iNKT activity in vivo.

<p>(<b>A</b>) In vivo cytokine secretion after i.p. administration of 2 µg α-GalCer in 200 µl of PBS to healthy, non-terminal and terminal sick 5T33MM mice. Serum was collected during a time course, responses were followed by determining IFN-γ and IL-4 levels by ELISA (mean ± SD is shown from 5 mice in each group). (<b>B</b>) Kaplan-Meier survival assay. Mice were intravenously injected with a single dose of matured DCs (6×10⁵ cell/mouse) loaded with α-GalCer (100 ng/mL) or unloaded at the same day of inoculation with 5T33MM cells (n = 8 mice/group, p = 0.0366). Vehicle mice had an average survival of 22 days, α-GalCer treated mice 29 days.</p

iNKT numbers in the 5T33MM model.

<p>(<b>A</b>) Representative FACS analysis of murine iNKT cells from blood, BM, spleen and liver in naive and 5T33MM mice. Live cells (7-AAD negative) were stained with α-GalCer/CD1d tetramer which specifically binds to Vα14 of the invariant TCR and with TCR-β. Double positive iNKT cells were gated. The percentages are indicated in each plot. (<b>B</b>) Box plots of the distribution of iNKT number data, obtained from 6 mice from independent experiments. Differences between naive and 5T33MM cells in BM, liver and spleen are significant (* and ** indicate p<0.05 and p<0.005, Mann-Whitney test).</p

iNKT numbers in MM patients.

<p>Human iNKT (number/mL) were analyzed in blood samples of 51 Patients (mean age is 64 years old) and 62 healthy donors (mean age is 66 years old) by flow cytometry. Total number of iNKT cells were calculated by determining the % iNKT cells (CD3, TCRVα24 and TCRVβ11+ cells) on total T lymphocyte number (* and ** indicate p<0.05 and p<0.01, Student t-test, ns =  not significant).</p

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).</p