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Antitumor monoclonal antibodies enhance cross-presentation ofcCellular antigens and the generation of myeloma-specific killer T cells by dendritic cells.

Dhodapkar KM, Krasovsky J, Williamson B, Dhodapkar MV - J. Exp. Med. (2002)

Bottom Line: The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood.Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells.Cross-presentation was inhibited by pretreatment of DCs with Fc gamma receptor blocking antibodies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumor Immunology and Immunotherapy, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood. Here we show that coating myeloma cells with anti-syndecan-1 antibody promotes cross-presentation of cellular antigens by dendritic cells (DCs) to autologous T cells from healthy donors. The tumor cells treated with anti-syndecan-1 or isotype-matched control antibody were fed to HLA-mismatched monocyte-derived immature DCs. Tumor cell-loaded mature DCs induced a strong CD8(+) T cell response that was specific for the cancer-testis (C-T) antigens expressed in the tumor. The CD8(+) T cells killed peptide-pulsed targets, as well as myeloma tumor cells. Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells. This enhanced cross-presentation was not due to enhanced tumor cell uptake or to DC maturation. When mixtures of NY-Eso-1-positive and -negative myeloma cells were captured by DCs, the anti-syndecan-1 antibody had to be on the NY-Eso-1-positive cells to elicit NY-Eso-1-specific response. Cross-presentation was inhibited by pretreatment of DCs with Fc gamma receptor blocking antibodies. Targeting of mAb-coated tumors to DCs may contribute to the efficacy of tumor-reactive mAb and offers a new strategy for immunotherapy.

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Related in: MedlinePlus

Generation of cancer testis antigen-specific IFN-γ–producing T cells by DCs. (A) Generation of MAGE-3 or NY-Eso-1 specific T cells by peptide-pulsed or tumor cell–loaded DCs. HLA A2.1 −ve myeloma cells (cag, arp) were killed either by γ-irradiation (30 Gy) (apoptosis), freeze thaw cycles (necrosis), or coated with anti–syndecan-1 antibody (mAb)/isotype control, irradiated (3 Gy), and added to HLA A2.1+ ve immature DCs as live (annexin V-negative) cells. DCs were matured using cytokine cocktail. Autologous T cells were stimulated with mature peptide-pulsed or tumor cell–loaded DCs, or tumor cells alone. After two stimulations, the number of HLA A*0201-restricted MAGE3/NY-Eso-1 peptide specific IFN-γ–producing cells were quantified with an ELISPOT assay, using peptide pulsed autologous DCs as APCs. (B) Summary of MAGE3/NY-Eso-1–specific T cells elicited in five experiments, using DCs loaded with dying (apoptotic/necrotic) or antibody-treated (anti–syndecan-1 mAb/isotype) myeloma cells (arp cells, MAGE3+, NY-Eso-1 –ve). (C) Stimulation using CD11c+ DCs from DC-tumor cocultures. CD11c+ DCs were purified using magnetic beads from 4-h cocultures of HLA A2.1 +ve DCs with dying HLA A2.1−ve myeloma (cag) cells (apoptosis/necrosis, as described earlier); or coated with anti–syndecan-1 antibody/isotype. These DCs were then matured using cytokine cocktail and used as APCs to either directly stimulate autologous T cells, or in transwell cultures separated from T cells and unpulsed DCs. After two stimulations, HLA A*0201-restricted MAGE-3 or NY-Eso-1 peptide-specific T cells were quantified by ELISPOT using autologous peptide-pulsed DCs as APCs. Data are representative of two similar experiments.
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fig3: Generation of cancer testis antigen-specific IFN-γ–producing T cells by DCs. (A) Generation of MAGE-3 or NY-Eso-1 specific T cells by peptide-pulsed or tumor cell–loaded DCs. HLA A2.1 −ve myeloma cells (cag, arp) were killed either by γ-irradiation (30 Gy) (apoptosis), freeze thaw cycles (necrosis), or coated with anti–syndecan-1 antibody (mAb)/isotype control, irradiated (3 Gy), and added to HLA A2.1+ ve immature DCs as live (annexin V-negative) cells. DCs were matured using cytokine cocktail. Autologous T cells were stimulated with mature peptide-pulsed or tumor cell–loaded DCs, or tumor cells alone. After two stimulations, the number of HLA A*0201-restricted MAGE3/NY-Eso-1 peptide specific IFN-γ–producing cells were quantified with an ELISPOT assay, using peptide pulsed autologous DCs as APCs. (B) Summary of MAGE3/NY-Eso-1–specific T cells elicited in five experiments, using DCs loaded with dying (apoptotic/necrotic) or antibody-treated (anti–syndecan-1 mAb/isotype) myeloma cells (arp cells, MAGE3+, NY-Eso-1 –ve). (C) Stimulation using CD11c+ DCs from DC-tumor cocultures. CD11c+ DCs were purified using magnetic beads from 4-h cocultures of HLA A2.1 +ve DCs with dying HLA A2.1−ve myeloma (cag) cells (apoptosis/necrosis, as described earlier); or coated with anti–syndecan-1 antibody/isotype. These DCs were then matured using cytokine cocktail and used as APCs to either directly stimulate autologous T cells, or in transwell cultures separated from T cells and unpulsed DCs. After two stimulations, HLA A*0201-restricted MAGE-3 or NY-Eso-1 peptide-specific T cells were quantified by ELISPOT using autologous peptide-pulsed DCs as APCs. Data are representative of two similar experiments.

