CD1d-Expressing Breast Cancer Cells Modulate NKT Cell-Mediated Antitumor Immunity in a Murine Model of Breast Cancer Metastasis
Figure 5
Gene knockdown of CD1d by shRNA in CD1d-expressing TM40D cells suppresses in vivo iNKT-mediated antitumor immunity and promotes increased spontaneous metastasis to lung.
(A) TM40D cells were transduced by a pLKO.1 lentivirus expressing shRNA against murine cd1d1. Real-time RT-PCR assay confirming downregulation of the cd1d gene in the TM40D-shCD1d cells, as compared to parental TM40D and TM40D-MB cells and scrambled shRNA control (TM40D-scr). TM40D-MB cells, as compared to parental TM40D (low metastatic) cells. GAPDH serves as an internal control. (B) FACS analysis of gene knockdown of CD1d using a PE-conjugated anti-CD1d mAb (1B1) or isotype IgG2b control. Mean fluorescence intensity (MFI) for IgG2b Isotype Control (black) = 127, TM40D-shCD1d (green) = 389, TM40D-MB (red) = 599, TM40D (blue) = 932. (C) Suppression of iNKT-regulated lymphocytes in vivo of TM40D-shCD1d tumor-implanted mice, as compared to parental TM40D control. Histograms quantifying the total percentage of live iNKT, and CD4+ and CD8+ T cell of total splenocytes. N = 5 (TM40D, TM40D-shCD1d). Data are mean ± SD. * P≤0.05. (D) Scatter plot depicting increased number of tumor foci counted per lung in TM40D-shCD1d tumor-implanted mice as compared to TM40D parental control. (* P≤0.05, one-way ANOVA test).