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Presentation of exogenous antigens on major histocompatibility complex (MHC) class I and MHC class II molecules is differentially regulated during dendritic cell maturation.

Delamarre L, Holcombe H, Mellman I - J. Exp. Med. (2003)

Bottom Line: Unlike MHC II, these events do not involve a marked redistribution of preexisting MHC I molecules from intracellular compartments to the DC surface.In contrast, formation of peptide-MHC I complexes from endogenous cytosolic antigens occurs even in unstimulated, immature DCs.Thus, the MHC I and MHC II pathways of antigen presentation are differentially regulated during DC maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Section of Immunobiology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, CT 06520-8002, USA.

ABSTRACT
During maturation, dendritic cells (DCs) regulate their capacity to process and present major histocompatibility complex (MHC) II-restricted antigens. Here we show that presentation of exogenous antigens by MHC I is also subject to developmental control, but in a fashion strikingly distinct from MHC II. Immature mouse bone marrow-derived DCs internalize soluble ovalbumin and sequester the antigen intracellularly until they receive an appropriate signal that induces cross presentation. At that time, peptides are generated in a proteasome-dependent fashion and used to form peptide-MHC I complexes that appear at the plasma membrane. Unlike MHC II, these events do not involve a marked redistribution of preexisting MHC I molecules from intracellular compartments to the DC surface. Moreover, out of nine stimuli well known to induce the phenotypic maturation of DCs and to promote MHC II presentation, only two (CD40 ligation, disruption of cell-cell contacts) activated cross presentation on MHC I. In contrast, formation of peptide-MHC I complexes from endogenous cytosolic antigens occurs even in unstimulated, immature DCs. Thus, the MHC I and MHC II pathways of antigen presentation are differentially regulated during DC maturation.

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CD11c-negative cells internalize and transport soluble OVA into the cytosol but do not exhibit cross presentation. (A) Immature B6D2F1 DC cultures were pulsed with FITC-OVA (5 mg/ml) for 30 min, then washed. FITC-OVA uptake and cell surface expression of CD11c were monitored by flow cytometry. (B) DC cultures were pulsed with OVA (5 mg/ml) for 30 min, washed, and chased for 30 min. CD11c-negative cells were separated from DCs using magnetic beads conjugated to anti-CD11c mAb. After homogenization of CD11c-negative cells, cytosolic and membrane/vesicle fractions were separated by ultracentrifugation, and probed for OVA and Cat L by Western blot. (C) DC cultures were pulsed with FITC-OVA (5 mg/ml) for 30 min, then washed. Cells were transferred on coverslips and incubated at 37°C for 30 min to allow their attachment. Cells were then fixed, permeabilized, and stained using a rabbit anti-OVA Ab, and TIB 120 (anti-MHC II), and analyzed by confocal microscopy. (D) DC cultures were pulsed with 1 mg/ml of OVA for 2 h, washed, activated with LPS and cluster disruption, and chased for 7 h. After fixation, CD11c-positive DCs were separated from CD11c-negative cells as in B. Mixed cultures before separation of OVA-pulsed cells (or BSA-pulsed cells as a control), CD11c-positive and CD11c-negative fractions (>95%) were then cultured with OT.1 T cells in the presence or absence of anti-CD28 mAb. T cell activation was monitored at 24 h by measuring IL-2 production. ND indicates “not done.” (E) Day 2 DC cultures were infected using a recombinant retrovirus encoding a cytoplasmic OVA construct. Cell surface expression of OVA/H2-Kb complexes (x-axis) and CD86 (y-axis) was monitored by flow cytometry on CD11c-negative and CD11c-positive cells at day 4. As a control noninfected cells were used. One representative experiment out of three is shown.
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fig3: CD11c-negative cells internalize and transport soluble OVA into the cytosol but do not exhibit cross presentation. (A) Immature B6D2F1 DC cultures were pulsed with FITC-OVA (5 mg/ml) for 30 min, then washed. FITC-OVA uptake and cell surface expression of CD11c were monitored by flow cytometry. (B) DC cultures were pulsed with OVA (5 mg/ml) for 30 min, washed, and chased for 30 min. CD11c-negative cells were separated from DCs using magnetic beads conjugated to anti-CD11c mAb. After homogenization of CD11c-negative cells, cytosolic and membrane/vesicle fractions were separated by ultracentrifugation, and probed for OVA and Cat L by Western blot. (C) DC cultures were pulsed with FITC-OVA (5 mg/ml) for 30 min, then washed. Cells were transferred on coverslips and incubated at 37°C for 30 min to allow their attachment. Cells were then fixed, permeabilized, and stained using a rabbit anti-OVA Ab, and TIB 120 (anti-MHC II), and analyzed by confocal microscopy. (D) DC cultures were pulsed with 1 mg/ml of OVA for 2 h, washed, activated with LPS and cluster disruption, and chased for 7 h. After fixation, CD11c-positive DCs were separated from CD11c-negative cells as in B. Mixed cultures before separation of OVA-pulsed cells (or BSA-pulsed cells as a control), CD11c-positive and CD11c-negative fractions (>95%) were then cultured with OT.1 T cells in the presence or absence of anti-CD28 mAb. T cell activation was monitored at 24 h by measuring IL-2 production. ND indicates “not done.” (E) Day 2 DC cultures were infected using a recombinant retrovirus encoding a cytoplasmic OVA construct. Cell surface expression of OVA/H2-Kb complexes (x-axis) and CD86 (y-axis) was monitored by flow cytometry on CD11c-negative and CD11c-positive cells at day 4. As a control noninfected cells were used. One representative experiment out of three is shown.

