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CD8alpha- CD11b+ dendritic cells present exogenous virus-like particles to CD8+ T cells and subsequently express CD8alpha and CD205 molecules.

Morón G, Rueda P, Casal I, Leclerc C - J. Exp. Med. (2002)

Bottom Line: PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population.In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished.These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.

View Article: PubMed Central - PubMed

Affiliation: Unité de Biologie des Régulations Immunitaires, Institut Pasteur, 75724 Paris, France.

ABSTRACT
Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8alpha- DCs, whereas 15 hours later they are presented mainly by CD8alpha+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8alpha and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8alpha in CD11b+ DCs suggests that CD8alpha expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished. These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.

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Differential kinetics of in vivo PPV-VLPs-OVA presentation by CD8α− and CD8α+ DCs. (A) Purification of CD8α− and CD8α+ CD11c+ spleen cells. Spleens from C57BL/6 mice were removed and after collagenase/DNase I digestion, cells were stained with MACS beads–anti-CD11c, PE–anti-CD11c, and FITC–anti-CD8α antibodies and passed through an AutoMACS and then immediately through a FACScan™ or MOFLO®. The percentages of the different cell populations obtained after each step of purification are indicated and correspond to naive mice. (B) In vitro antigen presentation assays. Spleen CD8α− (•) and CD8α+ (○) CD11c+ cells were incubated in vitro with OVA257–264 peptide (left) or PPV-VLPs-OVA (right) for 4 h. They were then washed and cultured overnight with 105 B3Z cells/well. (C) Ex vivo PPV-VLPs-OVA presentation assays. Mice were intravenously injected with 50 μg PPV-VLPs-OVA at 90 min (left) or 15 h (right) before DC purification. CD8α− (•) and CD8α+ (○) CD11c+ cells as well as CD11c− (⋄) spleen cells were purified and cultured overnight with 105 B3Z cells/well. In the insets, CD8α− (•) and CD8α+ (○) CD11c+ cells purified from PPV-VLPs-OVA–injected mice were cultured overnight with 105 B3Z cells/well in the presence of 10−1 nM OVA257–264 peptide. The presentation of the SIINFEKL peptide to B3Z cells was monitored by IL-2 production, measured by a CTLL proliferation assay, and expressed as mean ± SEM counts per minute (cpm) of duplicate wells. One representative experiment out of two (for B) or seven (for C) is depicted (three to five pooled mice per group).
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fig4: Differential kinetics of in vivo PPV-VLPs-OVA presentation by CD8α− and CD8α+ DCs. (A) Purification of CD8α− and CD8α+ CD11c+ spleen cells. Spleens from C57BL/6 mice were removed and after collagenase/DNase I digestion, cells were stained with MACS beads–anti-CD11c, PE–anti-CD11c, and FITC–anti-CD8α antibodies and passed through an AutoMACS and then immediately through a FACScan™ or MOFLO®. The percentages of the different cell populations obtained after each step of purification are indicated and correspond to naive mice. (B) In vitro antigen presentation assays. Spleen CD8α− (•) and CD8α+ (○) CD11c+ cells were incubated in vitro with OVA257–264 peptide (left) or PPV-VLPs-OVA (right) for 4 h. They were then washed and cultured overnight with 105 B3Z cells/well. (C) Ex vivo PPV-VLPs-OVA presentation assays. Mice were intravenously injected with 50 μg PPV-VLPs-OVA at 90 min (left) or 15 h (right) before DC purification. CD8α− (•) and CD8α+ (○) CD11c+ cells as well as CD11c− (⋄) spleen cells were purified and cultured overnight with 105 B3Z cells/well. In the insets, CD8α− (•) and CD8α+ (○) CD11c+ cells purified from PPV-VLPs-OVA–injected mice were cultured overnight with 105 B3Z cells/well in the presence of 10−1 nM OVA257–264 peptide. The presentation of the SIINFEKL peptide to B3Z cells was monitored by IL-2 production, measured by a CTLL proliferation assay, and expressed as mean ± SEM counts per minute (cpm) of duplicate wells. One representative experiment out of two (for B) or seven (for C) is depicted (three to five pooled mice per group).

Mentions: In mice, spleen DCs do not constitute a homogeneous cell population. On the basis of the CD8α chain expression, it is now accepted that two major subsets of DCs can be distinguished: CD8α− and CD8α+ DCs. To evaluate the capacity of these DC populations to process and present PPV-VLPs, CD8α− CD11c+ and CD8α+ CD11c+ cells were purified. A two-step method was used: first, an enrichment step of DCs by MACS anti-CD11c mAb and then a fluorescent-activated cell sorting, using PE–anti-CD11c and FITC–anti-CD8α. This proceeding allows the recovery of a high number of purified DCs in a short time with few steps and minimal manipulation. As shown in a representative experiment (Fig. 4 A), the purity of both cell populations was always >96%. Both DC subpopulations were equally able to stimulate B3Z cells after in vitro incubation with OVA257–264 peptide (Fig. 4 B, left), which shows that CD8α− and CD8α+ DCs have the same presentation capacity. These two DC subpopulations were incubated with PPV-VLPs-OVA and as shown in Fig. 4 B (right), CD8α− CD11c+ cells effectively presented the OVA257–264 epitope whereas CD8α+ CD11c+ cells weakly stimulated B3Z cells.


