Depletion of Tregs from <scp>CD4</scp>⁺<scp>CAR</scp>‐T cells enhances the tumoricidal effect of <scp>CD8</scp>⁺<scp>CAR</scp>‐T cells in anti‐<scp>CD19 CAR</scp>‐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.