This study evaluated the efficacy and safety of CD19 CAR-T cells manufactured from defined CD4+ and CD8+ T cell subsets in adults with B cell acute lymphoblastic leukemia (B-ALL) after lymphodepletion chemotherapy. The clinical trial aimed to assess the feasibility of selecting and engineering defined T cell subsets with a CD19 CAR and formulating a cell product with a consistent CD4+:CD8+ ratio. Key findings include: 1. **Feasibility and Efficacy**: CD19 CAR-T cells manufactured from defined CD4+ and CD8+ subsets were successfully infused to all patients, achieving a 93% remission rate in bone marrow (BM) by flow cytometry. 2. **Toxicity and Risk Stratification**: High CAR-T cell doses and tumor burden increased the risk of severe cytokine release syndrome (CRS) and neurotoxicity. Serum biomarkers, such as IL-6 and IFN-γ, were identified to predict severe toxicity. 3. **Proliferation and Persistence**: The kinetics of CAR-T cell expansion and persistence were influenced by cell dose and tumor burden. Risk-adapted dosing based on BM disease burden improved outcomes. 4. **Transgene Immune Responses**: CD8+ T cell-mediated immune responses to the CAR transgene limited the persistence and efficacy of second CAR-T cell infusions in some patients. 5. **Lymphodepletion Regimen**: Adding fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. The study highlights the importance of defining the composition of CAR-T cell products and optimizing lymphodepletion regimens to enhance the efficacy and safety of CD19 CAR-T therapy in B-ALL patients.This study evaluated the efficacy and safety of CD19 CAR-T cells manufactured from defined CD4+ and CD8+ T cell subsets in adults with B cell acute lymphoblastic leukemia (B-ALL) after lymphodepletion chemotherapy. The clinical trial aimed to assess the feasibility of selecting and engineering defined T cell subsets with a CD19 CAR and formulating a cell product with a consistent CD4+:CD8+ ratio. Key findings include: 1. **Feasibility and Efficacy**: CD19 CAR-T cells manufactured from defined CD4+ and CD8+ subsets were successfully infused to all patients, achieving a 93% remission rate in bone marrow (BM) by flow cytometry. 2. **Toxicity and Risk Stratification**: High CAR-T cell doses and tumor burden increased the risk of severe cytokine release syndrome (CRS) and neurotoxicity. Serum biomarkers, such as IL-6 and IFN-γ, were identified to predict severe toxicity. 3. **Proliferation and Persistence**: The kinetics of CAR-T cell expansion and persistence were influenced by cell dose and tumor burden. Risk-adapted dosing based on BM disease burden improved outcomes. 4. **Transgene Immune Responses**: CD8+ T cell-mediated immune responses to the CAR transgene limited the persistence and efficacy of second CAR-T cell infusions in some patients. 5. **Lymphodepletion Regimen**: Adding fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. The study highlights the importance of defining the composition of CAR-T cell products and optimizing lymphodepletion regimens to enhance the efficacy and safety of CD19 CAR-T therapy in B-ALL patients.