CD8alpha- CD11b+ dendritic cells present exogenous virus-like particles to CD8+ T cells and subsequently express CD8alpha and CD205 molecules.

Morón G, Rueda P, Casal I, Leclerc C - J. Exp. Med. (2002)

Differential kinetics of in vivo PPV-VLPs-OVA presentation by CD8α− and CD8α+ DCs. (A) Purification of CD8α− and CD8α+ CD11c+ spleen cells. Spleens from C57BL/6 mice were removed and after collagenase/DNase I digestion, cells were stained with MACS beads–anti-CD11c, PE–anti-CD11c, and FITC–anti-CD8α antibodies and passed through an AutoMACS and then immediately through a FACScan™ or MOFLO®. The percentages of the different cell populations obtained after each step of purification are indicated and correspond to naive mice. (B) In vitro antigen presentation assays. Spleen CD8α− (•) and CD8α+ (○) CD11c+ cells were incubated in vitro with OVA257–264 peptide (left) or PPV-VLPs-OVA (right) for 4 h. They were then washed and cultured overnight with 105 B3Z cells/well. (C) Ex vivo PPV-VLPs-OVA presentation assays. Mice were intravenously injected with 50 μg PPV-VLPs-OVA at 90 min (left) or 15 h (right) before DC purification. CD8α− (•) and CD8α+ (○) CD11c+ cells as well as CD11c− (⋄) spleen cells were purified and cultured overnight with 105 B3Z cells/well. In the insets, CD8α− (•) and CD8α+ (○) CD11c+ cells purified from PPV-VLPs-OVA–injected mice were cultured overnight with 105 B3Z cells/well in the presence of 10−1 nM OVA257–264 peptide. The presentation of the SIINFEKL peptide to B3Z cells was monitored by IL-2 production, measured by a CTLL proliferation assay, and expressed as mean ± SEM counts per minute (cpm) of duplicate wells. One representative experiment out of two (for B) or seven (for C) is depicted (three to five pooled mice per group).
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fig4: Differential kinetics of in vivo PPV-VLPs-OVA presentation by CD8α− and CD8α+ DCs. (A) Purification of CD8α− and CD8α+ CD11c+ spleen cells. Spleens from C57BL/6 mice were removed and after collagenase/DNase I digestion, cells were stained with MACS beads–anti-CD11c, PE–anti-CD11c, and FITC–anti-CD8α antibodies and passed through an AutoMACS and then immediately through a FACScan™ or MOFLO®. The percentages of the different cell populations obtained after each step of purification are indicated and correspond to naive mice. (B) In vitro antigen presentation assays. Spleen CD8α− (•) and CD8α+ (○) CD11c+ cells were incubated in vitro with OVA257–264 peptide (left) or PPV-VLPs-OVA (right) for 4 h. They were then washed and cultured overnight with 105 B3Z cells/well. (C) Ex vivo PPV-VLPs-OVA presentation assays. Mice were intravenously injected with 50 μg PPV-VLPs-OVA at 90 min (left) or 15 h (right) before DC purification. CD8α− (•) and CD8α+ (○) CD11c+ cells as well as CD11c− (⋄) spleen cells were purified and cultured overnight with 105 B3Z cells/well. In the insets, CD8α− (•) and CD8α+ (○) CD11c+ cells purified from PPV-VLPs-OVA–injected mice were cultured overnight with 105 B3Z cells/well in the presence of 10−1 nM OVA257–264 peptide. The presentation of the SIINFEKL peptide to B3Z cells was monitored by IL-2 production, measured by a CTLL proliferation assay, and expressed as mean ± SEM counts per minute (cpm) of duplicate wells. One representative experiment out of two (for B) or seven (for C) is depicted (three to five pooled mice per group).
Mentions: In mice, spleen DCs do not constitute a homogeneous cell population. On the basis of the CD8α chain expression, it is now accepted that two major subsets of DCs can be distinguished: CD8α− and CD8α+ DCs. To evaluate the capacity of these DC populations to process and present PPV-VLPs, CD8α− CD11c+ and CD8α+ CD11c+ cells were purified. A two-step method was used: first, an enrichment step of DCs by MACS anti-CD11c mAb and then a fluorescent-activated cell sorting, using PE–anti-CD11c and FITC–anti-CD8α. This proceeding allows the recovery of a high number of purified DCs in a short time with few steps and minimal manipulation. As shown in a representative experiment (Fig. 4 A), the purity of both cell populations was always >96%. Both DC subpopulations were equally able to stimulate B3Z cells after in vitro incubation with OVA257–264 peptide (Fig. 4 B, left), which shows that CD8α− and CD8α+ DCs have the same presentation capacity. These two DC subpopulations were incubated with PPV-VLPs-OVA and as shown in Fig. 4 B (right), CD8α− CD11c+ cells effectively presented the OVA257–264 epitope whereas CD8α+ CD11c+ cells weakly stimulated B3Z cells.

Bottom Line: PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population.In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished.These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.

View Article: PubMed Central - PubMed

Affiliation: Unité de Biologie des Régulations Immunitaires, Institut Pasteur, 75724 Paris, France.

ABSTRACT
Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8alpha- DCs, whereas 15 hours later they are presented mainly by CD8alpha+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8alpha and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8alpha in CD11b+ DCs suggests that CD8alpha expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished. These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.

Show MeSH
Related in: MedlinePlus