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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.


BCG and IFN-γ matured DC induce primary CD4+ Th1-type T cell responses as opposed to BCG matured or immature DC PBMC were stimulated with KLH-loaded DC matured with either BCG alone (1.6 × 106 CFU/ml) or BCG and IFN-γ (1000 U/ml). Every 10–14 days the cell lines were re-stimulated with the same DC used in the initial in vitro stimulation. After 3–5 rounds of such stimulation, the resulting cells were analyzed for KLH-specific proliferation in a 3H-thymidine incorporation assay as described in Materials and Methods (A). The KLH-specific T cells were non-specifically stimulated with anti-CD3 and PMA for 8 h, including GolgiPlug™ for last 6 hours. Subsequently the cells were stained for intracellular IFN-γ, IL-4 or IL-2 (B). The data are from one representative experiment out of four similar experiments.
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Figure 5: BCG and IFN-γ matured DC induce primary CD4+ Th1-type T cell responses as opposed to BCG matured or immature DC PBMC were stimulated with KLH-loaded DC matured with either BCG alone (1.6 × 106 CFU/ml) or BCG and IFN-γ (1000 U/ml). Every 10–14 days the cell lines were re-stimulated with the same DC used in the initial in vitro stimulation. After 3–5 rounds of such stimulation, the resulting cells were analyzed for KLH-specific proliferation in a 3H-thymidine incorporation assay as described in Materials and Methods (A). The KLH-specific T cells were non-specifically stimulated with anti-CD3 and PMA for 8 h, including GolgiPlug™ for last 6 hours. Subsequently the cells were stained for intracellular IFN-γ, IL-4 or IL-2 (B). The data are from one representative experiment out of four similar experiments.

Mentions: To assess the capacity of DC to induce responses in naïve T-cells, we stimulated PBMC three times with autologous DC loaded with KLH and matured with BCG, in the presence or absence of IFN-γ. The resulting T cell lines were tested for antigen specificity by standard proliferation assay. Only those lines that were generated using DC matured with BCG and IFN-γ proliferated in response to KLH, and not to the control antigen Influenza-A or unloaded DC (Figure 5A). When these lines were non-specifically stimulated with anti-CD3 and PMA, the T-cells that were expanded using DC matured with BCG and IFN-γ produced IFN-γ whereas those expanded using DC matured with BCG alone did not. No significant differences were seen on IL-2 and IL-4 production (Figure 5B).


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)

BCG and IFN-γ matured DC induce primary CD4+ Th1-type T cell responses as opposed to BCG matured or immature DC PBMC were stimulated with KLH-loaded DC matured with either BCG alone (1.6 × 106 CFU/ml) or BCG and IFN-γ (1000 U/ml). Every 10–14 days the cell lines were re-stimulated with the same DC used in the initial in vitro stimulation. After 3–5 rounds of such stimulation, the resulting cells were analyzed for KLH-specific proliferation in a 3H-thymidine incorporation assay as described in Materials and Methods (A). The KLH-specific T cells were non-specifically stimulated with anti-CD3 and PMA for 8 h, including GolgiPlug™ for last 6 hours. Subsequently the cells were stained for intracellular IFN-γ, IL-4 or IL-2 (B). The data are from one representative experiment out of four similar experiments.
© Copyright Policy
Related In: Results  -  Collection

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Figure 5: BCG and IFN-γ matured DC induce primary CD4+ Th1-type T cell responses as opposed to BCG matured or immature DC PBMC were stimulated with KLH-loaded DC matured with either BCG alone (1.6 × 106 CFU/ml) or BCG and IFN-γ (1000 U/ml). Every 10–14 days the cell lines were re-stimulated with the same DC used in the initial in vitro stimulation. After 3–5 rounds of such stimulation, the resulting cells were analyzed for KLH-specific proliferation in a 3H-thymidine incorporation assay as described in Materials and Methods (A). The KLH-specific T cells were non-specifically stimulated with anti-CD3 and PMA for 8 h, including GolgiPlug™ for last 6 hours. Subsequently the cells were stained for intracellular IFN-γ, IL-4 or IL-2 (B). The data are from one representative experiment out of four similar experiments.
Mentions: To assess the capacity of DC to induce responses in naïve T-cells, we stimulated PBMC three times with autologous DC loaded with KLH and matured with BCG, in the presence or absence of IFN-γ. The resulting T cell lines were tested for antigen specificity by standard proliferation assay. Only those lines that were generated using DC matured with BCG and IFN-γ proliferated in response to KLH, and not to the control antigen Influenza-A or unloaded DC (Figure 5A). When these lines were non-specifically stimulated with anti-CD3 and PMA, the T-cells that were expanded using DC matured with BCG and IFN-γ produced IFN-γ whereas those expanded using DC matured with BCG alone did not. No significant differences were seen on IL-2 and IL-4 production (Figure 5B).

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.