Signaling pathways induced by a tumor-derived vaccine in antigen presenting cells

Abstract

We have previously reported on the anti-tumoral potential of a chaperone-rich cell lysate (CRCL) vaccine. Immunization with CRCL generated from tumors elicits specific T and NK cell-dependent immune responses leading to protective immunity in numerous mouse tumor models. CRCL provides both a source of tumor antigens and danger signals leading to dendritic cell activation. In humans, tumor-derived CRCL induces dendritic cell activation and CRCL-loaded dendritic cells promote the generation of cytotoxic T lymphocytes in vitro. The current study was designed to identify the signaling events and modifications triggered by CRCL in antigen presenting cells. Our results indicate that tumor-derived CRCL not only promotes the activation of dendritic cells, but also significantly fosters the function of macrophages that thus appear as major targets of this vaccine. Activation of both cell types is associated with the induction of the MAP kinase pathway, the phosphorylation of STAT1, STAT5 and AKT and with transcription factor NF-kappaB activation in vitro and in vivo. These results thus provide important insights into the mechanisms by which CRCL-based vaccines exert their adjuvant effects on antigen presenting cells.

Figure 1. CRCL induces the upregulation of the co-stimulatory molecule CD70 by DC

(A) Day 5 bone marrow derived DC were treated with tumor-derived CRCL (25 μg/ml), or LPS (1 μg/ml) as positive control. (B) Similar as (A) but in presence of anti-CD40 antibody (5 μg/ml). After 48 hrs cells were stained with APC-conjugated anti-CD11c mAb and PE-conjugated anti-CD70 mAb and analyzed by flow cytometry. (A, B), Representative dot plots of three independent experiments. (C) Mean percent increase in CD70 expression by DC following the indicated treatments compared to untreated DC. *, a significant difference compared to DC treated with CRCL alone (p<0.05).

Figure 2. CRCL induces iNOS expression and NO production by macrophages

(A) RAW264.7 cells were plated on slide chambers and exposed for 24 hrs to tumor-derived CRCL (25 μg/ml). Cells were then washed and stained for iNOS expression by IHC. Upper panels, 200× magnification, lower panels 400× magnification. (B) Western blot detection of iNOS expression in RAW264.7 or peritoneal macrophage treated with CRCL (25 μg/ml). (C) RAW264.7 cells or peritoneal macrophages were incubated for 12 hrs with CRCL (25 μg/ml) and nitrite concentration was evaluated in the culture supernatants. In all the experiments LPS (1 μg/ml)was used as a positive control. *, a significant difference when compared to untreated control cells (p < 0.05).

Figure 3. CRCL triggers macrophage tumoricidal activity

Peritoneal macrophages (50,000 cells per well) were cultured with B16 tumor cells (20,000 cells per well) and treated or not with tumor-derived CRCL (25 μg/ml) or with LPS (1 μg/ml). After 24 hrs cell viability was determined using a crystal violet toxicity assay. B16 cells alone (Medium) and B16 cells cultured with macrophages (+mφ) were used as negative controls. The data are representative of three independent experiments. *, a significant difference when compared to tumor cells culured alone (p < 0.05).

Figure 4. Activation of NF-κB in CRCL treated DC and macrophages

(A) Day 5 bone marrow derived DC or peritoneal macrophages were incubated for 24 hrs with CRCL (25 μg/ml). Total cell extracts were performed and phospho-IκB or IκB expression was analyzed by Western blot. Untreated cells were used as a negative control and cells treated with LPS (1 μg/ml) were used as a positive control. (B) Day 5 bone marrow derived DC or peritoneal macrophages were incubated for 24 hrs with CRCL. Nuclear extracts were performed and the DNA binding activity of NF-κB p50 to a consensus DNA probe was assessed as described in materials and methods. DC or macrophages cultured alone were used as a negative control, and cells treated with LPS were used as a positive control. The data are shown as a mean ± SD of duplicate wells of NF-κB p50 activation determined as the OD value 450nm as indicated by the manufacturer. (C) RAW264.7 cells were transiently transfected with a NF-κB luciferase plasmid. After 36 hrs, cells were treated with LPS or CRCL for 12 hrs as indicated and a luciferase assay was performed as described in material and methods. Data are presented as Relative Luminescent Unit (RLU)/μg of protein. Results are representative of three independent experiments. *, a significant difference when compared to untreated control cells (p < 0.05).

Figure 5. CRCL induced phosphorylation of NF-κB p65 in vivo

Naïve mice (4 animals per group) were injected (s.c.) with CRCL (100 μl, 25 μg), LPS (100 μl, 1 μg) or PBS as control vehicle. Phospho-NF-κBp65 was detected in the draining lymph nodes by IHC staining. Upper panels, 200× magnification, lower panels 400× magnification. Histogram represents a double blind count of 10 frames of phospho-NF-κB p65 positive cells. *, a significant difference compared to untreated mice (p<0.002).

Figure 6. CRCL activates STAT1, STAT5, AKT and the MAP kinase pathway but impairs the phosphorylation of STAT3 in DC and macrophages

Day 5 bone marrow derived DC or peritoneal macrophages were incubated for 24 hrs with CRCL (25 μg/ml) or LPS (1 μg/ml) as a positive control. (A) Western blot analysis of total cell extracts was then performed probing for phospho STAT1 (P-STAT1), STAT1, phospho STAT5 (P-STAT5), STAT5, phospho ERK1/2 (P-ERK1/2), ERK1/2, phospho p38 (P-p38), p38, phospho AKT (P-AKT), AKT, phospho STAT3 (P-STAT3), or STAT3.

Figure 7. CRCL overcomes TGF-β-mediated DC suppression

Day 5 bone marrow derived DC were incubated for 24 hrs with CRCL (25 μg/ml) or LPS (1 μg/ml), with or without TGF-β (10 ng/ml). The supernatants were harvested after 24 hrs and tested for TNF-α production by ELISA. *, a significant difference when compared to DC treated with LPS without TGF-β (p<0.05). NS, non-significant difference when compated to DC treated with CRCL without TGF-β.