Fig. 3: Long term exposure to IL-35 leads to NK cell hyporesponsiveness and altered NK cell surface receptor expression.

a Schematic representation of the experimental protocol. NK cells were cultured in low dose IL-2 (100 UI/mL) with or without IL-35 (100 ng/ml) for 7 days and then used for phenotyping analysis, cocultured with K562 at 1:1, 1:2, 1:5 E/T ratio for 4 h (cytotoxicity assay) or with IL-12 and IL-18 for 16 h (cytokine production analysis). Images were provided by Servier Medical Art (https://smart.servier.com/), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). b Representative flow cytometry plots for IFN-γ expression (left) and quantification of IFN-γ production (% and MFI) (right) after 7 days in IL-2 with or without IL-35 followed by IL-12 and IL-18 for 16 h. Mean values ± S.D are shown (n = 5 individual donors). c Cytokines and chemokines were quantified by ECLIA assay in NK supernatants after 7d-culture in IL-2 with or without IL-35 followed by IL-12 and IL-18 for 16 h. Results are expressed as fold change in IL-35 compared to control without IL-35. Mean values ± S.D are shown (n = 6 individual donors). d Representative flow cytometry plots for AnnexinV/PI expression (left) and quantification of % dead K562 cells (right) after 7d-culture of NK cells in IL-2 with or without IL-35 followed by 4h-coculture NK/K562. Mean values ± S.D are shown (n = 5 individual donors). e Representative flow cytometry plots (left) and quantification of activating receptors’ expression (right) after 7d-culture, as indicated in Fig. 3a. Results are expressed as MFI in IL-35 relative to control without IL-35. Mean values ± S.D are shown (n = 7 individual donors). f Representative two-dimensional T-sne plots showing NK cell clustering based on NKp30, NKp44, NKp46, DNAM1, NKG2D expression (n = 7 individual donors). g Representative flow cytometry plots (left) and quantification of inhibitory receptors’ expression (right) after 7d-culture, as indicated in Fig. 3a. Results are expressed as MFI in IL-35 relative to control without IL-35. Mean values ± S.D are shown (n = 6 to 7 individual donors). h Representative two-dimensional T-sne plots showing NK cell clustering based on NKG2A, TIGIT, TIM3, PD-L1, CD96, LAG3 expression (n = 6 to 7 individual donors). Statistical significance was determined using paired T test. Source data are provided as a Source Data file.