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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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MHC I surface expression is up-regulated during DC maturation. (A) Immature CD11c-positive DCs from day 4 C57BL/6J cultures were purified using magnetic beads conjugated to anti-CD11c mAb. DCs were then replated and activated by addition of LPS. At various times, cells were harvested and the cell surface expression of MHC I H-2Kb, MHC II I-Ab, and CD86 was monitored by flow cytometry. Y-axis values represent the fold increase in surface expression of the different markers. The values were obtained by dividing the median fluorescence index (MFI) at the indicated time points by the MFI at time = 0. For the controls (nonstained cells and isotype control-stained cells), the values were obtained by dividing the MFI at the indicated time points by the MFI measured with the specific Ab at time = 0. In parallel, part of the cell samples were used for monitoring the expression of total MHC I HC by Western blot. One representative experiment out of four is shown. (B) Immature and mature (activated for 24 h by addition of LPS and cluster disruption) DCs were fixed, permeabilized, and stained using P8 (anti-HC-β2M complex), TIB 120 (anti-MHC II) and either anti-KDEL (ER-resident KDEL proteins) or anti-GM130 (Golgi marker), and analyzed by confocal microscopy. (C) Immature and mature (activated for 20 h by addition of LPS and cluster disruption) CD11c-positive DCs were purified, radiolabeled with 35S-methionine/cysteine, and chased in unlabeled medium. At the indicated times, cells were subject to surface biotinylation at 0°C. The resulting lysates were split into three unequal aliquots, two of which were used for immunoprecipitation of total HC and the assembled HC-β2M complex. Cell surface MHC I was determined by NeutrAvidin pull-down of immunoprecipitated HC-β2M complexes. Autoradiograms of one experiment (of two) are shown. Data from this experiment were quantified by digital scanning and plotted graphically as arbitrary units.
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fig1: MHC I surface expression is up-regulated during DC maturation. (A) Immature CD11c-positive DCs from day 4 C57BL/6J cultures were purified using magnetic beads conjugated to anti-CD11c mAb. DCs were then replated and activated by addition of LPS. At various times, cells were harvested and the cell surface expression of MHC I H-2Kb, MHC II I-Ab, and CD86 was monitored by flow cytometry. Y-axis values represent the fold increase in surface expression of the different markers. The values were obtained by dividing the median fluorescence index (MFI) at the indicated time points by the MFI at time = 0. For the controls (nonstained cells and isotype control-stained cells), the values were obtained by dividing the MFI at the indicated time points by the MFI measured with the specific Ab at time = 0. In parallel, part of the cell samples were used for monitoring the expression of total MHC I HC by Western blot. One representative experiment out of four is shown. (B) Immature and mature (activated for 24 h by addition of LPS and cluster disruption) DCs were fixed, permeabilized, and stained using P8 (anti-HC-β2M complex), TIB 120 (anti-MHC II) and either anti-KDEL (ER-resident KDEL proteins) or anti-GM130 (Golgi marker), and analyzed by confocal microscopy. (C) Immature and mature (activated for 20 h by addition of LPS and cluster disruption) CD11c-positive DCs were purified, radiolabeled with 35S-methionine/cysteine, and chased in unlabeled medium. At the indicated times, cells were subject to surface biotinylation at 0°C. The resulting lysates were split into three unequal aliquots, two of which were used for immunoprecipitation of total HC and the assembled HC-β2M complex. Cell surface MHC I was determined by NeutrAvidin pull-down of immunoprecipitated HC-β2M complexes. Autoradiograms of one experiment (of two) are shown. Data from this experiment were quantified by digital scanning and plotted graphically as arbitrary units.

Mentions: We first investigated whether maturation of mouse bone marrow-derived DCs was accompanied by an increase in cell surface expression of MHC I as observed previously for human DCs (16, 17) and the D1 mouse DC-like cell line (15). CD11c+ DCs were enriched (>95%) and then activated using LPS. After a lag of ∼7 h, MHC I began to increase such that by 30 h its expression was 7-fold higher than in immature DCs. MHC II and CD86 expression began to increase shortly after LPS addition, reaching levels 12-fold and >30-fold higher (respectively) at 30 h (Fig. 1 A).


