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CpG-matured murine plasmacytoid dendritic cells are capable of in vivo priming of functional CD8 T cell responses to endogenous but not exogenous antigens.

Salio M, Palmowski MJ, Atzberger A, Hermans IF, Cerundolo V - J. Exp. Med. (2004)

Bottom Line: In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed.In contrast, immature PDCs are unable to prime antigen-specific CTLs.Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Tumor Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headley Way, OX3 9DS Oxford, UK. mariolina.salio@imm.ox.ac.uk

ABSTRACT
Plasmacytoid dendritic cells (PDCs) are a unique leukocyte population capable of secreting high levels of type I interferon (IFN) in response to viruses and bacterial stimuli. In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed. We report that CpG-matured murine PDCs are capable of eliciting in naive mice antigen-specific CTLs against endogenous antigens as well as exogenous peptides, but not against an exogenous antigen. Type I IFN is not required for priming, as injection of CpG-matured PDCs into type I IFN receptor-deficient mice elicits functional CTL responses. Mature PDCs prime CTLs that secrete IFN-gamma and protect mice from a tumor challenge. In contrast, immature PDCs are unable to prime antigen-specific CTLs. However, mice injected with immature PDCs are fully responsive to secondary antigenic challenges, suggesting that PDCs have not induced long-lasting tolerance via anergic or regulatory T cells. Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.

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Intravenous injection of immature male PDCs does not prevent induction of subsequent UTY246–254 responses. C57BL/6 mice (n = 3) were primed as described in Fig. 1. 10 d after priming, cytolytic activity of the UTY246–254-specific cells was assessed in vivo against female or male syngeneic splenocytes unpulsed or peptide pulsed (as detailed in Fig. 1). The analysis of mean antigen-specific lysis 17 h (A) and 1 wk (B) after target cells injection is shown, calculated as described in Materials and Methods. The priming conditions are specified on the x axis. The first group of mice (controls) was injected with labeled splenocytes only and not with DC. The second group of mice was primed by female MDC, to control for responses to components of FCS used in the BM cultures. The high numbers of splenocytes used for the in vivo killing assay primes UTY246–254-specific CTL within 7 d (28), and this is reflected by the specific clearance of male splenocytes shown in B but absent at the earlier time point. The lack of clearance of peptide-pulsed female splenocytes reflects the short half life of the peptide on the surface of these cells, which, therefore, represent an important internal control for the experiment. (C) Mean proportions of tetramer+ cells as a percentage of CD8 cells (± SEM) for each group 7 d after PDC priming (white bar) and 1 wk after the in vivo killing assay (black bars). (D) IFN-γ ELISPOT performed on blood PBLs to assess responsiveness to 10 μg/ml UTY246–254 peptide 1 wk after the in vivo killing assay. All animals showed comparable responses to PHA stimulation (not depicted). The same groups of animals are shown in A–D. (E and F) Mean proportions of UTY246–254-H-2-Db tetramer+ cells as a percentage of CD8 cells (± SEM) after in vivo boosting with male immature DCs (E, staining performed at day 7) or UV-inactivated vaccinia-UTY246–254 minigene (F, staining performed at day 8).
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fig7: Intravenous injection of immature male PDCs does not prevent induction of subsequent UTY246–254 responses. C57BL/6 mice (n = 3) were primed as described in Fig. 1. 10 d after priming, cytolytic activity of the UTY246–254-specific cells was assessed in vivo against female or male syngeneic splenocytes unpulsed or peptide pulsed (as detailed in Fig. 1). The analysis of mean antigen-specific lysis 17 h (A) and 1 wk (B) after target cells injection is shown, calculated as described in Materials and Methods. The priming conditions are specified on the x axis. The first group of mice (controls) was injected with labeled splenocytes only and not with DC. The second group of mice was primed by female MDC, to control for responses to components of FCS used in the BM cultures. The high numbers of splenocytes used for the in vivo killing assay primes UTY246–254-specific CTL within 7 d (28), and this is reflected by the specific clearance of male splenocytes shown in B but absent at the earlier time point. The lack of clearance of peptide-pulsed female splenocytes reflects the short half life of the peptide on the surface of these cells, which, therefore, represent an important internal control for the experiment. (C) Mean proportions of tetramer+ cells as a percentage of CD8 cells (± SEM) for each group 7 d after PDC priming (white bar) and 1 wk after the in vivo killing assay (black bars). (D) IFN-γ ELISPOT performed on blood PBLs to assess responsiveness to 10 μg/ml UTY246–254 peptide 1 wk after the in vivo killing assay. All animals showed comparable responses to PHA stimulation (not depicted). The same groups of animals are shown in A–D. (E and F) Mean proportions of UTY246–254-H-2-Db tetramer+ cells as a percentage of CD8 cells (± SEM) after in vivo boosting with male immature DCs (E, staining performed at day 7) or UV-inactivated vaccinia-UTY246–254 minigene (F, staining performed at day 8).

Mentions: The inability of immature PDCs to prime antigen-specific CTLs could reflect the lack of expression of costimulatory or adhesion molecules essential to trigger naive T cell proliferation. Alternatively, immature PDCs could have induced the proliferation of anergic or regulatory cells nonreactive to further antigenic challenge or unable to bind UTY246–254-Db tetramers (32, 33). The possibility of anergy or negative regulation was ruled out by demonstrating that mice injected previously with immature PDCs developed cytolytic male-specific CD8 T cells upon boosting by splenocytes (Fig. 7, A and B). The acquisition of cytolytic activity correlated with the appearance of tetramer+ CTLs in the blood (Fig. 7 C), which also secreted IFN-γ in response to the cognate peptide in an ex vivo ELISPOT assay (Fig. 7 D).


