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In vitro generation of cytotoxic and regulatory T cells by fusions of human dendritic cells and hepatocellular carcinoma cells.

Koido S, Homma S, Hara E, Mitsunaga M, Namiki Y, Takahara A, Nagasaki E, Komita H, Sagawa Y, Ohkusa T, Fujise K, Gong J, Tajiri H - J Transl Med (2008)

Bottom Line: In addition, FCs were effective in activating CD4+ and CD8+ T cells able to produce IFN-gamma and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism.Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes.The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan. shigeo_koido@jikei.ac.jp

ABSTRACT

Background: Human hepatocellular carcinoma (HCC) cells express WT1 and/or carcinoembryonic antigen (CEA) as potential targets for the induction of antitumor immunity. In this study, generation of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) by fusions of dendritic cells (DCs) and HCC cells was examined.

Methods: HCC cells were fused to DCs either from healthy donors or the HCC patient and investigated whether supernatants derived from the HCC cell culture (HCCsp) influenced on the function of DCs/HCC fusion cells (FCs) and generation of CTL and Treg.

Results: FCs coexpressed the HCC cells-derived WT1 and CEA antigens and DCs-derived MHC class II and costimulatory molecules. In addition, FCs were effective in activating CD4+ and CD8+ T cells able to produce IFN-gamma and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism. However, HCCsp induced functional impairment of DCs as demonstrated by the down-regulation of MHC class I and II, CD80, CD86, and CD83 molecules. Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes. Interestingly, fusions of immature DCs generated in the presence of HCCsp and allogeneic HCC cells promoted the generation of CD4+ CD25high Foxp3+ Treg and inhibited CTL induction in the presence of HCCsp. Importantly, up-regulation of MHC class II, CD80, and CD83 on DCs was observed in the patient with advanced HCC after vaccination with autologous FCs. In addition, the FCs induced WT1- and CEA-specific CTL that were able to produce high levels of IFN-gamma.

Conclusion: The current study is one of the first demonstrating the induction of antigen-specific CTL and the generation of Treg by fusions of DCs and HCC cells. The local tumor-related factors may favor the generation of Treg through the inhibition of DCs maturation; however, fusion cell vaccination results in recovery of the DCs function and induction of antigen-specific CTL responses in vitro. The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.

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Induction of CEA- and WT1-reactive T cells by vaccination with autologous FCs. A, Nonadherent PBMCs obtained before (left panel) and after vaccination (right panel) were stimulated with auto-HCC cells lysates, DCs mixed with auto-HCC, or unirradiated DCs/auto-HCC. T cells were cocultured with the HCC cells at a ratio of 60: 1. B, Nonadherent PBMCs obtained before (left panel) and after vaccination (right panel) were stimulated by unirradiated DCs/auto-HCC. T cells were cocultured with the HCC cells, COLP-2, COLM-6, K562, or autologous monocytes at a ratio of 20:1. Percentage of cytotoxicity (mean ± SD of 3 replicates) was determined by flow cytometry-CTL assay. C, Nonadherent PBMCs (HLA-A2+/A24-) obtained before (left panel) and after vaccination (right pane) were stimulated with unirradiated DCs/auto-HCC. T cells were analyzed by HLA-A2/WT1 or HLA-A2/CEA pentameric assay. *, Significant differences.
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Figure 7: Induction of CEA- and WT1-reactive T cells by vaccination with autologous FCs. A, Nonadherent PBMCs obtained before (left panel) and after vaccination (right panel) were stimulated with auto-HCC cells lysates, DCs mixed with auto-HCC, or unirradiated DCs/auto-HCC. T cells were cocultured with the HCC cells at a ratio of 60: 1. B, Nonadherent PBMCs obtained before (left panel) and after vaccination (right panel) were stimulated by unirradiated DCs/auto-HCC. T cells were cocultured with the HCC cells, COLP-2, COLM-6, K562, or autologous monocytes at a ratio of 20:1. Percentage of cytotoxicity (mean ± SD of 3 replicates) was determined by flow cytometry-CTL assay. C, Nonadherent PBMCs (HLA-A2+/A24-) obtained before (left panel) and after vaccination (right pane) were stimulated with unirradiated DCs/auto-HCC. T cells were analyzed by HLA-A2/WT1 or HLA-A2/CEA pentameric assay. *, Significant differences.

Mentions: We next examined whether fusion cell vaccination could augment the induction of HCC cells-specific CTL in the patient. Before vaccination, coculture of nonadherent PBMCs with unirradiated DCs/auto-HCC resulted in low levels of CTL induction against autologous HCC cells in vitro (Figure 7A). However, the CTL responses against autologous HCC cells were significantly augmented after vaccination (Figure 7A and 7B). Preincubation of the autologous HCC cells with anti-HLA-ABC mAb inhibited the lysis, suggesting the MHC class I restriction (data not shown). In contrast, there was minimal lysis of autologous HCC cells by nonadherent PBMCs obtained before vaccination cocultured with the HCC cells lysates, an unfused mixture of DCs and the HCC cells (Figure 7A), DCs alone, or the HCC cells alone (data not shown). Moreover, nonadherent PBMCs obtained after vaccination stimulated with the HCC cells lysates have considerable cytotoxic activity while no cytotoxicity is observed using those obtained before vaccination (Figure 7A). These results suggest that vaccination with autologous FCs has the potential to increase CTL precursors against autologous HCC cells in the patient.


