HVEM costimulatory domain boosts CAR T cell efficacy against solid tumors via enhanced TRAF-mediated TNF signaling.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has shown success in hematological malignancies, but its efficacy against solid tumors remains limited. Herpesvirus entry mediator (HVEM), a co-stimulatory molecule, enhances CAR T cell activity, though the underlying mechanisms are unclear. This study investigates how HVEM-based CAR T cells outperform those using 4-1BB in solid tumor models. METHODS: Second-generation CAR T cells targeting carbonic anhydrase IX (CAIX) were engineered with either 4-1BB, wild type (WT) HVEM, or AVEE mutation (MUT) HVEM co-stimulatory domains. In vitro cytotoxicity, cytokine release, and signaling pathways were assessed using real-time cellular analysis, ELISA, and Western blotting. RNA sequencing and gene set enrichment analysis (GSEA) compared transcriptional profiles. Co-immunoprecipitation and mass spectrometry identified TNF receptor-associated factor (TRAF) protein interactions. In vivo efficacy was evaluated in orthotopic and subcutaneous renal cancer models using NPG mice. Metabolic activity was measured via Seahorse assays. RESULTS: HVEM-CAR T cells exhibited stronger cytotoxicity, cytokine release, and proliferation than 4-1BB-CAR T cells in response to target cancer cell stimulation in vitro. RNA sequencing and Western blotting revealed enhanced TNF signaling in HVEM-CAR T cells, with higher phosphorylation of AKT and ERK1/2, accompanied by elevated metabolic levels. HVEM recruited TRAF1, TRAF2, TRAF3, and TRAF5 via its AVEE motif, whereas 4-1BB only bound TRAF1-3. Mutation of the AVEE motif disrupted TRAF binding, reduced signaling activation, and impaired metabolic and antitumor functions. In vivo, HVEM-CAR T cells showed superior tumor control, prolonged survival, and increased intratumoral abundance compared to 4-1BB-CAR or AVEE-mutated HVEM-CAR T cells. CONCLUSIONS: HVEM enhances CAR T cell efficacy against solid tumors by robustly activating TNF signaling through TRAF recruitment, particularly via the AVEE motif. These findings highlight HVEM as a promising co-stimulatory domain for improving CAR T cell therapy in solid tumors, with implications for metabolic reprogramming and sustained antitumor activity.