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Cytokine-based log-scale expansion of functional murine dendritic cells.

Harada Y, Ueda Y, Kinoh H, Komaru A, Fuji-Ogawa T, Furuya A, Iida A, Hasegawa M, Ichikawa T, Yonemitsu Y - PLoS ONE (2009)

Bottom Line: Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells.The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Therapy, Chiba University Graduate School of Medicine, Chiba, Japan.

ABSTRACT

Background: Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies.

Methodology/principal findings: Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.

Conclusions/significance: The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.

Show MeSH

Related in: MedlinePlus

Assessment of functions that are typically seen in DCs.a. FITC-dextran uptake assay assessing endo-/phagocytotic activity, a typical feature of antigen-presenting cells such as DCs. This experiment was performed in triplicate, and showed similar results. b. A graph showing MLR activity for allo-antigen (C57BL6) by iDCs or activated DCs (C3H/He) by LPS derived from the conventional technique or expansion of HPs. c. Antitumor effect of subcutaneous vaccination with rSeV/dF-activated DCs that were derived from conventional or expansion techniques. Female C3H/He mice (7-week-old) were subcutaneously vaccinated via the left flank three times each weeks with 1×106 conventional/expanded DCs pulsed with tumor lysate. Two days after the final vaccination, 1×106 LM8 cells were inoculated intradermally into the left flank of mice. Note that 3-weeks expanded HPs treated with rSeV/dF did not show any effect on tumor growth. d. Antimetastatic activity via bolus intravenous injection of various DCs. Female C3H/He mice (7-week-old) were intravenously vaccinated with 1×106 conventional/expanded DCs once via the tail vein, and 2 days later, 1×106 of LM8 cells were inoculated intravenously. Seventeen days later, mice were sacrificed and the macroscopically recognized nodules on the surface of the bilateral lungs were counted.
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pone-0006674-g003: Assessment of functions that are typically seen in DCs.a. FITC-dextran uptake assay assessing endo-/phagocytotic activity, a typical feature of antigen-presenting cells such as DCs. This experiment was performed in triplicate, and showed similar results. b. A graph showing MLR activity for allo-antigen (C57BL6) by iDCs or activated DCs (C3H/He) by LPS derived from the conventional technique or expansion of HPs. c. Antitumor effect of subcutaneous vaccination with rSeV/dF-activated DCs that were derived from conventional or expansion techniques. Female C3H/He mice (7-week-old) were subcutaneously vaccinated via the left flank three times each weeks with 1×106 conventional/expanded DCs pulsed with tumor lysate. Two days after the final vaccination, 1×106 LM8 cells were inoculated intradermally into the left flank of mice. Note that 3-weeks expanded HPs treated with rSeV/dF did not show any effect on tumor growth. d. Antimetastatic activity via bolus intravenous injection of various DCs. Female C3H/He mice (7-week-old) were intravenously vaccinated with 1×106 conventional/expanded DCs once via the tail vein, and 2 days later, 1×106 of LM8 cells were inoculated intravenously. Seventeen days later, mice were sacrificed and the macroscopically recognized nodules on the surface of the bilateral lungs were counted.

Mentions: The function of the immunostimulating activity of expanded DCs was further assessed using two different syngeneic mouse models of cancer vaccines in vivo—namely, subcutaneous inoculation forming dermal tumor (major effector: cytotoxic T-lymphocytes, or CTLs) and lung metastasis by intravenous injection (major effector: natural killer cells, or NK cells) using syngeneic (C3H/He) LM8 osteosarcoma [12]. We here used rSeV/dF as a DC-stimulator, because it has been shown that DCs treated by this modality demonstrated strongly enhanced antitumor immunity in dermal [3], [4] and metastatic [13] tumors. As shown in Fig. 3c, three-times weekly prevaccination through an intradermal route of conventional DCs treated with rSeV/dF significantly prevented tumor formation (P<0.001). Under the same treatment regimen, rSeV/dF-DCs that were obtained from HPs expanded for 3 weeks also showed a similar effect; however, rSeV/dF-treated and 3-weeks' expanded HPs without GM-CSF/IL-4 for DC differentiation did not show a significant antitumor effect. When conventional DCs were administered intravenously via the tail vein 2 days before tumor inoculation, DCs activated either by LPS or rSeV/dF but not immature DCs significantly prevented the lung metastasis; the effect was more pronounced when rSeV/dF-DCs were used, as shown in Fig. 3d. The representative findings shown in this figure were obtained in our previous study using rat prostatic cancer AT6.3 cells [13]. Similar results were also obtained in the present study using expanded DCs, even those obtained from HPs after 2 weeks of expansion.