In vitro generation of cytotoxic and regulatory T cells by fusions of human dendritic cells and hepatocellular carcinoma cells.

Koido S, Homma S, Hara E, Mitsunaga M, Namiki Y, Takahara A, Nagasaki E, Komita H, Sagawa Y, Ohkusa T, Fujise K, Gong J, Tajiri H - J Transl Med (2008)

Induction of CEA- and WT1-reactive T cells by vaccination with autologous FCs. A, Nonadherent PBMCs obtained before (left panel) and after vaccination (right panel) were stimulated with auto-HCC cells lysates, DCs mixed with auto-HCC, or unirradiated DCs/auto-HCC. T cells were cocultured with the HCC cells at a ratio of 60: 1. B, Nonadherent PBMCs obtained before (left panel) and after vaccination (right panel) were stimulated by unirradiated DCs/auto-HCC. T cells were cocultured with the HCC cells, COLP-2, COLM-6, K562, or autologous monocytes at a ratio of 20:1. Percentage of cytotoxicity (mean ± SD of 3 replicates) was determined by flow cytometry-CTL assay. C, Nonadherent PBMCs (HLA-A2+/A24-) obtained before (left panel) and after vaccination (right pane) were stimulated with unirradiated DCs/auto-HCC. T cells were analyzed by HLA-A2/WT1 or HLA-A2/CEA pentameric assay. *, Significant differences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567290&req=5

Figure 7: Induction of CEA- and WT1-reactive T cells by vaccination with autologous FCs. A, Nonadherent PBMCs obtained before (left panel) and after vaccination (right panel) were stimulated with auto-HCC cells lysates, DCs mixed with auto-HCC, or unirradiated DCs/auto-HCC. T cells were cocultured with the HCC cells at a ratio of 60: 1. B, Nonadherent PBMCs obtained before (left panel) and after vaccination (right panel) were stimulated by unirradiated DCs/auto-HCC. T cells were cocultured with the HCC cells, COLP-2, COLM-6, K562, or autologous monocytes at a ratio of 20:1. Percentage of cytotoxicity (mean ± SD of 3 replicates) was determined by flow cytometry-CTL assay. C, Nonadherent PBMCs (HLA-A2+/A24-) obtained before (left panel) and after vaccination (right pane) were stimulated with unirradiated DCs/auto-HCC. T cells were analyzed by HLA-A2/WT1 or HLA-A2/CEA pentameric assay. *, Significant differences.
Mentions: We next examined whether fusion cell vaccination could augment the induction of HCC cells-specific CTL in the patient. Before vaccination, coculture of nonadherent PBMCs with unirradiated DCs/auto-HCC resulted in low levels of CTL induction against autologous HCC cells in vitro (Figure 7A). However, the CTL responses against autologous HCC cells were significantly augmented after vaccination (Figure 7A and 7B). Preincubation of the autologous HCC cells with anti-HLA-ABC mAb inhibited the lysis, suggesting the MHC class I restriction (data not shown). In contrast, there was minimal lysis of autologous HCC cells by nonadherent PBMCs obtained before vaccination cocultured with the HCC cells lysates, an unfused mixture of DCs and the HCC cells (Figure 7A), DCs alone, or the HCC cells alone (data not shown). Moreover, nonadherent PBMCs obtained after vaccination stimulated with the HCC cells lysates have considerable cytotoxic activity while no cytotoxicity is observed using those obtained before vaccination (Figure 7A). These results suggest that vaccination with autologous FCs has the potential to increase CTL precursors against autologous HCC cells in the patient.

Bottom Line: In addition, FCs were effective in activating CD4+ and CD8+ T cells able to produce IFN-gamma and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism.Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes.The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan. shigeo_koido@jikei.ac.jp

ABSTRACT

Background: Human hepatocellular carcinoma (HCC) cells express WT1 and/or carcinoembryonic antigen (CEA) as potential targets for the induction of antitumor immunity. In this study, generation of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) by fusions of dendritic cells (DCs) and HCC cells was examined.

Methods: HCC cells were fused to DCs either from healthy donors or the HCC patient and investigated whether supernatants derived from the HCC cell culture (HCCsp) influenced on the function of DCs/HCC fusion cells (FCs) and generation of CTL and Treg.

Results: FCs coexpressed the HCC cells-derived WT1 and CEA antigens and DCs-derived MHC class II and costimulatory molecules. In addition, FCs were effective in activating CD4+ and CD8+ T cells able to produce IFN-gamma and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism. However, HCCsp induced functional impairment of DCs as demonstrated by the down-regulation of MHC class I and II, CD80, CD86, and CD83 molecules. Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes. Interestingly, fusions of immature DCs generated in the presence of HCCsp and allogeneic HCC cells promoted the generation of CD4+ CD25high Foxp3+ Treg and inhibited CTL induction in the presence of HCCsp. Importantly, up-regulation of MHC class II, CD80, and CD83 on DCs was observed in the patient with advanced HCC after vaccination with autologous FCs. In addition, the FCs induced WT1- and CEA-specific CTL that were able to produce high levels of IFN-gamma.

Conclusion: The current study is one of the first demonstrating the induction of antigen-specific CTL and the generation of Treg by fusions of DCs and HCC cells. The local tumor-related factors may favor the generation of Treg through the inhibition of DCs maturation; however, fusion cell vaccination results in recovery of the DCs function and induction of antigen-specific CTL responses in vitro. The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.

Show MeSH
Related in: MedlinePlus