Background Adoptive cellular therapy (ACT), such as chimeric antigen receptor (CAR)-T cell therapy, has provided impressive clinical efficacy for treatment of patients with hematological malignancies.¹ However, its efficacy for solid tumours has been limited² largely due to immunosuppressive signals and competition for metabolic resources in the tumour microenvironment (TME) that can limit the efficacy and killing capacity of T cells.³ Overcoming these metabolic challenges is vital for enhancing the efficacy of ACT. Many strategies for reprogramming T cell metabolism have emerged in the literature, however the impact of the clinical activation strategy – bead-bound anti-CD3 and anti-CD28 antibodies – remains largely unexplored. This study aims to compare the metabolic programming, effector function, persistence and resilience to metabolic stress of T cells activated with bead-bound antibodies (beads) or with dendritic cells (DCs).

Methods We isolated CD8+ T cells from P14 mice that have a transgenic T cell receptor recognizing the gp33 peptide from lymphocytic choriomeningitis virus (LCMV). These T cells are co-cultured with either LPS-activated bone-marrow derived DCs pulsed with gp33 peptide, or with beads at ratios of 1:1 or 10:1 (beads:T cells). These T cells were activated for 72 hours followed by baseline analysis, transfer into in vitro stress conditions, or use for ACT in mice with established B16-gp33 melanoma tumours.

Results Increasing the ratio of beads resulted in increased oxidative and glycolytic metabolism to a level comparable with DC-activated T cells. Despite the comparable bioenergetic profile, DC-activated T cells demonstrated superior tumour clearance compared to all ratios of bead-activated T cells. Additionally, in vivo in the same TME, DC-activated T cells showed significantly improved survival, suggesting DC-activation provides additional signals that confer resiliency to stress and improved function. Similar resiliency was observed in DC-activated T cells in vitro after exposure to stress conditions such as IL-2 withdrawal. Metabolomics and RNA-sequencing analyses have revealed distinct signaling and phospholipid metabolism between DC and bead-activated T cells.

Conclusions DC-activation of T cells provides superior programming for efficacy, survival and resiliency to stress compared to bead-activation. These enhanced metrics are associated with unique metabolic and transcriptional programming. Current investigations are ongoing to determine if altering phospholipid metabolism in bead-activated T cells is sufficient to enhance their efficacy and resilience. This could then be incorporated into clinical protocols to improve ACT outcomes for patients.

References

1. Cappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol 2023;20:359–71.

2. Hou B, Tang Y, Li W, Zeng Q, Chang D. Efficiency of CAR-T therapy for treatment of solid tumor in clinical trials: a meta-analysis. Dis Markers 2019;2019:e3425291.

3. Hou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. Nat Rev Drug Discov 2021;20:531–50.

Ethics Approval This study was approved by the University Health Network Animal Care Committee approval number 929 and 6895.