Figure 4. Adipose iNKT cells express E4BP4 which induces IL-10 production.

(a) mRNA levels of E4BP4 (Nfil3) in iNKT cells from thymus, spleen, liver and adipose tissue, as wells as T_regs from adipose and spleen as comparison. (b) Representative histogram of intracellular staining for E4BP4 in iNKT cells in several organs and adipose (red), and isotype control (grey filled) (n=4 mice, same mice for each organ). (c) Representative histogram comparing intracellular staining of E4BP4 in adipose iNKT cells vs. adipose T_regs cells (B&C) representative of n=6. (d) Graph of E4BP4 expression (MFI) by flow cytometry gated on adipose and splenic CD4⁺ and CD8⁺ T cells after T_regs and iNKT cells were excluded, compared to adipose and splenic T_regs and iNKT cells (n=5). (e) Representative dot plot of Foxp3 staining and tetramer staining after gating on adipose TCRβ⁺ cells, and histogram of Foxp3 levels in iNKT cells and T_regs cells from adipose tissue (representative of n=8). (f) Intracellular staining of IL-10 in adipose iNKT cells after 4hrs stimulation in vivo following PBS or αGalCer injection, representative of n=6 mice. (g) Primary splenic iNKT cell lines were transfected with E4BP4 (red lines) or mock transfected (grey). Representative histogram of E4BP4 expression 18 hrs after transfection. (h) Intracellular levels of IL-10 levels and (i) mRNA levels of IL-10 in iNKT cells transfected with E4BP4 or mock transfected (n=3 cell lines transfected and mock transfected).