CpG-matured murine plasmacytoid dendritic cells are capable of in vivo priming of functional CD8 T cell responses to endogenous but not exogenous antigens.

Salio M, Palmowski MJ, Atzberger A, Hermans IF, Cerundolo V - J. Exp. Med. (2004)

Intravenous injection of immature male PDCs does not prevent induction of subsequent UTY246–254 responses. C57BL/6 mice (n = 3) were primed as described in Fig. 1. 10 d after priming, cytolytic activity of the UTY246–254-specific cells was assessed in vivo against female or male syngeneic splenocytes unpulsed or peptide pulsed (as detailed in Fig. 1). The analysis of mean antigen-specific lysis 17 h (A) and 1 wk (B) after target cells injection is shown, calculated as described in Materials and Methods. The priming conditions are specified on the x axis. The first group of mice (controls) was injected with labeled splenocytes only and not with DC. The second group of mice was primed by female MDC, to control for responses to components of FCS used in the BM cultures. The high numbers of splenocytes used for the in vivo killing assay primes UTY246–254-specific CTL within 7 d (28), and this is reflected by the specific clearance of male splenocytes shown in B but absent at the earlier time point. The lack of clearance of peptide-pulsed female splenocytes reflects the short half life of the peptide on the surface of these cells, which, therefore, represent an important internal control for the experiment. (C) Mean proportions of tetramer+ cells as a percentage of CD8 cells (± SEM) for each group 7 d after PDC priming (white bar) and 1 wk after the in vivo killing assay (black bars). (D) IFN-γ ELISPOT performed on blood PBLs to assess responsiveness to 10 μg/ml UTY246–254 peptide 1 wk after the in vivo killing assay. All animals showed comparable responses to PHA stimulation (not depicted). The same groups of animals are shown in A–D. (E and F) Mean proportions of UTY246–254-H-2-Db tetramer+ cells as a percentage of CD8 cells (± SEM) after in vivo boosting with male immature DCs (E, staining performed at day 7) or UV-inactivated vaccinia-UTY246–254 minigene (F, staining performed at day 8).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211835&req=5

fig7: Intravenous injection of immature male PDCs does not prevent induction of subsequent UTY246–254 responses. C57BL/6 mice (n = 3) were primed as described in Fig. 1. 10 d after priming, cytolytic activity of the UTY246–254-specific cells was assessed in vivo against female or male syngeneic splenocytes unpulsed or peptide pulsed (as detailed in Fig. 1). The analysis of mean antigen-specific lysis 17 h (A) and 1 wk (B) after target cells injection is shown, calculated as described in Materials and Methods. The priming conditions are specified on the x axis. The first group of mice (controls) was injected with labeled splenocytes only and not with DC. The second group of mice was primed by female MDC, to control for responses to components of FCS used in the BM cultures. The high numbers of splenocytes used for the in vivo killing assay primes UTY246–254-specific CTL within 7 d (28), and this is reflected by the specific clearance of male splenocytes shown in B but absent at the earlier time point. The lack of clearance of peptide-pulsed female splenocytes reflects the short half life of the peptide on the surface of these cells, which, therefore, represent an important internal control for the experiment. (C) Mean proportions of tetramer+ cells as a percentage of CD8 cells (± SEM) for each group 7 d after PDC priming (white bar) and 1 wk after the in vivo killing assay (black bars). (D) IFN-γ ELISPOT performed on blood PBLs to assess responsiveness to 10 μg/ml UTY246–254 peptide 1 wk after the in vivo killing assay. All animals showed comparable responses to PHA stimulation (not depicted). The same groups of animals are shown in A–D. (E and F) Mean proportions of UTY246–254-H-2-Db tetramer+ cells as a percentage of CD8 cells (± SEM) after in vivo boosting with male immature DCs (E, staining performed at day 7) or UV-inactivated vaccinia-UTY246–254 minigene (F, staining performed at day 8).
Mentions: The inability of immature PDCs to prime antigen-specific CTLs could reflect the lack of expression of costimulatory or adhesion molecules essential to trigger naive T cell proliferation. Alternatively, immature PDCs could have induced the proliferation of anergic or regulatory cells nonreactive to further antigenic challenge or unable to bind UTY246–254-Db tetramers (32, 33). The possibility of anergy or negative regulation was ruled out by demonstrating that mice injected previously with immature PDCs developed cytolytic male-specific CD8 T cells upon boosting by splenocytes (Fig. 7, A and B). The acquisition of cytolytic activity correlated with the appearance of tetramer+ CTLs in the blood (Fig. 7 C), which also secreted IFN-γ in response to the cognate peptide in an ex vivo ELISPOT assay (Fig. 7 D).

Bottom Line: In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed.In contrast, immature PDCs are unable to prime antigen-specific CTLs.Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Tumor Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headley Way, OX3 9DS Oxford, UK. mariolina.salio@imm.ox.ac.uk

ABSTRACT
Plasmacytoid dendritic cells (PDCs) are a unique leukocyte population capable of secreting high levels of type I interferon (IFN) in response to viruses and bacterial stimuli. In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed. We report that CpG-matured murine PDCs are capable of eliciting in naive mice antigen-specific CTLs against endogenous antigens as well as exogenous peptides, but not against an exogenous antigen. Type I IFN is not required for priming, as injection of CpG-matured PDCs into type I IFN receptor-deficient mice elicits functional CTL responses. Mature PDCs prime CTLs that secrete IFN-gamma and protect mice from a tumor challenge. In contrast, immature PDCs are unable to prime antigen-specific CTLs. However, mice injected with immature PDCs are fully responsive to secondary antigenic challenges, suggesting that PDCs have not induced long-lasting tolerance via anergic or regulatory T cells. Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.

Show MeSH
Related in: MedlinePlus