Abstract
Introduction: CAR T-cell immunotherapy involves the collection of T cells, followed by ex vivo genetic modification, expansion, and reinfusion into the patient to exert antitumor effects. Sequential autologous hematopoietic stem cell transplantation (auto-HSCT) followed by CAR T-cell therapy has emerged as an effective clinical approach for relapsed/refractory lymphoma, while allogeneic hematopoietic stem cell transplantation (allo-HSCT) combined with donor-derived CAR T-cell therapy has shown potent therapeutic effects in relapsed/refractory leukemia. These combined therapies typically require both peripheral stem cell collections (PSC) and peripheral lymphocyte collections (PLC) from the patient or donor. Notably, apheresis is an invasive and costly procedure, and PSC are detected containing a significant number of T cells. We wonder whether T cells from stem cell collections could be utilized for CAR-T cell manufacturing. This may eliminate the need for a separate peripheral lymphocyte collection, thereby reducing the physical and financial burden on patients and broadening access to HSCT combined with CAR T-cell therapy. In this study, we compared the functional properties and in vitro cytotoxic effects of CAR T-cells prepared from peripheral stem cell collections (PSC-CAR) and peripheral lymphocyte collections (PLC-CAR). These findings provide preliminary insights into optimizing the combination of HSCT and CAR T-cell immunotherapy for hematologic malignancies.
Methods: To investigate the functionality and cytotoxic effects, PSC and PLC products were obtained from patients scheduled for sequential auto-HSCT with CAR T-cell immunotherapy. T cells were isolated then used for CAR-T cell generation. The functional characterization was performed to characterize and compare the properties and functions of T cells, including immunophenotypic profiling of T-lymphocyte subsets, quantitative assessment of CAR transduction efficiency, evaluation of degranulation capacity, cytotoxic potential quantification against tumor targets at standardized effector-to-target ratios, and temporal monitoring of activation/exhaustion marker expression patterns.
Results: Analysis of CAR T-cells derived from 3 matched PSC/PLC products revealed no significant differences in T-cell subset distribution (CD8+/CD4+ ratio) or transduction efficiency between the two sources. Following CD19 CAR transduction, both PSC-CAR and PLC-CAR exhibited comparable CD8+/CD4+ T-cell ratios and degranulation capacity. To assess T-cell activation, surface expression of CD25 was evaluated. At baseline (without tumor cell interaction), PLC-CAR demonstrated significantly higher activation levels compared to PSC-CAR (p<0.01). This activation disparity was also evident during the early phase of co-culture but was eventually comparable between the two groups as the culture duration extended. Exhaustion marker expression, including PD-1, TIM-3, CTLA-4, and LAG-3, was analyzed. No significant differences were observed in the baseline expression of these markers between PSC-CAR and PLC-CAR. However, after tumor cell co-culture, PSC-CAR showed significantly higher LAG-3 expression compared to PLC-CAR (p<0.05). In cytotoxicity assays, PSC-CAR and PLC-CAR exhibited equivalent tumor-killing capacities across most effector-to-target (E:T) ratios. Only at the lowest E:T ratio (1:2) during the earliest timepoint (24 hours), did PSC-CAR show marginally reduced cytotoxic activity compared to PLC-CAR (p<0.05). Importantly, stem cell-derived CAR T-cells achieved equivalent tumoricidal activity to lymphocyte-derived counterparts across most E:T ratios, demonstrating only marginally reduced cytotoxicity at early timepoints (24h, E:T=1:2; p=0.02).
Conclusion: This study demonstrates that PSC-CAR and PLC-CAR exhibit comparable biological functionality and anti-tumor efficacy. These findings suggest that a single apheresis procedure may be sufficient to obtain both hematopoietic stem cells and T lymphocytes. this optimized strategy could reduce procedural burden, lower costs, and enhance clinical accessibility, thereby expanding the therapeutic potential of combined HSCT and CAR T-cell therapy for hematologic malignancies.
