CD8(+) chimeric antigen receptor T cells manufactured in absence of CD4(+) cells exhibit hypofunctional phenotype

BACKGROUND: Cell culture conditions during manufacturing can impact the clinical efficacy of chimeric antigen receptor (CAR) T cell products. Production methods have not been standardized because the optimal approach remains unknown. Separate CD4(+) and CD8(+) cultures offer a potential advantage but complicate manufacturing and may affect cell expansion and function. In a phase 1/2 clinical trial, we observed poor expansion of separate CD8(+) cell cultures and hypothesized that coculture of CD4(+) cells and CD8(+) cells at a defined ratio at culture initiation would enhance CD8(+) cell expansion and simplify manufacturing. METHODS: We generated CAR T cells either as separate CD4(+) and CD8(+) cells, or as combined cultures mixed in defined CD4:CD8 ratios at culture initiation. We assessed CAR T cell expansion, phenotype, function, gene expression, and in vivo activity of CAR T cells and compared these between separately expanded or mixed CAR T cell cultures. RESULTS: We found that the coculture of CD8(+) CAR T cells with CD4(+) cells markedly improves CD8(+) cell expansion, and further discovered that CD8(+) cells cultured in isolation exhibit a hypofunctional phenotype and transcriptional signature compared with those in mixed cultures with CD4(+) cells. Cocultured CAR T cells also confer superior antitumor activity in vivo compared with separately expanded cells. The positive impact of CD4(+) cells on CD8(+) cells was mediated through both cytokines and direct cell contact, including CD40L-CD40 and CD70-CD27 interactions. CONCLUSIONS: Our data indicate that CD4(+) cell help during cell culture maintains robust CD8(+) CAR T cell function, with implications for clinical cell manufacturing.