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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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Phenotypic analysis of DCs/allo-HCC fusion cells created in the presence of HCCsp. A, Four types of DC were analyzed by flow cytometry for expression of the indicated antigens (tinted area) B, Four types of FC preparation 1) DCs/allo-HCC; 2) DCs/allo-HCC/sp; 3) OK-DCs/allo-HCC; and 4) OK-DCs/allo-HCC/sp were analyzed by two-color flow cytometry for expression of WT1 and HLA-DR. Numbers represent cells positively staining for the indicated surface markers. C, Percentage of cells positive for WT1 and HLA-DR in four types of FC preparation from three healthy donors was analyzed. For each group, the mean ± SD is shown. *, Significant differences.
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Figure 2: Phenotypic analysis of DCs/allo-HCC fusion cells created in the presence of HCCsp. A, Four types of DC were analyzed by flow cytometry for expression of the indicated antigens (tinted area) B, Four types of FC preparation 1) DCs/allo-HCC; 2) DCs/allo-HCC/sp; 3) OK-DCs/allo-HCC; and 4) OK-DCs/allo-HCC/sp were analyzed by two-color flow cytometry for expression of WT1 and HLA-DR. Numbers represent cells positively staining for the indicated surface markers. C, Percentage of cells positive for WT1 and HLA-DR in four types of FC preparation from three healthy donors was analyzed. For each group, the mean ± SD is shown. *, Significant differences.

Mentions: To assess the effects of HCCsp on DCs/tumor fusion cell generation, we have created four types of FC preparation: 1) DCs/allo-HCC; 2) DCs/allo-HCC/sp; 3) OK-DCs/allo-HCC; and 4) OK-DCs/allo-HCC/sp. DCs displayed a characteristic phenotype with expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83 molecules (Figure 1A and 2A). However, the HCC cells used for fusion expressed high levels of WT1 and HLA-ABC and low levels of CEA but not HLA-DR, CD80, CD86, and CD83 molecules (Figure 1B, 2A, and 5A). Fusions of DCs to the HCC cells coexpressed the HCC cells-derived WT1 antigens and DCs-derived HLA-DR and costimulatory molecules (Figure 2B and 2C). The fusion efficiency was determined by dual expression of tumor marker, WT1, and DC marker, HLA-DR. The cells positive for both WT1 and HLA-DR in OK-DCs/allo-HCC increased when compared with those in DCs/allo-HCC (Figure 2B and 2C). These results support our previous finding that OK-432 promotes fusion efficiency [25]. However, the percentage of double-positive cells (WT1 and HLA-DR/CD86) in OK-DCs/allo-HCC/sp was significantly decreased. These results suggest that soluble factors derived from the HCC cells have detrimental effect on the expression of maturation molecules of DCs/tumor fusion cells.


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)

Phenotypic analysis of DCs/allo-HCC fusion cells created in the presence of HCCsp. A, Four types of DC were analyzed by flow cytometry for expression of the indicated antigens (tinted area) B, Four types of FC preparation 1) DCs/allo-HCC; 2) DCs/allo-HCC/sp; 3) OK-DCs/allo-HCC; and 4) OK-DCs/allo-HCC/sp were analyzed by two-color flow cytometry for expression of WT1 and HLA-DR. Numbers represent cells positively staining for the indicated surface markers. C, Percentage of cells positive for WT1 and HLA-DR in four types of FC preparation from three healthy donors was analyzed. For each group, the mean ± SD is shown. *, Significant differences.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Phenotypic analysis of DCs/allo-HCC fusion cells created in the presence of HCCsp. A, Four types of DC were analyzed by flow cytometry for expression of the indicated antigens (tinted area) B, Four types of FC preparation 1) DCs/allo-HCC; 2) DCs/allo-HCC/sp; 3) OK-DCs/allo-HCC; and 4) OK-DCs/allo-HCC/sp were analyzed by two-color flow cytometry for expression of WT1 and HLA-DR. Numbers represent cells positively staining for the indicated surface markers. C, Percentage of cells positive for WT1 and HLA-DR in four types of FC preparation from three healthy donors was analyzed. For each group, the mean ± SD is shown. *, Significant differences.
Mentions: To assess the effects of HCCsp on DCs/tumor fusion cell generation, we have created four types of FC preparation: 1) DCs/allo-HCC; 2) DCs/allo-HCC/sp; 3) OK-DCs/allo-HCC; and 4) OK-DCs/allo-HCC/sp. DCs displayed a characteristic phenotype with expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83 molecules (Figure 1A and 2A). However, the HCC cells used for fusion expressed high levels of WT1 and HLA-ABC and low levels of CEA but not HLA-DR, CD80, CD86, and CD83 molecules (Figure 1B, 2A, and 5A). Fusions of DCs to the HCC cells coexpressed the HCC cells-derived WT1 antigens and DCs-derived HLA-DR and costimulatory molecules (Figure 2B and 2C). The fusion efficiency was determined by dual expression of tumor marker, WT1, and DC marker, HLA-DR. The cells positive for both WT1 and HLA-DR in OK-DCs/allo-HCC increased when compared with those in DCs/allo-HCC (Figure 2B and 2C). These results support our previous finding that OK-432 promotes fusion efficiency [25]. However, the percentage of double-positive cells (WT1 and HLA-DR/CD86) in OK-DCs/allo-HCC/sp was significantly decreased. These results suggest that soluble factors derived from the HCC cells have detrimental effect on the expression of maturation molecules of DCs/tumor fusion cells.

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