays against Nalm6 and Raji cells, we observed enhanced anti-tumor efficacy and superior proliferative capacity in CAR-TCF1T cells. Consistent with these findings, CAR-TCF1T cells also exhibited improved anti-leukemic efficacy in NSG xenograft models compared to CAR-T cells. Gene set enrichment analysis (GSEA) revealed elevated activity of the apoptosis pathway in CAR-T cells, with Fas and FasL genes expressed at higher levels. Based on the RNA analysis, we further determined the surface expression of Apotracker by flow cytometry. The percentage of Apotracker-positive T cells was significantly lower in CAR-TCF1T cells than in CAR-T cells (CAR-T: 38.34% ± 3.3 vs. CAR-TCF1T: 24.3% ± 6.2, P < 0.05). In addition, western blot analysis revealed reduced levels of cleaved caspase 8 and cleaved caspase 3 in CAR-TCF1T cells. The intracellular level of ROS was alongside significantly lower upon antigen stimulation at 48 hours (CAR-T: 128.3 ± 1.5 vs CAR-TCF1T: 97.7 ± 2.6, p < 0.05). Next, we used an FasL inhibitor (APG101) to inhibit Fas-FasL binding in the tumor rechallenge assay. Interestingly, the FasL inhibitor largely increased the functionality of the CAR-T cells to a level close to that of the CAR-TCF1T cells. Collectively, these results suggest that TCF-1 overexpression augments CAR-T cell persistence and efficacy by suppressing activation-induced cell death (AICD).
Conclusion CD19.CAR-T cells engineered to overexpress TCF-1 demonstrated a less differentiated phenotype and showed long-lasting antitumor activities both in vitro and in vivo. Mechanistically, TCF-1 overexpression enabled greater CD19.CAR-T expansion and persistence via enhanced resistance to AICD.
References [1] Raghu D, Xue HH, Mielke LA. Control of Lymphocyte Fate, Infection, and Tumor Immunity by TCF-1.Trends Immunol 2019, 40(12):1149-1162.
[2] Chen GM, Chen C, Das RK et al. Integrative bulk and single-cell profiling of premanufacture T-cell populations reveals factors mediating long-term persistence of CAR T-cell therapy. Cancer Discov 2021, 11(9):2186–2199.
[3] Delpoux A, Marcel N, Hess Michelini R et al. FOXO1 con- strains activation and regulates senescence in CD8 T cells. Cell Rep 2021, 34(4):108674.
