Skip to main content
. 2024 Jan 2;15:89. doi: 10.1038/s41467-023-44310-y

Fig. 1. NKTs expressing IL-12 acquire pro-inflammatory properties.

Fig. 1

a Schematic timeline of the protocol used to select, transduce, and expand human NKTs obtained from peripheral blood. b Schematics of the retroviral vectors used to engineer human NKTs. Gamma retroviral vectors encoding either GFP only (GFP) or the p40 and p35 subunits of IL-12 connected by a flexible linker and GFP (IL12(I)GFP). c Representative flow cytometry plots of NKTs showing the transduction efficiency measured as the percentage of GFP+ cells in non-transduced NKTs (NT) and NKTs transduced with GFP or IL12(I)GFP vectors. d Representative flow cytometry plots showing NKT purity measured as percentages of iNKT+CD3+ cells in NT, GFP, and IL12(I)GFP NKTs. e Quantification of IL-12 produced by NT, GFP, and IL12(I)GFP NKTs activated with control IgG Ab or with the iNKT Ab (Vα24). IL-12 was measured in supernatants collected 24 h after plating 5 ×105 cells/well in 24 well plate in 2 mL of complete media without cytokines. Mean is shown; n = 5 donors. Source data are provided as a Source Data file. f Representative flow cytometry plots showing the expression of CD212 (IL-12Rβ) on the cell membrane of NT, GFP, and IL12(I)GFP NKTs at day 14 of culture. Empty lines represent the isotype control. g Representative western blot illustrating the STAT4 phosphorylation in NT, GFP, and IL12(I)GFP NKTs at day 14 of culture; n = 4. Source data are provided as a Source Data file. h Quantification of IFN-γ and IL-4 produced by NT, GFP, and IL12(I)GFP NKTs after activation with the control IgG Ab or with the iNKT antibody (Vα24). Cytokines were measured in supernatants collected 24 h after plating 5 × 105 cells/well in 24 well plate in 2 mL of complete media without cytokines. Mean is shown; n = 5 donors; *p = 0.0286; **p = 0.0031 NT vs IL12(i)GFP; **p = 0.0025 GFP vs IL12(i)GFP; ***p = 0.0006; two-way ANOVA. Source data are provided as a Source Data file. i Relative expression of IL-12B, IFN-γ, and T-bet assessed by qPCR in IL12(I)GFP vs NT NKTs. Mean is shown; n = 5 donors. Source data are provided as a Source Data file. j NKTs were transduced with retroviral vectors in which GFP was switched with ΔNGFR to allow sorting of the transduced NKTs25. The panel illustrates the PAT-PCA visualization of control and IL-12-expressing NKTs after activation with the control IgG Ab or the iNKT Ab (Vα24) by IsoPlexis IsoCode Secretome. Each dot represents a single cell. NKTs were categorized into circles representing polyfunctional groups with the co-secretion of different cytokines; n = 4.