Eliciting cytotoxic T lymphocytes against human laryngeal cancer-derived antigens: evaluation of dendritic cells pulsed with a heat-treated tumor lysate and other antigen-loading strategies for dendritic-cell-based vaccination

Abstract

Background: Dendritic cells (DCs) have been used successfully in clinical pilot studies. However, tumor-specific immunity and clinical responses were only induced in certain cancer patients. It has been well documented that immunotherapy efficacy can be optimized for responses using antigen pulsing.

Methods: The human laryngeal squamous cell cancer (LSCC) cell line SNU899 was used to evaluate the in vitro anti-tumor efficacy of three different preparations of dendritic cell (DC) vaccines consisting of either whole tumor cells or their derivatives including: i) DCs pulsed with a tumor cell supernatant (DC-TCS), ii) DCs pulsed with whole-cell tumor stressed lysate (DC-TSL), and iii) DCs pulsed with irradiated tumor cells (DC-ITC).

Results: Our results showed that DC-TSL is an effective source of tumor-associated antigens (TAAs) for pulsing DCs. DC-TSL induced the highest expansion of TAA-specific T cells, the strongest Th1 cytokine response, and the most potent cytotoxic T lymphocyte (CTL) activity. DC-TCS and DC-ITC inhibited T cell activation but induced a certain extent of CTL activity.

Conclusions: These data suggest that DC-TSL is a more potent inducer of antitumor immunity against laryngeal cancer than other antigen-loading strategies using whole tumor cell materials. This strategy provides an alternative approach for DC-based immunotherapy for laryngeal cancer.

Fig. 1

Photomicrographs of DC cultures (4×). DCs cultured for 5 days were pulsed for 24 h with SNU899 Ags using three different methods. All methods employed a tumor:DC ratio of 3:1. Compared with the DC control (≤200 μm), DC-TCS or DC-TSLs appeared to form larger adherent aggregates (>200 μm), whereas DC-ITC had a higher density

Fig. 2

Phenotypes of DCs loaded with SNU899-derived Ags by different methods. To induce maturation, control DCs and SNU899-derived Ag-loaded DCs were treated with 1 μg/ml LPS for 48 h. A DC maturation marker (CD83), MHC-II molecule (HLA-DR), and co-stimulatory molecules (CD80, CD86, and CD40) were measured by flow cytometry. DC-TSL expressed the highest level of HLA-DR and CD86. DC-ITC also exhibited elevated HLA-DR expression, but expressed the lowest level of co-stimulatory molecules. HLA-DR expression on the surface of DC-TCSs remained at the same level as in unpulsed DCs. Data shown are representative of three experiments

Fig. 3

Induction of TAA-specific T cell responses by DCs loaded with SNU899-derived Ags by various methods. a Induction of TAA-specific CD4⁺ T cells. TAA-loaded mature DCs were added to autologous PBLs at a ratio of 1:20. On day 6 after the second stimulation, the number of CD4⁺ T cells was assessed by flow cytometry. The methods used to load the DCs induced differential class II cross-presentation of TAAs to the T cells. However, the stressed lysis strategy resulted in the highest efficiency of T cell activation, reflecting a higher level of TAAs after loading. b Induction of TAA-specific CD8⁺ T cells. Similar to the cross-presentation by MHC class II, TCL-DCs were also much more efficient at cross-presentation of TAAs by MHC-I, and thus T cell activation, than DC-TCS or DC-ITC. Data are mean values ± SD of triplicate determinations. Statistical comparisons were performed using Student’s t-test. *P < 0.05 vs. DC control; **P < 0.01 vs. DC control

Fig. 4

Induction of TAA-specific Th1 cytokines by three DC Ag-loading strategies. DCs were pulsed with either SNU899 tumor cell supernatant, stressed lysate, or irradiated tumor cells. Autologous PBLs were stimulated twice with Ag-loaded mature DCs. On day 6 after the second stimulation, production of IFN-γ, IL-2, and TNF-α by TAA-specific CD4⁺ T cells was detected by intracellular staining. a Numbers indicate the percentage of gated CD4⁺ T cells. When PBLs were stimulated by TCL-DCs, the percentage of IFN-γ and IL-2-secreting CD4⁺ T cells was higher than in other groups, whereas DC-ITC induced a higher percentage of TNF-α-secreting CD4⁺ T cells. b Number of Th1 cytokine (IFN-γ, IL-2, and TNF-α)-positive cells were assessed by flow cytometry. Compared with the DC control, TCL-DCs stimulated an effective TAA-specific T cell response, whereas DC-TCS and DC-ITC inhibited such responses. These results clearly demonstrate the efficacy of stressed lysis as a means of tumor cell preparation for DC loading. Data are mean values ± SD of triplicate determinations. Statistical comparisons were performed using Student’s t-test. *P < 0.05 vs. DC control; **P < 0.01 vs. DC control

Fig. 5

Induction of TAA-specific CTL activity by three DC Ag-loading strategies. a Ag-loaded DCs were used as stimulators of autologous PBLs to compare their efficacy in CTL stimulation against Ag-pulsed target cells. PBLs and DCs were generated from a single donor. Data are from one representative experiment. b TAA-specific cytotoxicity was determined by flow cytometry. Data are the mean percentages of cytotoxicity ± SD of triplicate determinations. TCL-DCs induced a more potent CTL response compared with DC-ITC as shown by the increased cytotoxicity from SNU899-derived Ag-pulsed autologous DCs used as targets at a 10:1 E:T. Statistical comparisons were performed using Student’s t-test