Single-cell profiling of CAR-T CD19 cell phenotypes and immune system dynamics in pediatric BCP-ALL.

Background Chimeric Antigen Receptor T-cell (CAR-T) therapy targeting CD19 has transformed the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the phenotypic heterogeneity of CAR+ T-cells and their interactions with the immune system remain poorly understood. Here, we characterize CAR+ T-cell subsets and persistence in patients receiving standard-of-care tisagenlecleucel in an effort to optimize therapeutic efficacy in pediatric BCP-ALL. Methods Nineteen pediatric patients with relapsed/refractory BCP-ALL treated with tisagenlecleucel in Poland were included in the study. CAR+ T-cell composition and the peripheral blood mononuclear cell (PBMC) immune system were assessed using mass cytometry, with qPCR validation of CAR+ T-cells. Machine learning algorithms in R classified phenotypes. Statistical analyses examined associations between CAR+ T-cell subsets, immune system changes, and CAR-T therapy outcomes, including cytokine release syndrome (CRS). Results Infusion product analysis showed a predominance of CD4+ Central Memory (16.6%), MAIT/NKT (7.04%), and Treg memory (63.3%) cells, with high consistency between paired products. Post-infusion, CAR+ Treg memory cells declined to <1%, while CD8+ subsets expanded. PBMC immune system analysis revealed increased monocyte and NK cell counts post-infusion. Patients who experienced CRS had fewer classical monocytes at day 7 and fewer transitional monocytes at day 28. Early and late NK cells increased post-infusion in all patients. However, pre-infusion levels were lower in patients experiencing CRS. CRS correlated with higher CAR+ CD8+ Terminal Effector and MAIT/NKT frequencies in infusion products. While CAR+ composition was not linked to disease burden, prior treatments, or therapy outcomes, changes in monocyte and NK cell dynamics were associated with disease burden, relapse, and CRS. Conclusions This study provides a detailed characterization of CAR-T CD19 cell composition and post-infusion dynamics in pediatric BCP-ALL patients treated with tisagenlecleucel. Infusion products were predominantly CAR+ CD4+ T cells with a Central Memory phenotype, including a notable Treg memory subset that declined post-infusion. CAR+ CD8+ subsets expanded, coinciding with immune system shifts. These changes correlated with clinical responses, including CRS, highlighting the role of the immune system in CAR-T therapy outcomes and informing strategies to optimize treatment.