CD79A/CD40 intracellular domain uses a 4-1BB-like metabolic pathway driven by cholesterol biosynthesis

Chimeric antigen receptor (CAR)-T cell therapy targeting CD19 has transformed the treatment of hematologic malignancies. The costimulatory domain (CSD) of CAR constructs plays a crucial role in determining T cell metabolism, persistence, and antitumor function. We previously developed a novel CSD combining CD79A and CD40, which conferred superior proliferation and antitumor efficacy compared with CD28-based or 4-1BB-based CAR-T cells. The metabolic mechanisms underlying these effects remain to be elucidated.

We compared CD28, 4-1BB, and CD79A/CD40 CAR-T cells using transcriptomic analysis, metabolic flux assays, and metabolomics. Patient samples treated with 4-1BB-based or CD28-based CAR-T therapies were analyzed to assess correlations between serum lipids and CAR-T expansion.

Transcriptomic profiling revealed that CD79A/CD40 CAR-T cells shared gene expression patterns with 4-1BB CAR-T cells, particularly in pathways related to oxidative phosphorylation (OXPHOS) and T cell memory differentiation, but were distinct from CD28 CAR-T cells. Both 4-1BB and CD79A/CD40 CAR-T cells relied on OXPHOS and exhibited greater mitochondrial fitness, as evidenced by higher spare respiratory capacity and mitochondrial mass. Notably, CD79A/CD40 CAR-T cells displayed significantly enhanced glycolysis during the early phase following antigen stimulation, distinguishing them from 4-1BB CAR-T cells and supporting rapid initial expansion. Metabolomic profiling showed upregulation of cholesterol biosynthesis enzymes in both CD79A/CD40 and 4-1BB CAR-T cells, suggesting a shared reliance on cholesterol metabolism. Importantly, in patients treated with 4-1BB-based CAR-T therapy, higher serum low-density lipoprotein cholesterol levels positively correlated with CAR-T expansion in the late phase, particularly within CD4+T cell subsets. This relationship was not observed in patients receiving CD28-based CAR-T therapy. These findings indicate that cholesterol availability may influence CAR-T persistence in vivo, and that the metabolic phenotype of CD79A/CD40 CAR-T cells is optimized for both early proliferation and long-term survival.

CD79A/CD40 CAR-T cells exhibit unique metabolic adaptations, including early glycolytic activation, sustained OXPHOS, and upregulated cholesterol metabolism, which together may underpin their enhanced proliferation and persistence. Targeting cholesterol metabolism may represent a novel strategy to optimize CAR-T cell function and improve therapeutic outcomes.