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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

T cell stimulation by DC matured with BCG alone or in combination with IFN-γ is mediated via identical co-stimulatory molecules DC were treated with 4.1 × 105 CFU/ml of BCG alone, BCG plus 1000 U/ml IFN-γ or left untreated (immature) for 24 h. The DC were harvested, washed, and placed in mixed leukocyte culture with allogeneic T cells at 1:100 ratio in triplicate wells of a microculture plate. Blocking antibodies were added to the DC at 1 μg/well for 1 h prior to the addition of T cells. Five days later culture supernatants collected for evaluating IFN-γ secretion by ELISA (B). The cells were pulsed with 3H-thymidine for an additional 24 h cells before harvest and quantification of incorporated radioactivity in a scintillation counter (A). Results shown in each panel are from one of two identical experiments.
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Figure 7: T cell stimulation by DC matured with BCG alone or in combination with IFN-γ is mediated via identical co-stimulatory molecules DC were treated with 4.1 × 105 CFU/ml of BCG alone, BCG plus 1000 U/ml IFN-γ or left untreated (immature) for 24 h. The DC were harvested, washed, and placed in mixed leukocyte culture with allogeneic T cells at 1:100 ratio in triplicate wells of a microculture plate. Blocking antibodies were added to the DC at 1 μg/well for 1 h prior to the addition of T cells. Five days later culture supernatants collected for evaluating IFN-γ secretion by ELISA (B). The cells were pulsed with 3H-thymidine for an additional 24 h cells before harvest and quantification of incorporated radioactivity in a scintillation counter (A). Results shown in each panel are from one of two identical experiments.

Mentions: Another level of signal modulation by IFN-γ during BCG-induced maturation is possibly via the induction of additional co-stimulatory cell surface molecules that, in turn, mediate the potentiation of the ensuing immune responses by these DC. To address this, MLR cultures were set up as earlier, and certain co-stimulatory molecules, or a combinations of them, were blocked using specific antibodies. Immature or matured DC were exposed to anti-CD54, anti-CD80, anti-CD86, or isotype-matched control blocking antibodies for 1 hour prior to addition of T-cells. Proliferation (Figure 7A) and IFN-γ secretion (Figure 7B) were measured. Blocking CD54, CD80, and CD86 together abrogated proliferation by over 95%, independent of the type of DC maturation protocol used. Similarly, blocking these molecules inhibited 75% of the IFN-γ secreted, indicating that the same co-stimulatory molecules may be responsible for T cell activation. This data, however, does not exclude the induction or upregulation of new co-stimulatory molecules. As expected, there was a reduction of IFN-γ production by the T cells in the presence of anti-IL-12 antibodies (Figure 7B), confirming that IL-12 modulated the qualitative responses T cells in this system.


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)

T cell stimulation by DC matured with BCG alone or in combination with IFN-γ is mediated via identical co-stimulatory molecules DC were treated with 4.1 × 105 CFU/ml of BCG alone, BCG plus 1000 U/ml IFN-γ or left untreated (immature) for 24 h. The DC were harvested, washed, and placed in mixed leukocyte culture with allogeneic T cells at 1:100 ratio in triplicate wells of a microculture plate. Blocking antibodies were added to the DC at 1 μg/well for 1 h prior to the addition of T cells. Five days later culture supernatants collected for evaluating IFN-γ secretion by ELISA (B). The cells were pulsed with 3H-thymidine for an additional 24 h cells before harvest and quantification of incorporated radioactivity in a scintillation counter (A). Results shown in each panel are from one of two identical experiments.
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Related In: Results  -  Collection

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Figure 7: T cell stimulation by DC matured with BCG alone or in combination with IFN-γ is mediated via identical co-stimulatory molecules DC were treated with 4.1 × 105 CFU/ml of BCG alone, BCG plus 1000 U/ml IFN-γ or left untreated (immature) for 24 h. The DC were harvested, washed, and placed in mixed leukocyte culture with allogeneic T cells at 1:100 ratio in triplicate wells of a microculture plate. Blocking antibodies were added to the DC at 1 μg/well for 1 h prior to the addition of T cells. Five days later culture supernatants collected for evaluating IFN-γ secretion by ELISA (B). The cells were pulsed with 3H-thymidine for an additional 24 h cells before harvest and quantification of incorporated radioactivity in a scintillation counter (A). Results shown in each panel are from one of two identical experiments.
Mentions: Another level of signal modulation by IFN-γ during BCG-induced maturation is possibly via the induction of additional co-stimulatory cell surface molecules that, in turn, mediate the potentiation of the ensuing immune responses by these DC. To address this, MLR cultures were set up as earlier, and certain co-stimulatory molecules, or a combinations of them, were blocked using specific antibodies. Immature or matured DC were exposed to anti-CD54, anti-CD80, anti-CD86, or isotype-matched control blocking antibodies for 1 hour prior to addition of T-cells. Proliferation (Figure 7A) and IFN-γ secretion (Figure 7B) were measured. Blocking CD54, CD80, and CD86 together abrogated proliferation by over 95%, independent of the type of DC maturation protocol used. Similarly, blocking these molecules inhibited 75% of the IFN-γ secreted, indicating that the same co-stimulatory molecules may be responsible for T cell activation. This data, however, does not exclude the induction or upregulation of new co-stimulatory molecules. As expected, there was a reduction of IFN-γ production by the T cells in the presence of anti-IL-12 antibodies (Figure 7B), confirming that IL-12 modulated the qualitative responses T cells in this system.

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