Depletion of Tregs from CD4 + CAR ‐T cells enhances the tumoricidal effect of CD8 + CAR ‐T cells in anti‐ CD19 CAR ‐T therapy

Chimeric antigen receptor T (CAR‐T) cell therapy, which targets CD19 for hematological malignancies, represents a breakthrough in cancer immunotherapy. However, some patients may develop resistance to CAR‐T treatment, underscoring the importance of optimizing CAR‐T design to enhance responsiveness. Here, we investigated the impact of different subpopulations in anti‐CD19 CAR‐T cells on the tumoricidal effect. Different populations of anti‐CD19 CAR‐T cells were isolated by magnetic‐activated cell sorting (MACS). Their lytic activities on the acute lymphocytic leukemia cell line SUP‐B15 and diffuse large B‐cell lymphoma EB‐3 cell line were examined in a co‐culture system. The anti‐tumorigenic outcome of different CAR‐T cell compositions was evaluated in a xenograft mouse model of EB‐3 cells. CD8 + CAR‐T cells exhibited the most potent tumoricidal activity against SUP‐B15 and EB‐3 cells. Additionally, CD4 + T helper cells enhanced the lytic effects of CD8 + CAR‐T cells by increasing the availability of interleukin‐2 (IL‐2). Depleting CD25 + Treg (T regulatory) cells from CD4 + CAR‐T population further augmented the tumoricidal activity of CD8 + CAR‐T cells by preventing IL‐2 deprivation. Consistently, in vivo experiments demonstrated that the CD4 + CD25 + Treg population dampened the antitumor activity of CD8 + CAR‐T cells, while depletion of Tregs from CD4 + CAR‐T cells enhanced the tumoricidal effect. These findings emphasize the potential role of CAR Treg cells in therapeutic resistance, suggesting that the depletion of Tregs in the anti‐CD19 CAR‐T population may serve as a strategy to augment the anticancer effect of CD8 + CAR‐T cells.