iNKT frequency and activity during the development of the disease in the 5T33MM model.

<p><b>(A, upper panel)</b> Frequency of iNKTs in liver and spleen during the course of the disease, analyzed by flow cytometry as was done in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065075#pone-0065075-g001" target="_blank">figure 1A</a>. Mice were isolated at different time points and liver and MACS-sorted splenic iNKTs were stained with α-GalCer/CD1d tetramer. Plasmacytosis (tumor load) was assessed on May-Grünwald Giemsa stained cytosmears of the isolated BM. The mean ± SD of 3 mice in independent experiment is shown. <b>(A, lower panel)</b> The mean % iNKT cells in liver and spleen of naive and end stage 5T33 mice (n = 6, ** indicates p<0.01, Mann-Whitney test). <b>(B, upper panel)</b> In vitro iNKT activity. Mice were isolated at different time points and liver iNKTs were co-cultured with α-GalCer loaded or unloaded DCs for 72 h. IFN-γ secretion in the co-culture was measured by ELISA. The mean ± SD of 3 mice in independent experiment is shown. <b>(B, lower panel)</b> The mean IFN-γ secretion of DC co-cultures with liver iNKT cells of naïve and end stage 5T33 mice (n = 6, ** indicates p<0.01, Mann-Whitney test). (<b>C</b>) Gene expression of the Vα14 receptor of liver iNKTs of healthy and diseased 5T33MM was assessed by RT-PCR (n = 6). <b>(D, upper panel)</b> Expression of CD1d in live spleen and BM cells in the 5T33MM model during the course of the MM disease compared to tumor load. Mice were isolated at different time points of the disease and spleen and BM cells were double stained for CD1d and 5T33MM. The mean ± SD of 3 mice in independent experiment is shown. <b>(D, lower panel)</b> The mean CD1d expression in spleen and BM cells of naive and end stage 5T33 mice (n = 6, ** indicates p<0.01, Mann-Whitney test).