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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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Inhibition of the differentiation of DCs by HCCsp. A, We have created four types of DC from four healthy donors; 1) DCs; 2) DCs/sp; 3) OK-DCs; and 4) OK-DCs/sp. MFIs of HLA-ABC, HLA-DR, CD80, CD86, and CD83 in four types of DC were analyzed. For each group of DCs, the mean ± SD is shown. *, Significant differences. P value (OK-DCs vs OK-DCs/sp) is represented. B, MFIs of isotype control, HLA-ABC, HLA-DR, CD80, CD86, and CD83 in the HCC cells were analyzed.
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Figure 1: Inhibition of the differentiation of DCs by HCCsp. A, We have created four types of DC from four healthy donors; 1) DCs; 2) DCs/sp; 3) OK-DCs; and 4) OK-DCs/sp. MFIs of HLA-ABC, HLA-DR, CD80, CD86, and CD83 in four types of DC were analyzed. For each group of DCs, the mean ± SD is shown. *, Significant differences. P value (OK-DCs vs OK-DCs/sp) is represented. B, MFIs of isotype control, HLA-ABC, HLA-DR, CD80, CD86, and CD83 in the HCC cells were analyzed.

Mentions: Monocyte-derived DCs from healthy donors were generated in the presence of GM-CSF and IL-4. To assess the effects of HCCsp on DCs generation, we have prepared four types of DC preparation; 1) DCs; 2) DCs/sp; 3) OK-DCs; and 4) OK-DCs/sp. Mean fluorescence intensity (MFI) of HLA-ABC, HLA-DR, CD80, CD86, and CD83 by four types of DC was determined by FACS analysis. The DCs displayed a characteristic phenotype with expression of HLA-ABC, HLA-DR, costimulatory molecules (CD80 and CD86), but low levels of the maturation marker, CD83 (Figure 1A). OK-DCs, as compared with DCs, expressed much higher levels of HLA-DR, CD80, CD86, and CD83 (Figure 1A). Interestingly, DCs generated in the presence of the supernatants (DCs/sp) were associated with down-regulation of antigen presenting molecules, including HLA-DR, CD80, and CD86 (Figure 1A). To evaluate the effects of HCCsp on the activation of DCs, DCs/sp were also subsequently stimulated with the Toll-like receptor (TLR) 4 agonist, OK-432 for 3 days (OK-DCs/sp). OK-DCs/sp, as compared with OK-DCs, exhibited much lower expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. Therefore, even if DCs/sp were exposed to a stimulus such as OK-432, these DCs could not express full levels of costimulatory molecules and the maturation marker in the presence of HCCsp.


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)

Inhibition of the differentiation of DCs by HCCsp. A, We have created four types of DC from four healthy donors; 1) DCs; 2) DCs/sp; 3) OK-DCs; and 4) OK-DCs/sp. MFIs of HLA-ABC, HLA-DR, CD80, CD86, and CD83 in four types of DC were analyzed. For each group of DCs, the mean ± SD is shown. *, Significant differences. P value (OK-DCs vs OK-DCs/sp) is represented. B, MFIs of isotype control, HLA-ABC, HLA-DR, CD80, CD86, and CD83 in the HCC cells were analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Inhibition of the differentiation of DCs by HCCsp. A, We have created four types of DC from four healthy donors; 1) DCs; 2) DCs/sp; 3) OK-DCs; and 4) OK-DCs/sp. MFIs of HLA-ABC, HLA-DR, CD80, CD86, and CD83 in four types of DC were analyzed. For each group of DCs, the mean ± SD is shown. *, Significant differences. P value (OK-DCs vs OK-DCs/sp) is represented. B, MFIs of isotype control, HLA-ABC, HLA-DR, CD80, CD86, and CD83 in the HCC cells were analyzed.
Mentions: Monocyte-derived DCs from healthy donors were generated in the presence of GM-CSF and IL-4. To assess the effects of HCCsp on DCs generation, we have prepared four types of DC preparation; 1) DCs; 2) DCs/sp; 3) OK-DCs; and 4) OK-DCs/sp. Mean fluorescence intensity (MFI) of HLA-ABC, HLA-DR, CD80, CD86, and CD83 by four types of DC was determined by FACS analysis. The DCs displayed a characteristic phenotype with expression of HLA-ABC, HLA-DR, costimulatory molecules (CD80 and CD86), but low levels of the maturation marker, CD83 (Figure 1A). OK-DCs, as compared with DCs, expressed much higher levels of HLA-DR, CD80, CD86, and CD83 (Figure 1A). Interestingly, DCs generated in the presence of the supernatants (DCs/sp) were associated with down-regulation of antigen presenting molecules, including HLA-DR, CD80, and CD86 (Figure 1A). To evaluate the effects of HCCsp on the activation of DCs, DCs/sp were also subsequently stimulated with the Toll-like receptor (TLR) 4 agonist, OK-432 for 3 days (OK-DCs/sp). OK-DCs/sp, as compared with OK-DCs, exhibited much lower expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. Therefore, even if DCs/sp were exposed to a stimulus such as OK-432, these DCs could not express full levels of costimulatory molecules and the maturation marker in the presence of HCCsp.

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