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Interferon-gamma Added During Bacillus Calmette-Guerin Induced Dendritic Cell Maturation Stimulates Potent Th1 Immune Responses.

Shankar G, Pestano LA, Bosch ML - J Transl Med (2003)

Bottom Line: In MLR or recall immune responses, the addition of IFN-gamma at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-gamma-producing Th1 cells.IFN-gamma co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC.These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Northwest Biotherapeutics, Inc, 21720-23rd Dr, SE, Suite 100, Bothell, WA, U,S,A. gshanka3@cntus.jnj.com

ABSTRACT
Dendritic cells (DC) are increasingly prepared in vitro for use in immunotherapy trials. Mature DC express high levels of surface molecules needed for T cell activation and are superior at antigen-presentation than immature DC. Bacillus Calmette-Guerin (BCG) is one of several products known to induce DC maturation, and interferon (IFN)-gamma has been shown to enhance the activity of DC stimulated with certain maturation factors. In this study, we investigated the use of IFN-gamma in combination with the powerful maturation agent, BCG. The treatment of immature DC with IFN-gamma plus BCG led to the upregulation of CD54, CD80, and CD86 in comparison with BCG treatment alone. In MLR or recall immune responses, the addition of IFN-gamma at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-gamma-producing Th1 cells. In primary immune responses, on the other hand, BCG and IFN-gamma co-treated DC stimulated higher proportions of specific T cells as well as IFN-gamma secretion by these T cells. Thus the use of IFN-gamma during BCG-induced DC maturation differentially affects the nature of recall versus naïve antigen-specific T-cell responses. IFN-gamma co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC. These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.

No MeSH data available.


IFN-γ in combination with BCG stimulates enhanced DC surface expression of CD54, CD80, and CD86 DC, derived from 6-day monocyte cultures with GM-CSF and IL-4, were left untreated, matured with 4.1 × 105 CFU/ml of BCG alone, or in combination with various concentrations of IFN-γ for an additional 2 days in the presence of GM-CSF and IL-4. Cells were harvested and analyzed by flow cytometry for CD54, CD80, and CD86 expression. DC-gated (based on light scatter properties) data are shown (from a representative experiment out of three similar experiments). Isotype control staining is overlaid and shown by the light gray curve. Percent positive cells are shown in each panel and mean fluorescence intensity indicated within parentheses.
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Figure 1: IFN-γ in combination with BCG stimulates enhanced DC surface expression of CD54, CD80, and CD86 DC, derived from 6-day monocyte cultures with GM-CSF and IL-4, were left untreated, matured with 4.1 × 105 CFU/ml of BCG alone, or in combination with various concentrations of IFN-γ for an additional 2 days in the presence of GM-CSF and IL-4. Cells were harvested and analyzed by flow cytometry for CD54, CD80, and CD86 expression. DC-gated (based on light scatter properties) data are shown (from a representative experiment out of three similar experiments). Isotype control staining is overlaid and shown by the light gray curve. Percent positive cells are shown in each panel and mean fluorescence intensity indicated within parentheses.

Mentions: We had previously found that DC respond to killed BCG mycobacteria with increased expression of MHC class I and class II molecules, CD54, CD80 and CD86, and CD83. These phenotypic consequences of maturation induced by BCG have also been described by others, and are similar to those obtained using other maturation factors such as LPS or monocyte-conditioned media. To investigate the consequences of IFN-γ addition to BCG-induced DC maturation, we first tested the expression of maturation-associated markers. Dendritic cells matured for 48 h with BCG alone, or in combination with various concentrations of IFN-γ were analyzed by flow cytometry for CD54, CD80, and CD86 expression. Figure 1 shows the upregulation of DC cell surface molecules in response to a 48 h exposure to increasing amounts of IFN-γ added to a fixed concentration of inactivated BCG. As expected, compared to immature DC a larger percentage of BCG-matured DC expressed co-stimulatory molecules, and this percentage was not affected by IFN-γ co-treatment. In contrast, the level of expression of these molecules on the cells was upregulated by IFN-γ. At the highest dose tested (1000 U/ml), there was an approximately 2-fold increase in CD54 and CD80 expression, and a 3-fold increase in CD86 expression, as measured by mean fluorescence intensity (MFI). The expression levels of CD83, MHC class-I, and MHC class-II were not different between BCG alone versus BCG and IFN-γ co-treatment, but higher than that of immature DC by at least 2-fold (data not shown).


