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

Abstract

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.

Keywords: CD4-CD8 Ratio; CD8-Positive T-Lymphocytes; Cell Engineering; Immunotherapy, Adoptive; Receptors, Chimeric Antigen.