Figure 1.
The paradoxical functions of WSX1 between in-vivo and in-vitro assays.
(a) Detection of WSX1 expression via western blot (WB) analysis (left) or flow cytometry (right) from LLC tumor cells transduced with retrovirus containing either control GFP or WSX1 gene. Cell extracts of the established cell lines were analyzed using western blot techniques and probed with mouse WSX1 and actin antibodies. (b) Detection of the role of WSX1 in tumor cell proliferation. GFP or WSX1 positive LLC cells were harvested on the indicated days and were analyzed for ATP release (left, N = 4, error bars are smaller than symbols). Comparative analysis of soft agar growth assay between LLC-GFP and LLC-WSX1 cells (right, data is representative of two independent experiments, each experiment performed in triplicate). (c) Comparison of tumor growth between LLC-GFP and LLC-WSX1 (left, N = 4, representative of four independent experiments) or AGS-GFP and AGS-fWSX1 (right, N = 5). (d) Detection of WSX1 localization in the cell. Images of TC1-WSX1 (left) or LLC-WSX1 (right) cells expressing WSX1 gene were captured using a confocal microscope. Points, mean; bars, SE. *, P<0.05 comparing GFP- to WSX1-positive tumors.
Figure 2.
IL27-independent and WSX1-dependent tumor growth promotion.
(a) Comparison of IL27 signaling function between WSX1- and DN-WSX1 cells. Equal amounts of cell extracts of LLC cells expressing GFP, WSX1, or DN-WSX1 were treated with IL27 for 10 minutes (10′), overnight (24 hr), or left untreated (-), and then analyzed using WB techniques and probed with pSTAT1 and STAT1 antibodies. (b) Comparison of tumor growth between LLC-GFP, LLC-WSX1, and LLC-MUT in wildtype C57Bl/6 mice (N = 5). Representative of two independent experiments. (c) Comparison of tumor growth between LLC-GFP and LLC-WSX1 in TCCR−/− mice (N = 4). (d) Comparison of tumor growth between LLC-WSX1 and LLC-MUT in TCCR−/− mice (N = 4). Points, mean; bars, SE. *, P<0.05 between LLC-WSX1 and LLC-GFP. #, P<0.05 between LLC-MUT and LLC-GFP.
Figure 3.
NKG2D-independent and WSX1-dependent tumor growth.
(a) Comparison of NKG2D ligand expression in LLC cells transduced with either GFP (shaded gray) or WSX1 (not shaded) stained with NKG2D-Fc followed by anti-human IgG-PE. (b) Comparison of NKG2D expression in NK+, CD8+, and CD4+ cells in the splenocytes of mice bearing LLC-GFP or LLC-WSX1 tumors, or mice without tumors. Splenocytes from tumor bearing mice were pooled (5 mice/group), processed, and stained with the following antibodies: NK1.1-FITC, CD8-FITC, CD4-APC, and NKG2D-PE. (c) Comparison of membrane-only or total cellular expression of NKG2D in the splenocytes of mice bearing LLC-GFP (dotted line) or LLC-WSX1 tumors (solid line) or mice without tumors (shaded in gray). Processed splenocytes were either permeabilized (bottom panel, ii) or left untreated (top panel, i) and stained with NKG2D-PE antibody.
Figure 4.
WSX1-mediated immunosuppression in the tumor microenvironment.
(a) Comparison of tumor growth between LLC-GFP and LLC-WSX1 in nude mice (N = 5–6). (b) Comparison of the number of T cells in the tumor microenvironment between LLC-GFP and LLC-WSX1 tumor-bearing mice. Tumors from 5 mice were pooled, processed, and then stained with CD3-APC or CD4-PE. Circled area represents T cell population. Points, mean; bars, SE. *, P<0.05.
Figure 5.
Induction of immunosuppression by WSX1 expression in LLC tumor cells.
Comparison of the effect of LLC-GFP vs. LLC-WSX1 (a) tumor cells on the number of immune cells in-vitro. Splenocytes were co-incubated with the tumor cells mentioned above in the presence of anti-CD3 and anti-CD28 antibodies for 72 hours and stained with the following antibodies: CD45-FITC, CD8-PeCy7, and CD4-PeCy7 (b) IFNγ expression in CD4+ and CD8+, cells was compared in splenocytes co-cultured with either LLC-GFP vs. LLC-WSX1. Purified NK cells in the presence or absence of IL2 (c, left and right, respectively) were cultured in the presence or absence (control) of the indicated tumor target cell lines, and IFN-γ release was determined by specific ELISA after 24 hours. Detection of the effects of AGS-GFP vs. AGS-fWSX1 on immune cells. Splenocytes and AGS tumor cells were mixed and analyzed as in (a, b).
Figure 6.
Cell contact-dependent and WSX1-mediated immunosuppression.
Detection of the effect of LLC-GFP or LLC-WSX1 tumor cells on CD4+ T cells number (a) and IFNγ expression (b). Splenocytes and tumor cells were either mixed and seeded either in the bottom of the transwell (NO BARRIER) or separated and seeded in the top and bottom of the transwell, respectively (BARRIER). The splenocytes without tumor cells were used as control (SPLEEN). Cells were analyzed similarly as in Figure 5a.
Figure 7.
Effect of WSX1 on T cell numbers.
(a) Comparison of apoptosis markers in CD4+ and CD8+ T cells when co-incubated with LLC-GFP or LLC-WSX1 tumor cells. Splenocytes and tumor cells are co-incubated as in Figure 5A, and then cells gated for CD4+ (left) or CD8+ (right) markers were analyzed for Annexin V and propidium iodine (PI) expression, Caspase 8, and Caspase 9. Representative of two independent experiments. (b) Comparison the differences in splenocytes cell numbers in the presence of either LLC-GFP or LLC-WSX1 tumor cells. Cells were seeded as in Figure 5A, and viable splenocytes were counted via tryptophan blue exclusion. (c) Comparison of purified and CFSE-labeled CD3+ T cell numbers in the presence of either LLC-GFP or LLC-WSX1 tumor cells. Data is presented as ratio of gated CFSE-positive lymphocytes (gating based on size in FSC vs. SSC) to tumor cells. (d) Detection of CFSE-positive lymphocytes within the tumor microenvironment 6 days post-transfer of tail-vein injection of CFSE- labeled CD3+T into LLC-GFP or LLC-WSX1 tumor-bearing mice. N = 4. Representative of two independent experiments. Points, mean; bars, SE. *, P<0.05.