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)

MHC I surface expression is up-regulated during DC maturation. (A) Immature CD11c-positive DCs from day 4 C57BL/6J cultures were purified using magnetic beads conjugated to anti-CD11c mAb. DCs were then replated and activated by addition of LPS. At various times, cells were harvested and the cell surface expression of MHC I H-2Kb, MHC II I-Ab, and CD86 was monitored by flow cytometry. Y-axis values represent the fold increase in surface expression of the different markers. The values were obtained by dividing the median fluorescence index (MFI) at the indicated time points by the MFI at time = 0. For the controls (nonstained cells and isotype control-stained cells), the values were obtained by dividing the MFI at the indicated time points by the MFI measured with the specific Ab at time = 0. In parallel, part of the cell samples were used for monitoring the expression of total MHC I HC by Western blot. One representative experiment out of four is shown. (B) Immature and mature (activated for 24 h by addition of LPS and cluster disruption) DCs were fixed, permeabilized, and stained using P8 (anti-HC-β2M complex), TIB 120 (anti-MHC II) and either anti-KDEL (ER-resident KDEL proteins) or anti-GM130 (Golgi marker), and analyzed by confocal microscopy. (C) Immature and mature (activated for 20 h by addition of LPS and cluster disruption) CD11c-positive DCs were purified, radiolabeled with 35S-methionine/cysteine, and chased in unlabeled medium. At the indicated times, cells were subject to surface biotinylation at 0°C. The resulting lysates were split into three unequal aliquots, two of which were used for immunoprecipitation of total HC and the assembled HC-β2M complex. Cell surface MHC I was determined by NeutrAvidin pull-down of immunoprecipitated HC-β2M complexes. Autoradiograms of one experiment (of two) are shown. Data from this experiment were quantified by digital scanning and plotted graphically as arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196081&req=5

fig1: MHC I surface expression is up-regulated during DC maturation. (A) Immature CD11c-positive DCs from day 4 C57BL/6J cultures were purified using magnetic beads conjugated to anti-CD11c mAb. DCs were then replated and activated by addition of LPS. At various times, cells were harvested and the cell surface expression of MHC I H-2Kb, MHC II I-Ab, and CD86 was monitored by flow cytometry. Y-axis values represent the fold increase in surface expression of the different markers. The values were obtained by dividing the median fluorescence index (MFI) at the indicated time points by the MFI at time = 0. For the controls (nonstained cells and isotype control-stained cells), the values were obtained by dividing the MFI at the indicated time points by the MFI measured with the specific Ab at time = 0. In parallel, part of the cell samples were used for monitoring the expression of total MHC I HC by Western blot. One representative experiment out of four is shown. (B) Immature and mature (activated for 24 h by addition of LPS and cluster disruption) DCs were fixed, permeabilized, and stained using P8 (anti-HC-β2M complex), TIB 120 (anti-MHC II) and either anti-KDEL (ER-resident KDEL proteins) or anti-GM130 (Golgi marker), and analyzed by confocal microscopy. (C) Immature and mature (activated for 20 h by addition of LPS and cluster disruption) CD11c-positive DCs were purified, radiolabeled with 35S-methionine/cysteine, and chased in unlabeled medium. At the indicated times, cells were subject to surface biotinylation at 0°C. The resulting lysates were split into three unequal aliquots, two of which were used for immunoprecipitation of total HC and the assembled HC-β2M complex. Cell surface MHC I was determined by NeutrAvidin pull-down of immunoprecipitated HC-β2M complexes. Autoradiograms of one experiment (of two) are shown. Data from this experiment were quantified by digital scanning and plotted graphically as arbitrary units.
Mentions: We first investigated whether maturation of mouse bone marrow-derived DCs was accompanied by an increase in cell surface expression of MHC I as observed previously for human DCs (16, 17) and the D1 mouse DC-like cell line (15). CD11c+ DCs were enriched (>95%) and then activated using LPS. After a lag of ∼7 h, MHC I began to increase such that by 30 h its expression was 7-fold higher than in immature DCs. MHC II and CD86 expression began to increase shortly after LPS addition, reaching levels 12-fold and >30-fold higher (respectively) at 30 h (Fig. 1 A).

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