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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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Cross presentation of soluble OVA follows the classical MHC I pathway (A) Left panel: immature B6D2F1 DCs were pulsed with indicated concentrations of OVA (or BSA as a control) for 2 h, washed, activated by LPS addition and cluster disruption. After a 7 h chase, the cells were fixed and cultured with OT.1 CD8+ T cells. Right panel: after a 2 h pulse with OVA 1 mg/ml (or BSA as a control), immature DCs were washed, stimulated by LPS addition and cluster disruption, chased for the indicated times, then fixed and added to OT.1 T cells. T cell responses were monitored by measuring IL-2 secretion. (B and C) Immature DCs were pulsed with FITC-OVA (5 mg/ml) for 30 min, washed, transferred to coverslips after disrupting DC clusters and incubated at 37°C for 30 min to allow cell attachment. DCs were then fixed, permeabilized, and stained using a rabbit anti-OVA Ab, and TIB 120 (anti-MHC II), and analyzed by confocal microscopy. (D) DCs were pulsed with OVA (5mg/ml) for 30 min, washed, and chased for 30 min after stimulation by LPS addition and cluster disruption. CD11c-positive DCs were purified using magnetic beads conjugated to anti-CD11c mAb. After homogenization, cytosolic and membrane/vesicle fractions were separated by ultracentrifugation, and probed for OVA and Cat L by Western blot. One representative experiment out of three is shown.
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fig2: Cross presentation of soluble OVA follows the classical MHC I pathway (A) Left panel: immature B6D2F1 DCs were pulsed with indicated concentrations of OVA (or BSA as a control) for 2 h, washed, activated by LPS addition and cluster disruption. After a 7 h chase, the cells were fixed and cultured with OT.1 CD8+ T cells. Right panel: after a 2 h pulse with OVA 1 mg/ml (or BSA as a control), immature DCs were washed, stimulated by LPS addition and cluster disruption, chased for the indicated times, then fixed and added to OT.1 T cells. T cell responses were monitored by measuring IL-2 secretion. (B and C) Immature DCs were pulsed with FITC-OVA (5 mg/ml) for 30 min, washed, transferred to coverslips after disrupting DC clusters and incubated at 37°C for 30 min to allow cell attachment. DCs were then fixed, permeabilized, and stained using a rabbit anti-OVA Ab, and TIB 120 (anti-MHC II), and analyzed by confocal microscopy. (D) DCs were pulsed with OVA (5mg/ml) for 30 min, washed, and chased for 30 min after stimulation by LPS addition and cluster disruption. CD11c-positive DCs were purified using magnetic beads conjugated to anti-CD11c mAb. After homogenization, cytosolic and membrane/vesicle fractions were separated by ultracentrifugation, and probed for OVA and Cat L by Western blot. One representative experiment out of three is shown.

Mentions: Although mature DCs are known to stimulate CD8+ T cells better than immature DCs (16, 18, 19, 26), it is unknown if the formation of peptide–MHC I complexes from exogenous antigen is subject to regulation. To address this question, immature DCs were pulsed with soluble OVA for 2 h, washed, and stimulated by adding LPS. After dispersal of cell clusters by pipetting, the DCs were chased for up to 24 h. The DCs were then fixed and cultured with OT.1 CD8+ T cells specific for the H-2Kb/OVA complex. After being pulsed with >0.5/ml of OVA, DCs efficiently activated OT.1 T cells, with an optimal response at 7 h after inducing DC maturation (Fig. 2 A), as previously observed in D1 cells (6). Presentation was also >95% inhibited by the proteasome inhibitors lactacystin and epoxomicin, and in DCs isolated from TAP1−/− mice (unpublished data). Thus, presentation of exogenous soluble OVA by MHC I follows the classical MHC I pathway (5, 27, 28).


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)

Cross presentation of soluble OVA follows the classical MHC I pathway (A) Left panel: immature B6D2F1 DCs were pulsed with indicated concentrations of OVA (or BSA as a control) for 2 h, washed, activated by LPS addition and cluster disruption. After a 7 h chase, the cells were fixed and cultured with OT.1 CD8+ T cells. Right panel: after a 2 h pulse with OVA 1 mg/ml (or BSA as a control), immature DCs were washed, stimulated by LPS addition and cluster disruption, chased for the indicated times, then fixed and added to OT.1 T cells. T cell responses were monitored by measuring IL-2 secretion. (B and C) Immature DCs were pulsed with FITC-OVA (5 mg/ml) for 30 min, washed, transferred to coverslips after disrupting DC clusters and incubated at 37°C for 30 min to allow cell attachment. DCs were then fixed, permeabilized, and stained using a rabbit anti-OVA Ab, and TIB 120 (anti-MHC II), and analyzed by confocal microscopy. (D) DCs were pulsed with OVA (5mg/ml) for 30 min, washed, and chased for 30 min after stimulation by LPS addition and cluster disruption. CD11c-positive DCs were purified using magnetic beads conjugated to anti-CD11c mAb. After homogenization, cytosolic and membrane/vesicle fractions were separated by ultracentrifugation, and probed for OVA and Cat L by Western blot. One representative experiment out of three is shown.
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Related In: Results  -  Collection

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fig2: Cross presentation of soluble OVA follows the classical MHC I pathway (A) Left panel: immature B6D2F1 DCs were pulsed with indicated concentrations of OVA (or BSA as a control) for 2 h, washed, activated by LPS addition and cluster disruption. After a 7 h chase, the cells were fixed and cultured with OT.1 CD8+ T cells. Right panel: after a 2 h pulse with OVA 1 mg/ml (or BSA as a control), immature DCs were washed, stimulated by LPS addition and cluster disruption, chased for the indicated times, then fixed and added to OT.1 T cells. T cell responses were monitored by measuring IL-2 secretion. (B and C) Immature DCs were pulsed with FITC-OVA (5 mg/ml) for 30 min, washed, transferred to coverslips after disrupting DC clusters and incubated at 37°C for 30 min to allow cell attachment. DCs were then fixed, permeabilized, and stained using a rabbit anti-OVA Ab, and TIB 120 (anti-MHC II), and analyzed by confocal microscopy. (D) DCs were pulsed with OVA (5mg/ml) for 30 min, washed, and chased for 30 min after stimulation by LPS addition and cluster disruption. CD11c-positive DCs were purified using magnetic beads conjugated to anti-CD11c mAb. After homogenization, cytosolic and membrane/vesicle fractions were separated by ultracentrifugation, and probed for OVA and Cat L by Western blot. One representative experiment out of three is shown.
Mentions: Although mature DCs are known to stimulate CD8+ T cells better than immature DCs (16, 18, 19, 26), it is unknown if the formation of peptide–MHC I complexes from exogenous antigen is subject to regulation. To address this question, immature DCs were pulsed with soluble OVA for 2 h, washed, and stimulated by adding LPS. After dispersal of cell clusters by pipetting, the DCs were chased for up to 24 h. The DCs were then fixed and cultured with OT.1 CD8+ T cells specific for the H-2Kb/OVA complex. After being pulsed with >0.5/ml of OVA, DCs efficiently activated OT.1 T cells, with an optimal response at 7 h after inducing DC maturation (Fig. 2 A), as previously observed in D1 cells (6). Presentation was also >95% inhibited by the proteasome inhibitors lactacystin and epoxomicin, and in DCs isolated from TAP1−/− mice (unpublished data). Thus, presentation of exogenous soluble OVA by MHC I follows the classical MHC I pathway (5, 27, 28).

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