Mentions: FITC-OVA was also internalized by ∼60% of the CD11c-negative, MHC II-negative cells (e.g., progenitors or macrophages) that contaminate all DC cultures (Fig. 3 A). Surprisingly, in up to 75% of these cells, OVA was detected in the cytosol (Fig. 3, B and C), although the pattern of OVA fragments detected differed from those seen in DCs (Fig. 2 D). Thus, egress of OVA or OVA fragments to the cytosol did not appear to be a unique property of CD11c-positive DCs.


Presentation of exogenous antigens on major histocompatibility complex (MHC) class I and MHC class II molecules is differentially regulated during dendritic cell maturation.

Delamarre L, Holcombe H, Mellman I - J. Exp. Med. (2003)

CD11c-negative cells internalize and transport soluble OVA into the cytosol but do not exhibit cross presentation. (A) Immature B6D2F1 DC cultures were pulsed with FITC-OVA (5 mg/ml) for 30 min, then washed. FITC-OVA uptake and cell surface expression of CD11c were monitored by flow cytometry. (B) DC cultures were pulsed with OVA (5 mg/ml) for 30 min, washed, and chased for 30 min. CD11c-negative cells were separated from DCs using magnetic beads conjugated to anti-CD11c mAb. After homogenization of CD11c-negative cells, cytosolic and membrane/vesicle fractions were separated by ultracentrifugation, and probed for OVA and Cat L by Western blot. (C) DC cultures were pulsed with FITC-OVA (5 mg/ml) for 30 min, then washed. Cells were transferred on coverslips and incubated at 37°C for 30 min to allow their attachment. Cells were then fixed, permeabilized, and stained using a rabbit anti-OVA Ab, and TIB 120 (anti-MHC II), and analyzed by confocal microscopy. (D) DC cultures were pulsed with 1 mg/ml of OVA for 2 h, washed, activated with LPS and cluster disruption, and chased for 7 h. After fixation, CD11c-positive DCs were separated from CD11c-negative cells as in B. Mixed cultures before separation of OVA-pulsed cells (or BSA-pulsed cells as a control), CD11c-positive and CD11c-negative fractions (>95%) were then cultured with OT.1 T cells in the presence or absence of anti-CD28 mAb. T cell activation was monitored at 24 h by measuring IL-2 production. ND indicates “not done.” (E) Day 2 DC cultures were infected using a recombinant retrovirus encoding a cytoplasmic OVA construct. Cell surface expression of OVA/H2-Kb complexes (x-axis) and CD86 (y-axis) was monitored by flow cytometry on CD11c-negative and CD11c-positive cells at day 4. As a control noninfected cells were used. One representative experiment out of three is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196081&req=5

