Fig 1.
Mesothelioma tumor cells modulate human MoDC lipid content and function.
Human blood monocytes cultured with GM-CSF and IL-4 were exposed to varying ratios of JU77 mesothelioma tumor cells (A). At day 7, iMoDCs stained for CD11c expression and lipid levels using BODIPY were analysed by flow cytometry. Large cells were first gated (B) and CD11c+ DCs identified (unfilled line), relative to the isotype control (shaded area; C). The BODIPY mean fluorescence intensity (MFI) of CD11c+ DCs is proportional to intracellular lipid content (D). Pooled data shows lipid levels of iMoDCs co-cultured with varying ratios of JU77 tumor cells (E). During differentiation, DCs were also exposed to Met5A cells (at a ratio of 1DC: 10Met5A cells), and DC lipid content measured as BODIPY MFI (F). The DQ-OVA assay was used to measure the antigen processing ability of iMoDCs exposed to JU77 tumor cells (G). Data in (E) and (G) is from 6 individuals and data in (F) is from 2 individuals; all data shown as mean ± SEM. * = p < 0.05; ** = p < 0.005; *** = p < 0.0005.
Fig 2.
Mesothelioma tumor cells promote partial DC maturation.
Differentiating iMoDCs exposed to varying ratios of JU77 cells were stained for CD11c and maturation markers for flow cytometric analysis. CD11c+ DCs were identified as described in Fig 1. Expression of each surface marker was analyzed on gated CD11c+ cells; representative gating strategy is shown (A). Pooled data of the percent of iMoDCs expressing CD1a (B), CD86 (C), HLA-DR (E) and CD80 (G) and surface expression levels (shown as MFIs) of CD86 (D), HLA-DR (F) and CD80 (H) on iMoDCs is from 6 individuals and shown as mean ± SEM. * = p < 0.05; ** = p < 0.005.
Fig 3.
Mesothelioma cells promote production of tolerogenic cytokines by iMoDCs.
Cytokine concentrations were measured in conditioned medium collected from co-cultures of immature MoDCs and JU77 tumor cells using a cytometric bead array; pooled data from 3 individuals is expressed as mean ± SEM (A). Cytokine concentrations were also measured in conditioned medium collected from cultured JU77 tumor cells; data is from one experiment (B). * = concentration below detection limit of assay.
Fig 4.
Mesothelioma-derived soluble factors modulate immature MoDC lipid content.
Different concentrations of JU77 tumor cell-conditioned medium (TCM) were included in the human DC differentiation protocol (A) and lipid levels measured using BODIPY (shown as MFI; B). The antigen processing capacity of iMoDCs exposed to JU77 TCM was assessed using the DQ-OVA assay and is shown as DQ-OVA MFI (C). Expression of DC maturation markers was also measured. Pooled data shows the percent of iMoDCs positive for CD1a (D) and CD86 (E), and surface expression levels of CD86 on iMoDCs (shown as MFI; F) after exposure to varying concentrations of TCM. Pooled data in (B-F) is from 6 individuals. All data is shown as mean ± SEM. * = p < 0.05; ** = p < 0.005; *** = p < 0.0005.
Fig 5.
Triglyceride-rich lipoproteins and mesothelioma-derived soluble factors modulate immature MoDCs.
During differentiation, immature MoDCs were exposed to 50% JU77 TCM, with the addition of either triglyceride-rich lipoproteins (TG) or low-density lipoproteins (LDL). The lipid content of MoDCs exposed to JU77 TCM +/- TG (A) or JU77 TCM +/- LDL (B) was measured using BODIPY MFI. The DQ-OVA assay was used to assess the antigen processing capacity of MoDCs exposed to JU77 TCM +/- TG (C) or JU77 TCM +/- LDL (D). Expression of CD1a and CD86 was also examined on MoDCs exposed to JU77 TCM +/- TG (E and G) or JU77 TCM +/- LDL (F and H). Data is from 4 individuals and is shown as mean ± SEM. * = p < 0.05.