Interferon-gamma Added During Bacillus Calmette-Guerin Induced Dendritic Cell Maturation Stimulates Potent Th1 Immune Responses.

Shankar G, Pestano LA, Bosch ML - J Transl Med (2003)

IFN-γ in combination with BCG stimulates enhanced DC surface expression of CD54, CD80, and CD86 DC, derived from 6-day monocyte cultures with GM-CSF and IL-4, were left untreated, matured with 4.1 × 105 CFU/ml of BCG alone, or in combination with various concentrations of IFN-γ for an additional 2 days in the presence of GM-CSF and IL-4. Cells were harvested and analyzed by flow cytometry for CD54, CD80, and CD86 expression. DC-gated (based on light scatter properties) data are shown (from a representative experiment out of three similar experiments). Isotype control staining is overlaid and shown by the light gray curve. Percent positive cells are shown in each panel and mean fluorescence intensity indicated within parentheses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC239912&req=5

Figure 1: IFN-γ in combination with BCG stimulates enhanced DC surface expression of CD54, CD80, and CD86 DC, derived from 6-day monocyte cultures with GM-CSF and IL-4, were left untreated, matured with 4.1 × 105 CFU/ml of BCG alone, or in combination with various concentrations of IFN-γ for an additional 2 days in the presence of GM-CSF and IL-4. Cells were harvested and analyzed by flow cytometry for CD54, CD80, and CD86 expression. DC-gated (based on light scatter properties) data are shown (from a representative experiment out of three similar experiments). Isotype control staining is overlaid and shown by the light gray curve. Percent positive cells are shown in each panel and mean fluorescence intensity indicated within parentheses.
Mentions: We had previously found that DC respond to killed BCG mycobacteria with increased expression of MHC class I and class II molecules, CD54, CD80 and CD86, and CD83. These phenotypic consequences of maturation induced by BCG have also been described by others, and are similar to those obtained using other maturation factors such as LPS or monocyte-conditioned media. To investigate the consequences of IFN-γ addition to BCG-induced DC maturation, we first tested the expression of maturation-associated markers. Dendritic cells matured for 48 h with BCG alone, or in combination with various concentrations of IFN-γ were analyzed by flow cytometry for CD54, CD80, and CD86 expression. Figure 1 shows the upregulation of DC cell surface molecules in response to a 48 h exposure to increasing amounts of IFN-γ added to a fixed concentration of inactivated BCG. As expected, compared to immature DC a larger percentage of BCG-matured DC expressed co-stimulatory molecules, and this percentage was not affected by IFN-γ co-treatment. In contrast, the level of expression of these molecules on the cells was upregulated by IFN-γ. At the highest dose tested (1000 U/ml), there was an approximately 2-fold increase in CD54 and CD80 expression, and a 3-fold increase in CD86 expression, as measured by mean fluorescence intensity (MFI). The expression levels of CD83, MHC class-I, and MHC class-II were not different between BCG alone versus BCG and IFN-γ co-treatment, but higher than that of immature DC by at least 2-fold (data not shown).

Bottom Line: In MLR or recall immune responses, the addition of IFN-gamma at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-gamma-producing Th1 cells.IFN-gamma co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC.These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Northwest Biotherapeutics, Inc, 21720-23rd Dr, SE, Suite 100, Bothell, WA, U,S,A. gshanka3@cntus.jnj.com

ABSTRACT
Dendritic cells (DC) are increasingly prepared in vitro for use in immunotherapy trials. Mature DC express high levels of surface molecules needed for T cell activation and are superior at antigen-presentation than immature DC. Bacillus Calmette-Guerin (BCG) is one of several products known to induce DC maturation, and interferon (IFN)-gamma has been shown to enhance the activity of DC stimulated with certain maturation factors. In this study, we investigated the use of IFN-gamma in combination with the powerful maturation agent, BCG. The treatment of immature DC with IFN-gamma plus BCG led to the upregulation of CD54, CD80, and CD86 in comparison with BCG treatment alone. In MLR or recall immune responses, the addition of IFN-gamma at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-gamma-producing Th1 cells. In primary immune responses, on the other hand, BCG and IFN-gamma co-treated DC stimulated higher proportions of specific T cells as well as IFN-gamma secretion by these T cells. Thus the use of IFN-gamma during BCG-induced DC maturation differentially affects the nature of recall versus naïve antigen-specific T-cell responses. IFN-gamma co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC. These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.

No MeSH data available.