fig3: CD11c-negative cells internalize and transport soluble OVA into the cytosol but do not exhibit cross presentation. (A) Immature B6D2F1 DC cultures were pulsed with FITC-OVA (5 mg/ml) for 30 min, then washed. FITC-OVA uptake and cell surface expression of CD11c were monitored by flow cytometry. (B) DC cultures were pulsed with OVA (5 mg/ml) for 30 min, washed, and chased for 30 min. CD11c-negative cells were separated from DCs using magnetic beads conjugated to anti-CD11c mAb. After homogenization of CD11c-negative cells, cytosolic and membrane/vesicle fractions were separated by ultracentrifugation, and probed for OVA and Cat L by Western blot. (C) DC cultures were pulsed with FITC-OVA (5 mg/ml) for 30 min, then washed. Cells were transferred on coverslips and incubated at 37°C for 30 min to allow their attachment. Cells were then fixed, permeabilized, and stained using a rabbit anti-OVA Ab, and TIB 120 (anti-MHC II), and analyzed by confocal microscopy. (D) DC cultures were pulsed with 1 mg/ml of OVA for 2 h, washed, activated with LPS and cluster disruption, and chased for 7 h. After fixation, CD11c-positive DCs were separated from CD11c-negative cells as in B. Mixed cultures before separation of OVA-pulsed cells (or BSA-pulsed cells as a control), CD11c-positive and CD11c-negative fractions (>95%) were then cultured with OT.1 T cells in the presence or absence of anti-CD28 mAb. T cell activation was monitored at 24 h by measuring IL-2 production. ND indicates “not done.” (E) Day 2 DC cultures were infected using a recombinant retrovirus encoding a cytoplasmic OVA construct. Cell surface expression of OVA/H2-Kb complexes (x-axis) and CD86 (y-axis) was monitored by flow cytometry on CD11c-negative and CD11c-positive cells at day 4. As a control noninfected cells were used. One representative experiment out of three is shown.
Mentions: FITC-OVA was also internalized by ∼60% of the CD11c-negative, MHC II-negative cells (e.g., progenitors or macrophages) that contaminate all DC cultures (Fig. 3 A). Surprisingly, in up to 75% of these cells, OVA was detected in the cytosol (Fig. 3, B and C), although the pattern of OVA fragments detected differed from those seen in DCs (Fig. 2 D). Thus, egress of OVA or OVA fragments to the cytosol did not appear to be a unique property of CD11c-positive DCs.

Bottom Line: Unlike MHC II, these events do not involve a marked redistribution of preexisting MHC I molecules from intracellular compartments to the DC surface.In contrast, formation of peptide-MHC I complexes from endogenous cytosolic antigens occurs even in unstimulated, immature DCs.Thus, the MHC I and MHC II pathways of antigen presentation are differentially regulated during DC maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Section of Immunobiology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, CT 06520-8002, USA.

ABSTRACT
During maturation, dendritic cells (DCs) regulate their capacity to process and present major histocompatibility complex (MHC) II-restricted antigens. Here we show that presentation of exogenous antigens by MHC I is also subject to developmental control, but in a fashion strikingly distinct from MHC II. Immature mouse bone marrow-derived DCs internalize soluble ovalbumin and sequester the antigen intracellularly until they receive an appropriate signal that induces cross presentation. At that time, peptides are generated in a proteasome-dependent fashion and used to form peptide-MHC I complexes that appear at the plasma membrane. Unlike MHC II, these events do not involve a marked redistribution of preexisting MHC I molecules from intracellular compartments to the DC surface. Moreover, out of nine stimuli well known to induce the phenotypic maturation of DCs and to promote MHC II presentation, only two (CD40 ligation, disruption of cell-cell contacts) activated cross presentation on MHC I. In contrast, formation of peptide-MHC I complexes from endogenous cytosolic antigens occurs even in unstimulated, immature DCs. Thus, the MHC I and MHC II pathways of antigen presentation are differentially regulated during DC maturation.

Show MeSH
Related in: MedlinePlus