Mentions: After phagocytosis and ex vivo maturation using cytokine cocktail, the tumor-loaded, peptide-pulsed, or unpulsed DCs, as well as tumor cells alone, were used as APCs to stimulate T cells in culture. No T cell reactivity to either MAGE3 or NY-Eso-1 peptide was noted at baseline before in vitro expansion in any subject (data not shown). However, DCs loaded with apoptotic-, necrotic-, or antibody-coated myeloma cells expanded C-T antigen-specific T cells using two cycles of stimulation in vitro (Fig. 3 A). If the tumor expressed both MAGE-3 and NY-Eso-1 (cag cells), then both MAGE-3 and NY-Eso-1–specific T cells developed. In contrast, expansion of only MAGE-3 but not NY-Eso-1–specific T cells was seen using DCs loaded with arp cells, consistent with the pattern of antigen expression on these cells. Peptide-pulsed DCs also led to the expansion of peptide-specific T cells, while no expansion was seen using tumor cells alone. The antigen-specific T cell responses were lost after depletion with anti-CD8 antibody before the ELISPOT, indicating the CD8+ nature of the IFN-γ–secreting cells (data not shown).


Antitumor monoclonal antibodies enhance cross-presentation ofcCellular antigens and the generation of myeloma-specific killer T cells by dendritic cells.

Dhodapkar KM, Krasovsky J, Williamson B, Dhodapkar MV - J. Exp. Med. (2002)

Generation of cancer testis antigen-specific IFN-γ–producing T cells by DCs. (A) Generation of MAGE-3 or NY-Eso-1 specific T cells by peptide-pulsed or tumor cell–loaded DCs. HLA A2.1 −ve myeloma cells (cag, arp) were killed either by γ-irradiation (30 Gy) (apoptosis), freeze thaw cycles (necrosis), or coated with anti–syndecan-1 antibody (mAb)/isotype control, irradiated (3 Gy), and added to HLA A2.1+ ve immature DCs as live (annexin V-negative) cells. DCs were matured using cytokine cocktail. Autologous T cells were stimulated with mature peptide-pulsed or tumor cell–loaded DCs, or tumor cells alone. After two stimulations, the number of HLA A*0201-restricted MAGE3/NY-Eso-1 peptide specific IFN-γ–producing cells were quantified with an ELISPOT assay, using peptide pulsed autologous DCs as APCs. (B) Summary of MAGE3/NY-Eso-1–specific T cells elicited in five experiments, using DCs loaded with dying (apoptotic/necrotic) or antibody-treated (anti–syndecan-1 mAb/isotype) myeloma cells (arp cells, MAGE3+, NY-Eso-1 –ve). (C) Stimulation using CD11c+ DCs from DC-tumor cocultures. CD11c+ DCs were purified using magnetic beads from 4-h cocultures of HLA A2.1 +ve DCs with dying HLA A2.1−ve myeloma (cag) cells (apoptosis/necrosis, as described earlier); or coated with anti–syndecan-1 antibody/isotype. These DCs were then matured using cytokine cocktail and used as APCs to either directly stimulate autologous T cells, or in transwell cultures separated from T cells and unpulsed DCs. After two stimulations, HLA A*0201-restricted MAGE-3 or NY-Eso-1 peptide-specific T cells were quantified by ELISPOT using autologous peptide-pulsed DCs as APCs. Data are representative of two similar experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196013&req=5

