Fig 2: IL-15 armored CAR-T cells exhibit higher proliferative and less-differentiated phenotype in vitro. A The extent of T cell proliferation was reflected through direct cell counting over time. CAR-T cells were stimulated every 7 days with fresh NALM-6-eGFP cells (10:1), and T cells were counted before the addition of NALM-6-eGFP cells. B CAR-T cells were co-cultured with NALM-6-eGFP (2:1) without IL-2 in the culture medium for 24 h. The concentration of IL-2 in the supernatant was measured via ELISA. C CAR-T cells were co-cultured with NALM-6-eGFP (10:1) for 7 days, the T subsets were detected using flow cytometry. D CAR-T cells were co-cultured with NALM-6-eGFP (2:1) for 24 h. The supernatant was collected to detect the concentration of IFN-γ via ELISA. E CAR-T cells were co-cultured with NALM-6-eGFP (10:1). After 7 days, all cells were collected and the percentages of apoptotic cells and cell viability were detected through Annexin V and 7-ADD staining. Results were analyzed by student’s t-test followed by parametric test. * p < 0.05; ** p < 0.01; *** p < 0.001

Fig 3: IL-15 armored CAR-T cells expressed with IL-15Ra exhibit enhanced anti-tumor activity and reduced toxicity in vivo. A Schematic diagram of mouse experimental processes. 1 × 106 NALM-6-eGFP cells were injected into NOD-SCID mice intravenously to construct the xenograft mouse model. One days after tumor cell injection, 1 × 107 CAR-T cells (2 × 106 CAR positive cells) were injected into tail vein once a day for 3 days. Tumor development was monitored using IVIS. B, C Quantitative bioluminescence (radiance = photons/cm2/sr) imaging data for all mice are shown. Statistic analysis of quantitative bioluminescence of day 34 is shown. D Overall survival rates of mice with NALM-6-eGFP xenografts are shown. E Livers from CAR-T treated mice were collected to stain hematoxylin and eosin. Yellow arrow shows the numerous of infiltrated neutrophils, the black arrow shows the area of necrotic lesions. Right panel shows the GVHD scores of the livers. F Sera from mice were extracted and the concentrations of human IL-15 were measured using ELISA. Results were analyzed by student’s t-test followed by parametric test or Mann–Whitney test. The survival curve was analyzed using Mantel-Cox test. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s., not significant

Fig 4: IL-15Ra reduces the IL-15-induced toxicity in vitro. A CAR-T cells were co-cultured with NALM-6-eGFP (2:1) for 24 h. The supernatant was collected to detect the concentration of IL-15 via ELISA. B CAR-T cells were co-cultured with NALM-6-eGFP (10:1) for 7 days, the expression of CD132 on the cell surface was detected using flow cytometry. C CAR-T cells were co-cultured with NALM-6-eGFP cells (2:1) in 24-well plate for 24 h. Flow cytometry was used to detect the live NALM-6-eGFP. D CAR-T cells were co-cultured with NALM-6-eGFP cells (2:1) in 96-well plate for 24 h. Luciferase activity was used to determine the tumor cell viability. Left panel shows the picture obtained from IVIS imaging system, right panel shows the statistical analysis. Control represents NALM-6-eGFP cells. Results were analyzed by student’s t-test followed by parametric test. * p < 0.05; ** p < 0.01; *** p < 0.001