Cytokine-based log-scale expansion of functional murine dendritic cells.

Harada Y, Ueda Y, Kinoh H, Komaru A, Fuji-Ogawa T, Furuya A, Iida A, Hasegawa M, Ichikawa T, Yonemitsu Y - PLoS ONE (2009)

Assessment of functions that are typically seen in DCs.a. FITC-dextran uptake assay assessing endo-/phagocytotic activity, a typical feature of antigen-presenting cells such as DCs. This experiment was performed in triplicate, and showed similar results. b. A graph showing MLR activity for allo-antigen (C57BL6) by iDCs or activated DCs (C3H/He) by LPS derived from the conventional technique or expansion of HPs. c. Antitumor effect of subcutaneous vaccination with rSeV/dF-activated DCs that were derived from conventional or expansion techniques. Female C3H/He mice (7-week-old) were subcutaneously vaccinated via the left flank three times each weeks with 1×106 conventional/expanded DCs pulsed with tumor lysate. Two days after the final vaccination, 1×106 LM8 cells were inoculated intradermally into the left flank of mice. Note that 3-weeks expanded HPs treated with rSeV/dF did not show any effect on tumor growth. d. Antimetastatic activity via bolus intravenous injection of various DCs. Female C3H/He mice (7-week-old) were intravenously vaccinated with 1×106 conventional/expanded DCs once via the tail vein, and 2 days later, 1×106 of LM8 cells were inoculated intravenously. Seventeen days later, mice were sacrificed and the macroscopically recognized nodules on the surface of the bilateral lungs were counted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2723913&req=5

pone-0006674-g003: Assessment of functions that are typically seen in DCs.a. FITC-dextran uptake assay assessing endo-/phagocytotic activity, a typical feature of antigen-presenting cells such as DCs. This experiment was performed in triplicate, and showed similar results. b. A graph showing MLR activity for allo-antigen (C57BL6) by iDCs or activated DCs (C3H/He) by LPS derived from the conventional technique or expansion of HPs. c. Antitumor effect of subcutaneous vaccination with rSeV/dF-activated DCs that were derived from conventional or expansion techniques. Female C3H/He mice (7-week-old) were subcutaneously vaccinated via the left flank three times each weeks with 1×106 conventional/expanded DCs pulsed with tumor lysate. Two days after the final vaccination, 1×106 LM8 cells were inoculated intradermally into the left flank of mice. Note that 3-weeks expanded HPs treated with rSeV/dF did not show any effect on tumor growth. d. Antimetastatic activity via bolus intravenous injection of various DCs. Female C3H/He mice (7-week-old) were intravenously vaccinated with 1×106 conventional/expanded DCs once via the tail vein, and 2 days later, 1×106 of LM8 cells were inoculated intravenously. Seventeen days later, mice were sacrificed and the macroscopically recognized nodules on the surface of the bilateral lungs were counted.
Mentions: The function of the immunostimulating activity of expanded DCs was further assessed using two different syngeneic mouse models of cancer vaccines in vivo—namely, subcutaneous inoculation forming dermal tumor (major effector: cytotoxic T-lymphocytes, or CTLs) and lung metastasis by intravenous injection (major effector: natural killer cells, or NK cells) using syngeneic (C3H/He) LM8 osteosarcoma [12]. We here used rSeV/dF as a DC-stimulator, because it has been shown that DCs treated by this modality demonstrated strongly enhanced antitumor immunity in dermal [3], [4] and metastatic [13] tumors. As shown in Fig. 3c, three-times weekly prevaccination through an intradermal route of conventional DCs treated with rSeV/dF significantly prevented tumor formation (P<0.001). Under the same treatment regimen, rSeV/dF-DCs that were obtained from HPs expanded for 3 weeks also showed a similar effect; however, rSeV/dF-treated and 3-weeks' expanded HPs without GM-CSF/IL-4 for DC differentiation did not show a significant antitumor effect. When conventional DCs were administered intravenously via the tail vein 2 days before tumor inoculation, DCs activated either by LPS or rSeV/dF but not immature DCs significantly prevented the lung metastasis; the effect was more pronounced when rSeV/dF-DCs were used, as shown in Fig. 3d. The representative findings shown in this figure were obtained in our previous study using rat prostatic cancer AT6.3 cells [13]. Similar results were also obtained in the present study using expanded DCs, even those obtained from HPs after 2 weeks of expansion.

Bottom Line: Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells.The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Therapy, Chiba University Graduate School of Medicine, Chiba, Japan.

ABSTRACT

Background: Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies.

Methodology/principal findings: Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.

Conclusions/significance: The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.

Show MeSH
Related in: MedlinePlus