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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.


Related in: MedlinePlus

IFN-γ treatment in combination with BCG or BCG alone induces similar proportions of antigen-specific Vβ17+ T cells in recall responses, but which differ in IFN-γ producing ability DC were loaded with inactivated Influenza A virus or control protein (KLH) by pulsing with these proteins for 1 h followed by 2 hours of incubation before maturation with BCG (1.6 × 106 CFU/ml), BCG and IFN-γ (1000 U/ml), or left untreated (immature). After 20 hours of maturation, the DC were washed and autologous PBMC were added at a 10:1 (PBMC:DC) ratio in AIM-V media containing IL-2 and IL-15. Seven or eight days later the resulting cell lines were counted, analyzed for the percentage of Vβ17+CD8+ expression on T cells by flow cytometry, and the results calculated as a percent of the response seen using Influenza A-pulsed immature DC as the control (A). The influenza A virus-specific Vβ17+CD8+ T cells were re-stimulated for 40 h (including addition of GolgiPlug™ in the last 16 h) with immature or BCG ± IFN-γ matured DC loaded with Influenza M1 protein and evaluated for intracellular IFN-γ (B). Data in panel A are combined from three separate experiments and data in panel B are from one representative experiment out of four similar experiments.
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Figure 4: IFN-γ treatment in combination with BCG or BCG alone induces similar proportions of antigen-specific Vβ17+ T cells in recall responses, but which differ in IFN-γ producing ability DC were loaded with inactivated Influenza A virus or control protein (KLH) by pulsing with these proteins for 1 h followed by 2 hours of incubation before maturation with BCG (1.6 × 106 CFU/ml), BCG and IFN-γ (1000 U/ml), or left untreated (immature). After 20 hours of maturation, the DC were washed and autologous PBMC were added at a 10:1 (PBMC:DC) ratio in AIM-V media containing IL-2 and IL-15. Seven or eight days later the resulting cell lines were counted, analyzed for the percentage of Vβ17+CD8+ expression on T cells by flow cytometry, and the results calculated as a percent of the response seen using Influenza A-pulsed immature DC as the control (A). The influenza A virus-specific Vβ17+CD8+ T cells were re-stimulated for 40 h (including addition of GolgiPlug™ in the last 16 h) with immature or BCG ± IFN-γ matured DC loaded with Influenza M1 protein and evaluated for intracellular IFN-γ (B). Data in panel A are combined from three separate experiments and data in panel B are from one representative experiment out of four similar experiments.

Mentions: Next, we investigated whether the qualitative immunopotentiating effect of IFN-γ on BCG-induced DC maturation also applies to antigen-specific T cell responses. The recall T-cell response to influenza was used as the model system to address this question. DC from an HLA-A2.1+ donor were loaded with inactivated influenza-A virus or KLH, followed by maturation with BCG, in the presence or absence of IFN-γ. After maturation the DC were washed and mixed with autologous PBMC at a 10:1 (PBMC: DC) ratio. Seven-to-eight days later the resulting cell lines were counted and analyzed by flow cytometry for the percentage of Vβ17+CD8+ T cells, which are a population of T-cells that react specifically with the immunodominant HLA-A2*01-restricted matrix (M1) protein peptide [34]. Figure 4A shows the data expressed as a percentage of the response seen using Influenza A-pulsed immature DC ("percent of control"). We found that both maturation conditions led to the expansion of Vβ17+ T-cells to the same degree, but were both significantly higher than that induced by immature DC. From this it is apparent that memory T-cells will expand in response to mature antigen-loaded DC, and the level of expansion was not dependent on the presence of IFN-γ during maturation.


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 treatment in combination with BCG or BCG alone induces similar proportions of antigen-specific Vβ17+ T cells in recall responses, but which differ in IFN-γ producing ability DC were loaded with inactivated Influenza A virus or control protein (KLH) by pulsing with these proteins for 1 h followed by 2 hours of incubation before maturation with BCG (1.6 × 106 CFU/ml), BCG and IFN-γ (1000 U/ml), or left untreated (immature). After 20 hours of maturation, the DC were washed and autologous PBMC were added at a 10:1 (PBMC:DC) ratio in AIM-V media containing IL-2 and IL-15. Seven or eight days later the resulting cell lines were counted, analyzed for the percentage of Vβ17+CD8+ expression on T cells by flow cytometry, and the results calculated as a percent of the response seen using Influenza A-pulsed immature DC as the control (A). The influenza A virus-specific Vβ17+CD8+ T cells were re-stimulated for 40 h (including addition of GolgiPlug™ in the last 16 h) with immature or BCG ± IFN-γ matured DC loaded with Influenza M1 protein and evaluated for intracellular IFN-γ (B). Data in panel A are combined from three separate experiments and data in panel B are from one representative experiment out of four similar experiments.
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Related In: Results  -  Collection

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Figure 4: IFN-γ treatment in combination with BCG or BCG alone induces similar proportions of antigen-specific Vβ17+ T cells in recall responses, but which differ in IFN-γ producing ability DC were loaded with inactivated Influenza A virus or control protein (KLH) by pulsing with these proteins for 1 h followed by 2 hours of incubation before maturation with BCG (1.6 × 106 CFU/ml), BCG and IFN-γ (1000 U/ml), or left untreated (immature). After 20 hours of maturation, the DC were washed and autologous PBMC were added at a 10:1 (PBMC:DC) ratio in AIM-V media containing IL-2 and IL-15. Seven or eight days later the resulting cell lines were counted, analyzed for the percentage of Vβ17+CD8+ expression on T cells by flow cytometry, and the results calculated as a percent of the response seen using Influenza A-pulsed immature DC as the control (A). The influenza A virus-specific Vβ17+CD8+ T cells were re-stimulated for 40 h (including addition of GolgiPlug™ in the last 16 h) with immature or BCG ± IFN-γ matured DC loaded with Influenza M1 protein and evaluated for intracellular IFN-γ (B). Data in panel A are combined from three separate experiments and data in panel B are from one representative experiment out of four similar experiments.
Mentions: Next, we investigated whether the qualitative immunopotentiating effect of IFN-γ on BCG-induced DC maturation also applies to antigen-specific T cell responses. The recall T-cell response to influenza was used as the model system to address this question. DC from an HLA-A2.1+ donor were loaded with inactivated influenza-A virus or KLH, followed by maturation with BCG, in the presence or absence of IFN-γ. After maturation the DC were washed and mixed with autologous PBMC at a 10:1 (PBMC: DC) ratio. Seven-to-eight days later the resulting cell lines were counted and analyzed by flow cytometry for the percentage of Vβ17+CD8+ T cells, which are a population of T-cells that react specifically with the immunodominant HLA-A2*01-restricted matrix (M1) protein peptide [34]. Figure 4A shows the data expressed as a percentage of the response seen using Influenza A-pulsed immature DC ("percent of control"). We found that both maturation conditions led to the expansion of Vβ17+ T-cells to the same degree, but were both significantly higher than that induced by immature DC. From this it is apparent that memory T-cells will expand in response to mature antigen-loaded DC, and the level of expansion was not dependent on the presence of IFN-γ during maturation.

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.


Related in: MedlinePlus