fig3: Generation of cancer testis antigen-specific IFN-γ–producing T cells by DCs. (A) Generation of MAGE-3 or NY-Eso-1 specific T cells by peptide-pulsed or tumor cell–loaded DCs. HLA A2.1 −ve myeloma cells (cag, arp) were killed either by γ-irradiation (30 Gy) (apoptosis), freeze thaw cycles (necrosis), or coated with anti–syndecan-1 antibody (mAb)/isotype control, irradiated (3 Gy), and added to HLA A2.1+ ve immature DCs as live (annexin V-negative) cells. DCs were matured using cytokine cocktail. Autologous T cells were stimulated with mature peptide-pulsed or tumor cell–loaded DCs, or tumor cells alone. After two stimulations, the number of HLA A*0201-restricted MAGE3/NY-Eso-1 peptide specific IFN-γ–producing cells were quantified with an ELISPOT assay, using peptide pulsed autologous DCs as APCs. (B) Summary of MAGE3/NY-Eso-1–specific T cells elicited in five experiments, using DCs loaded with dying (apoptotic/necrotic) or antibody-treated (anti–syndecan-1 mAb/isotype) myeloma cells (arp cells, MAGE3+, NY-Eso-1 –ve). (C) Stimulation using CD11c+ DCs from DC-tumor cocultures. CD11c+ DCs were purified using magnetic beads from 4-h cocultures of HLA A2.1 +ve DCs with dying HLA A2.1−ve myeloma (cag) cells (apoptosis/necrosis, as described earlier); or coated with anti–syndecan-1 antibody/isotype. These DCs were then matured using cytokine cocktail and used as APCs to either directly stimulate autologous T cells, or in transwell cultures separated from T cells and unpulsed DCs. After two stimulations, HLA A*0201-restricted MAGE-3 or NY-Eso-1 peptide-specific T cells were quantified by ELISPOT using autologous peptide-pulsed DCs as APCs. Data are representative of two similar experiments.
Mentions: After phagocytosis and ex vivo maturation using cytokine cocktail, the tumor-loaded, peptide-pulsed, or unpulsed DCs, as well as tumor cells alone, were used as APCs to stimulate T cells in culture. No T cell reactivity to either MAGE3 or NY-Eso-1 peptide was noted at baseline before in vitro expansion in any subject (data not shown). However, DCs loaded with apoptotic-, necrotic-, or antibody-coated myeloma cells expanded C-T antigen-specific T cells using two cycles of stimulation in vitro (Fig. 3 A). If the tumor expressed both MAGE-3 and NY-Eso-1 (cag cells), then both MAGE-3 and NY-Eso-1–specific T cells developed. In contrast, expansion of only MAGE-3 but not NY-Eso-1–specific T cells was seen using DCs loaded with arp cells, consistent with the pattern of antigen expression on these cells. Peptide-pulsed DCs also led to the expansion of peptide-specific T cells, while no expansion was seen using tumor cells alone. The antigen-specific T cell responses were lost after depletion with anti-CD8 antibody before the ELISPOT, indicating the CD8+ nature of the IFN-γ–secreting cells (data not shown).

Bottom Line: The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood.Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells.Cross-presentation was inhibited by pretreatment of DCs with Fc gamma receptor blocking antibodies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumor Immunology and Immunotherapy, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood. Here we show that coating myeloma cells with anti-syndecan-1 antibody promotes cross-presentation of cellular antigens by dendritic cells (DCs) to autologous T cells from healthy donors. The tumor cells treated with anti-syndecan-1 or isotype-matched control antibody were fed to HLA-mismatched monocyte-derived immature DCs. Tumor cell-loaded mature DCs induced a strong CD8(+) T cell response that was specific for the cancer-testis (C-T) antigens expressed in the tumor. The CD8(+) T cells killed peptide-pulsed targets, as well as myeloma tumor cells. Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells. This enhanced cross-presentation was not due to enhanced tumor cell uptake or to DC maturation. When mixtures of NY-Eso-1-positive and -negative myeloma cells were captured by DCs, the anti-syndecan-1 antibody had to be on the NY-Eso-1-positive cells to elicit NY-Eso-1-specific response. Cross-presentation was inhibited by pretreatment of DCs with Fc gamma receptor blocking antibodies. Targeting of mAb-coated tumors to DCs may contribute to the efficacy of tumor-reactive mAb and offers a new strategy for immunotherapy.

Show MeSH
Related in: MedlinePlus