Impact of CD4+ T cell help on CD8+ CAR T cell gene expression during ex-vivo manufacturing

Cell culture conditions impact the clinical efficacy of chimeric antigen receptor (CAR) T cell products, but the optimal approach remains unknown. Separate CD4+ and CD8+ cultures offer a potential advantage but complicate manufacturing and may affect cell expansion and function. We evaluated the co-culture of CD4+ and CD8+ cells at a defined ratio at culture initiation. We observed that the presence of CD4+ cells markedly improves expansion of CD8+ CAR T cells, and CD8+ cells cultured in isolation exhibit a hypofunctional phenotype and transcriptional signature compared with those co-cultured with CD4+ cells. Mixed-culture CAR T cells also confer superior anti-tumor activity in vivo compared with separately expanded, co-infused cells. CD4+ cell effects on CD8+ cells are mediated through both cytokines and direct cell contact, including CD40L-CD40 and CD70-CD27 interactions. Overall design: We analyzed transcriptional profiles of CD8+ CAR T cells from 6 subjects (a mix of healthy donors and patients with B-cell lymphoma) expanded in mixed cultures with CD4+ cells compared with those in separate CD8-only cell cultures. Total RNA was isolated from FACS-sorted CD8+ T cells expressing a 3rd generation CD20-targeted chimeric antigen receptor (CAR) containing CD28 and 41BB costimulatory domains, at either day 8 (without restimulation) or day 14 (after restimulation with CD20+ lymphoblastoid cells at day 7).