Fig 6.
Mesothelioma-derived factors may promote lipid accumulation in immature bone marrow-derived murine DCs.
Murine bone marrow (BM) progenitor cells cultured with GM-CSF and IL-4 were exposed to TCM from the AE17 mesothelioma cell line (A). At day 10, immature BMDC lipid levels were measured by BODIPY staining (B). Immature BMDCs were matured for 2 days using LPS and were co-exposed to 50% AE17 TCM (C) before lipid levels were analyzed (D). Pooled data in (B) and (D) is from 2 experiments. All data is shown as mean ± SEM.
Fig 7.
Tumor-DCs accumulate lipid and reduce numerically with disease progression.
Mice were inoculated with 5 x 105 AE17 mesothelioma cells and tumors allowed to develop into small (< 40 mm2) or large (> 80 mm2) tumors. Total CD11c+ DCs within tumors and lymphoid organs were identified (A) and lipid levels measured using BODIPY staining shown as MFIs of CD11c+ DCs; representative samples shown (B). Pooled data show the proportions of CD11c+ DCs within tumors (C), spleens (D), dLN (E) and ndLN (F). Pooled data show the lipid content of CD11c+ DCs in AE17 tumors (G), spleens (H), dLN (I) and ndLN (J). Lymphoid organs from tumor-bearing mice were compared to those from healthy mice: n = 18 mice with small tumors, n = 9 mice with large tumors and n = 8 healthy control mice. All pooled data are shown as mean ± SEM.
Fig 8.
Lipid levels of three DC subsets increase in large mesothelioma tumors.
DC subsets were identified by first gating on CD11c+ DCs (A). Expression of CD4 versus CD8α was used to identify CD4+CD8α -, CD8α +CD4- and CD4-CD8α - DC subsets (B). Plasmacytoid DCs were identified as B220+Gr1+ cells (C). Lipid levels, shown as BODIPY MFI, were measured for CD8α +CD4- DCs (D), CD4+CD8α- DCs (E), CD4-CD8α- DCs (F) and plasmacytoid DCs (G) in small and large AE17 tumors: n = 18 mice with small tumors and n = 9 mice with large tumors. Pooled data are shown as mean ± SEM. ** = p < 0.005.
Fig 9.
CD8α+CD4- DC proportions decrease in mice with large mesothelioma tumors.
DC subsets were identified as described in Fig 8. The proportions of CD8α+CD4- DCs (A), CD4+CD8α- DCs (B), CD4-CD8α- DCs (C) and plasmacytoid DCs (D) within small versus large AE17 tumors were compared. The proportion of CD8α+CD4- DCs in dLN (E) and spleens (F) of tumor-bearing mice were compared to healthy mice: n = 18 mice with small tumors, n = 9 mice with large tumors and n = 8 healthy control mice. Pooled data are shown as mean ± SEM. ** = p < 0.005.
Fig 10.
Tumor-antigen-specific CD8+ T cell proliferation in draining lymph nodes decreases with increasing tumor burden.
C57BL/6J mice were inoculated with 5 x105 AE17 tumor cells, which were used as negative controls (B) or with AE17sOVA tumor cells (growth rate shown in A; n = 10 mice; data shown as mean ± SEM) on day 0. CFSE-labelled, CD8+, class I restricted, OVA-specific T cells from OT-1 mice were adoptively transferred at days 4, 11, 18 and 25 into the tumor-bearing mice. The dLNs were harvested from recipient mice three days post transfer such that FACS analysis was on days 7 (C, G), 14 (D, G), 21 (E, G) and 28 (F, G) post tumor cell inoculation. The lymph nodes were prepared as single cell suspensions and stained for CD8 for re-isolation of CFSE-labelled OT-1 cells. FACS analysis was performed by gating on CD8+ T cells. Representative FACS profiles shown in B-F. Results from individual mice from 1 experiment with 3–6 mice/group (G).