The immunostimulatory effects of retinoblastoma cell supernatant on dendritic cells

Abstract

Dendritic cells (DCs) are crucial for the induction and maintenance of tumor-specific immune responses. Studies have shown that tumor-associated DCs are immunosuppressed in some human tumors. However, phenotype and function of DCs in retinoblastoma (RB) remain unclear. RB cell supernatant (RBcs) was used to treat DCs in vitro to explore the effect of RB cells on DCs. DCs were generated from peripheral blood mononuclear cells of healthy donors. On day 5 of culture, DCs were treated with RBcs for 24 h, and then purified using magnetic beads. The maturation of DCs was induced by TNF-α or LPS. After treatment with RBcs, expression of co-stimulatory molecules CD80 and CD86 was elevated in DCs, accompanied by increased production of IL-12p70, TNF-α, IL-6, IL-1β, and IL-8 but decreased production of IL-10. RBcs neither inhibited DC maturation nor promoted DC apoptosis. Moreover, RBcs-exposed DCs stimulated allogenetic T cell proliferation and T cell-derived cytokine production. These results indicate that RBcs can improve DCs' antigen presenting function and capability to activate T cells, suggesting that RB cells may have an immunostimulatory effect on DCs, and DC-based immunotherapy may be adopted in the treatment of RB.

Figure 1

The photomicrograph of DC cultures (200×). Control DCs or RBcs-exposed DCs were treated with 20 ng/mL TNF-α (A) or 1 μg/mL LPS (B) for 24 h. Y79 DC: RBcs-exposed DCs

Figure 2

Expression of DC markers, MHC and co-stimulatory molecules in RBcs-exposed DC. Control DCs or RBcs-exposed DCs were treated with 20 ng/mL TNF-α (A) or 1 μg/mL LPS (B) for 24 h. The cells were then harvested for immunofluorescence staining and flow cytometry. Bold lines denote fluorescence when stained with fluorochrome-conjugated antibody to the indicated antigen, and fine lines denote fluorescence when stained with isotype control mAb. Data shown are a representative experiment of five. Y79 DC: RBcs-exposed DCs

Figure 3

Production of cytokines IL-12, IL-10, TNF-α, IL-1β, IL-6, and IL-8 by RBcs-exposed DCs. Control DCs or RBcs-exposed DCs (4 × 10⁴ cells/well) were treated with 20 ng/mL TNF-α (A) or 1 μg/mL LPS (B) for 24 h. Concentrations of cytokines in cell-free supernatants were analyzed by CBA Human Inflammation Kit that could identify all six kinds of cytokines in a single sample. Quantitative production of a particular kind of cytokine is indicated by PE-labeled-specific Ab staining. The data are mean values ± SD of triplicate determinations. Data shown are a representative experiment of three. *P < 0.05, vs. control DCs. **P < 0.01, vs. control DCs. Y79 DC: RBcs-exposed DCs

Figure 4

Apoptosis of RBcs-exposed DCs. Control DCs or RBcs-exposed DCs were treated with 20 ng/mL TNF-α (A) or 1 μg/mL LPS (B) for 24 h. DCs were stained with FITC-Annexin-V and propidium iodide (PI), and the proportion of apoptotic cells (Annexin⁺/PI^-) was determined by flow cytometry (a and b). Apoptosis was also assessed by a FACS-based TUNEL assay utilizing FITC-dUTP. The proportion of apoptotic cells (dUTP⁺) was determined by flow cytometry (c and d). Data shown are one representative experiment of three. Y79 DC: RBcs-exposed DCs

Figure 5

Enhanced allogenetic T cell proliferation stimulated by RBcs-exposed DCs. After treatment with TNF-α (A) or LPS (B) for 24 h, varying numbers (400, 2000, or 10000) of irradiated (30 Gy) DCs were added to triplicate wells containing 2 × 10⁵ purified allogeneic T lymphocytes and incubated for 4 days. Cultures were pulsed during the final 8 h of incubation, and incorporation of [³H]-thymidine was measured. The data are mean values ± SD of triplicate determinations. Data shown are a representative experiment of three. *P < 0.05, vs control DCs. **P < 0.01, vs control DCs. Y79 DC: RBcs-exposed DCs

Figure 6

Increased cytokine production of by allogenetic T cell stimulated with RBcs-exposed DCs. After treatment with TNF-α (A) or LPS (B) for 24 h, varying numbers (400, 2000, or 10000) of irradiated (30 Gy) DCs were added to triplicate wells containing 2 × 10⁵ purified allogeneic T lymphocytes and incubated for 3 days. The concentrations of cytokines producted in cell-free supernatants were analyzed by CBA Human Th1/Th2/Th17 Cytokine Kit that could identify all seven kinds of cytokines in a single sample. Quantitative production of a particular kind of cytokine is indicated by PE-labeled-specific Ab staining. The data are mean values ± SD of triplicate determinations. Data shown are a representative experiment of three. *P < 0.05, vs. control DCs. **P < 0.01, vs. control DCs. ***P < 0.001, vs. control DCs. Y79 DC: RBcs-exposed DCs

Figure 6

Increased cytokine production of by allogenetic T cell stimulated with RBcs-exposed DCs. After treatment with TNF-α (A) or LPS (B) for 24 h, varying numbers (400, 2000, or 10000) of irradiated (30 Gy) DCs were added to triplicate wells containing 2 × 10⁵ purified allogeneic T lymphocytes and incubated for 3 days. The concentrations of cytokines producted in cell-free supernatants were analyzed by CBA Human Th1/Th2/Th17 Cytokine Kit that could identify all seven kinds of cytokines in a single sample. Quantitative production of a particular kind of cytokine is indicated by PE-labeled-specific Ab staining. The data are mean values ± SD of triplicate determinations. Data shown are a representative experiment of three. *P < 0.05, vs. control DCs. **P < 0.01, vs. control DCs. ***P < 0.001, vs. control DCs. Y79 DC: RBcs-exposed DCs