Figure S1: Correlation Analysis Between Serum Leptin Levels (Ng/Ml) and Body Mass Index

SUPPLEMENTARY FIGURES

Figure S1: Correlation analysis between serum leptin levels (ng/ml) and body mass index in healthy control, MGUS and NDMM patients.

Figure S2: Leptin and its receptor on iNKT cells in the 5T33MM murine model. A) Leptin serum levels measured in 5T33MM WT mice at week 1, 2 and 3 after inoculation (n=4) compared to naive C57BL/KaLwRij mice (n=4). B) Mean Fluorescence Intensity (MFI) of leptin receptor expression on iNKT cells in liver, bone marrow, spleen and blood at week 1, 2 and 3 after inoculation with 5T33MM cells compared to leptin receptor levels in naive C57BL/KaLwRij mice (n=3).* p<0.05, ** p<0.01, *** p<0.001

Figure S3: iNKT co-culture experiments with leptin and the 2.17-mAlb Leptin receptor antagonist. A) Mean IFN-γ levels of murine iNKT co-culture experiments are illustrated. Experiments were performed with different murine MM cell lines: 5T33MMvt and 5T2MM. Respective conditions are iNKT cells + α-GalCer-loaded DC’s; iNKT cells + α-GC- loaded DC’s + MM cells; iNKT cells + α-GC- loaded DC’s + MM cells + 2.17-mAlb; iNKT cells + α-GC- loaded DC’s + 2.17-mAlb; iNKT cells + α-GC- loaded DC’s + leptin; iNKT cells + α-GC- loaded DC’s + leptin + MM; iNKT cells + α-GC- loaded DC’s + leptin + 2.17-mAlb. B) IFN-γ levels of human iNKT co-culture experiment are illustrated. Human co-culture experiments were performed using the same set-up as the murine co-culture experiments. Different human MM cell lines were tested including RPMI 8226 and U-266. Natural killer T cells (NKT); Vivo (vv); Vitro (vt); Dendritic cells (DC); Alpha- Galactosylceramide (α-GC); Multiple myeloma cells (MM); Leptin Receptor antagonist (2.17-mAlb); Leptin (L). * p<0.05, ** p<0.01, *** p<0.001

Figure S4:In vivo treatment with 2.17-mAlb Leptin receptor antagonist and α-GalCer. A) Mice were weighted daily. Weight curves from one 5T33MM WT mouse representative for each group are shown. Similar weight curves were seen for 5T33MM CD1d-/- mice (data not shown). B) Fat pad weight at the end of the experiment for both 5T33MM WT and 5T33MM CD1d-/- mice.Alpha-Galactosylceramide (α-GC); Leptin Receptor antagonist (2.17-mAlb); Tumor necrosis factor alpha (TNFα); Interferon gamma (IFN-γ); Interleukin 6 (IL-6); Wild type (WT). * p<0.05, ** p<0.01, *** p<0.001

Figure S5: The combination effects of leptin receptor blockade and iNKT cell activation in 5T33MM C57BL/KaLwRij CD1d-/-. A) IFN-γ levels in the serum 16h after the first stimulation and second stimulation (one week later) with α-GalCer are represented. B)Serum TNFα and IL-6 levels were determined by ELISA at the end of the experiment for each treatment group in 5T33MM WT mice.C) Serum TNFα and IL-6 levels at the end of the experiment for each group in 5T33MM C57BL/KaLwRij CD1d-/- mice. Alpha- Galactosylceramide (α-GC); Leptin Receptor antagonist (2.17-mAlb); Multiple myeloma (MM); Tumor necrosis factor alpha (TNFα); Interferon gamma (IFN-γ); Interleukine 6 (IL-6).

* p<0.05, ** p<0.01, *** p<0.001

Figure S6: Evaluation of CD69 and CD25 expression oniNKT and NK cells after leptin receptor blockade and iNKT cell activation. a)Percentage expression levels of activation marker CD69 on NK cells in liver, spleen, bone marrow and blood. b) Percentage expression levels of activation marker CD25 are illustrated for iNKT and NK cells in liver, spleen, bone marrow and blood of the different treatment groups. Natural killer T cells (NKT); Natural killer cells (NK); Alpha- Galactosylceramide (α-GC); Multiple myeloma (MM) Leptin Receptor antagonist (2.17-mAlb)

Figure S7: Evaluation of BrdU incorporation of iNKT and NK cells after leptin receptor blockade and iNKT cell activation. A) Fluorescence histogram representing BrdU incorporation of NK cells in the liver of 5T33MM WT mice.B) Table with an overview of the % BrdU incorporation ± SD of iNKT and NK cells in the liver for each treatment group in 5T33MM C57BL/KaLwRij WT and C57BL/KaLwRij CD1d-/- mice. Natural killer T cells (NKT); Natural killer cells (NK); Alpha- Galactosylceramide (α-GC); Multiple myeloma (MM) Leptin Receptor antagonist (2.17-mAlb)