Fig 2: NaMiX increased degranulation and IFN-γ secretion of PBMCs against ACH-2 cells. Human PBMCs were pre-incubated for 24 h (for IFN-γ expression) or 48 h (for CD107a expression) with the NaMiX and stimulated with Raji cells for 5 h in the presence of anti-CD107a mAb. A Cells were further stained for extracellular markers to identify CD107a expression on CD3−CD56+CD16+ NK cells (upper panel) and CD3+CD8+ T cells (lower panel) using anti-CD3, CD8, CD14, CD16, CD19 and CD56 antibodies. B Cells were also permeabilized and further stained for IFN-γ (NK cells on upper panel and CD8+ T cells on lower panel). C After 48 h incubation, supernatant was collected and analyzed by ELISA for IFN-γ (left panel), granzyme B (middle panel) and perforin secretion (right panel). Figure A to C represent three independent experiments. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p < 0.05, **p < 0.005). D Representative expression of CD107a and INF- γ on NK cells pre-activated by NaMiX for 48 h and 24 h, respectively, for one donor included in panel A and B. NaMiX engrafted with IL-15Rα/IL-15 using C4bpα or C4bpβ and expressing the anti-NKG2A or the anti-KIR scFv are so called: α.anti-NKG2A.IL-15, β.anti-NKG2A.IL-15, α.anti-KIR.IL-15 and β.anti-KIR.IL-15, respectively while the control NaMiX without IL-15Rα/IL-15 expressing the anti-NKG2A scFv is termed β.anti-NKG2A, the recombinant human IL-15 as rhIL-15, and the control condition without any molecules as medium

Fig 3: NaMiX increased cytokine production and cytolytic activity of PBMCs but did not increase Ki-67 expression. Human PBMCs were pre-incubated for 24 h (IFN-γ expression) or 48 h (CD107a expression and Ki67 expression) with NaMiX. Cells were further stained for extracellular markers to identify CD3−CD56+CD16+ NK cells (A) and CD3+CD8+. T cells (B) using anti-CD3, CD8, CD14, CD16, CD19 and CD56 antibodies. C After 48 h incubation, the supernatant was tested for the presence of IFN-γ (left panel), granzyme B (middle panel) and perforin (right panel) by ELISA. The figures represent three independent experiments with three different healthy donors. NaMiX engrafted with IL-15Rα/IL-15 using C4bpα or C4bpβ and expressing the anti-NKG2A or the anti-KIR scFv are so called: α.anti-NKG2A.IL-15, β.anti-NKG2A.IL-15, α.anti-KIR.IL-15 and β.anti-KIR.IL-15, respectively while the control NaMiX without IL-15Rα/IL-15 expressing the anti-NKG2A scFv is termed β.anti-NKG2A, the recombinant human IL-15 as rhIL-15, and the control condition without any molecules as medium. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001)

Fig 4: NaMiX increased degranulation and IFN-γ secretion of NK cells against resistant Raji cells. Human PBMCs were pre-incubated for 24 h (for IFN-γ expression) or 48 h (for CD107a expression) with NaMiX and stimulated with Raji cells for 5 h in the presence of anti-CD107a mAb. A Cells were further stained for extracellular markers to identify CD107a expression on CD3−CD56+CD16+ NK cells (upper panel) and CD3+CD8+ T cells (lower panel) using anti-CD3, CD8, CD14, CD16, CD19 and CD56 antibodies. B Cells were also permeabilized and further stained for IFN-γ (NK cells on upper panel and CD8+. T cells on lower panel). C After 48h incubation, supernatant was collected and analysed by ELISA for IFN-γ (left panel), granzyme B (middle panel) and perforin secretion (right panel). NaMiX engrafted with IL-15Rα/IL-15 using C4bpα or C4bpβ and expressing the anti-NKG2A or the anti-KIR scFv are so called: α.anti-NKG2A.IL-15, β.anti-NKG2A.IL-15, α.anti-KIR.IL-15 and β.anti-KIR.IL-15, respectively while the control NaMiX without IL-15Rα/IL-15 expressing the anti-NKG2A scFv is termed β.anti-NKG2A, the recombinant human IL-15 as rhIL-15, and the control condition without any molecules as medium. Figure A to C represent three independent experiments. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p < 0.05, **p < 0.005)

Fig 5: NaMiX enhanced the cytotoxic killing activity of NK and CD8+ T cells against Raji and ACH-2 cells. Human PBMCs were pre-incubated for 48 h with the different NaMiX. A Stimulated PBMCs were incubated with Raji (left panel) or ACH-2 (right panel) cells pre-stained with CellTrace Violet for 5 h. Cells were further stained using a Live/Dead marker and analyzed by flow cytometry. B Stimulated PBMCs were incubated with Raji (left panel) or ACH-2 (right panel) cells for 5 h and the CCK8 reagent was added after 2 h. Absorbance was read at 450 nm. The figures represent three independent experiments with three different donors. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p < 0.05, **p < 0.005, ***p < 0.001). C Primary NK cells were pre-stimulated for 24 h with the different NaMiX and incubated or not with Raji cells pre-stained with CellTrace Violet for 5 h in the presence of CD107a (right panel). Cells were further stained using a Live/Dead marker and analyzed by flow cytometry (left panel). Both panels show the data of one representative donor due to the high variability of the response in primary NK cells. NaMiX engrafted with IL-15Rα/IL-15 using C4bpα or C4bpβ and expressing the anti-NKG2A or the anti-KIR scFv are so called: α.anti-NKG2A.IL-15, β.anti-NKG2A.IL-15, α.anti-KIR.IL-15 and β.anti-KIR.IL-15, respectively while the control NaMiX without IL-15Rα/IL-15 expressing the anti-NKG2A scFv is termed β.anti-NKG2A, the recombinant human IL-15 as rhIL-15, and the control condition without any molecules as medium