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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 is differentially regulated by DC maturation. (A) Left panel: immature B6D2F1 DCs were pulsed with OVA or BSA (1 mg/ml) for 2h in 24 well-plates where the cells were originally plated, washed carefully to avoid disruption of cell clusters (by adding media slowly along the wall of each well and then aspirating gently media at the same spot, repeatedly), and chased for 7 h with or without stimulation before fixation and culture with OT.1 T cells. Right panel: OVA-pulsed cells and T cells were cultured in the presence of OVA peptide. T cell responses were monitored at 24 h by measuring IL-2 release. (B) After the pulse-chase, MHC I H-2Kb surface expression was evaluated by flow cytometry on the CD11c-positive population of “LPS-treated and cluster disrupted” (solid black line) or unstimulated OVA-pulsed cells (dashed black line). The solid and dashed gray lines depict staining with an isotype control on stimulated and unstimulated cells, respectively. On the x-axis, the fluorescence intensity is given, whereas the y-axis depicts the relative cell number. (C) DCs were pulsed with OVA (5 mg/ml) for 30 min, washed, and chased for 30 min. CD11c-positive cells purified using magnetic beads conjugated to anti-CD11c mAb. After homogenization, cytosolic and membrane/vesicle fractions were separated, and probed for OVA and Cat L by Western blot. (D) After 7 h in culture, maturation state of “unstimulated” 2 h OVA-pulsed DCs (i.e., not treated with added LPS and not subjected to cluster disruption) and “mock-treated” DCs was examined by measuring surface expression of MHC II (x-axis) and CD86 (y-axis) by flow cytometry on the CD11c-positive population. (E) The capacity of the stimuli LPS and “cluster disruption” to trigger DC maturation was monitored on the CD11c-positive population of unpulsed cultures activated for 7 h as described above. One representative experiment out of three is shown.
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fig4: Cross presentation of soluble OVA is differentially regulated by DC maturation. (A) Left panel: immature B6D2F1 DCs were pulsed with OVA or BSA (1 mg/ml) for 2h in 24 well-plates where the cells were originally plated, washed carefully to avoid disruption of cell clusters (by adding media slowly along the wall of each well and then aspirating gently media at the same spot, repeatedly), and chased for 7 h with or without stimulation before fixation and culture with OT.1 T cells. Right panel: OVA-pulsed cells and T cells were cultured in the presence of OVA peptide. T cell responses were monitored at 24 h by measuring IL-2 release. (B) After the pulse-chase, MHC I H-2Kb surface expression was evaluated by flow cytometry on the CD11c-positive population of “LPS-treated and cluster disrupted” (solid black line) or unstimulated OVA-pulsed cells (dashed black line). The solid and dashed gray lines depict staining with an isotype control on stimulated and unstimulated cells, respectively. On the x-axis, the fluorescence intensity is given, whereas the y-axis depicts the relative cell number. (C) DCs were pulsed with OVA (5 mg/ml) for 30 min, washed, and chased for 30 min. CD11c-positive cells purified using magnetic beads conjugated to anti-CD11c mAb. After homogenization, cytosolic and membrane/vesicle fractions were separated, and probed for OVA and Cat L by Western blot. (D) After 7 h in culture, maturation state of “unstimulated” 2 h OVA-pulsed DCs (i.e., not treated with added LPS and not subjected to cluster disruption) and “mock-treated” DCs was examined by measuring surface expression of MHC II (x-axis) and CD86 (y-axis) by flow cytometry on the CD11c-positive population. (E) The capacity of the stimuli LPS and “cluster disruption” to trigger DC maturation was monitored on the CD11c-positive population of unpulsed cultures activated for 7 h as described above. One representative experiment out of three is shown.

Mentions: Because peptide–MHC I complexes could form from endogenous OVA in immature DCs, we next asked if cross presentation by DCs is similarly independent of DC maturation. Cultures were pulsed with OVA for 2 h and washed carefully to avoid disruption of cell clusters, itself a potential maturation stimulus (14). After a 7 h chase, the cells were then fixed to prevent maturation and cultured with OT.1 CD8+ T cells. As shown in Fig. 4 A (left panel), the unstimulated DCs were unable to cross present OVA, suggesting that DC maturation was in fact required to activate cross presentation. This was in marked contrast to DCs subjected to dispersal of the cell clusters by repeated pipetting and addition of LPS before the 7 h culture period. These “LPS-treated and cluster disrupted” DCs were efficient at cross presentation despite expressing no more MHC I than unstimulated cells (Fig. 4 B); presentation of preprocessed OVA peptide was similar in all cases (Fig. 4 A, right panel). In fact, cluster disruption alone was found to be a potent stimulus of cross presentation while LPS alone (in the absence of cluster disruption) was almost totally ineffective (Fig. 4 A, left panel).


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 is differentially regulated by DC maturation. (A) Left panel: immature B6D2F1 DCs were pulsed with OVA or BSA (1 mg/ml) for 2h in 24 well-plates where the cells were originally plated, washed carefully to avoid disruption of cell clusters (by adding media slowly along the wall of each well and then aspirating gently media at the same spot, repeatedly), and chased for 7 h with or without stimulation before fixation and culture with OT.1 T cells. Right panel: OVA-pulsed cells and T cells were cultured in the presence of OVA peptide. T cell responses were monitored at 24 h by measuring IL-2 release. (B) After the pulse-chase, MHC I H-2Kb surface expression was evaluated by flow cytometry on the CD11c-positive population of “LPS-treated and cluster disrupted” (solid black line) or unstimulated OVA-pulsed cells (dashed black line). The solid and dashed gray lines depict staining with an isotype control on stimulated and unstimulated cells, respectively. On the x-axis, the fluorescence intensity is given, whereas the y-axis depicts the relative cell number. (C) DCs were pulsed with OVA (5 mg/ml) for 30 min, washed, and chased for 30 min. CD11c-positive cells purified using magnetic beads conjugated to anti-CD11c mAb. After homogenization, cytosolic and membrane/vesicle fractions were separated, and probed for OVA and Cat L by Western blot. (D) After 7 h in culture, maturation state of “unstimulated” 2 h OVA-pulsed DCs (i.e., not treated with added LPS and not subjected to cluster disruption) and “mock-treated” DCs was examined by measuring surface expression of MHC II (x-axis) and CD86 (y-axis) by flow cytometry on the CD11c-positive population. (E) The capacity of the stimuli LPS and “cluster disruption” to trigger DC maturation was monitored on the CD11c-positive population of unpulsed cultures activated for 7 h as described above. One representative experiment out of three is shown.
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fig4: Cross presentation of soluble OVA is differentially regulated by DC maturation. (A) Left panel: immature B6D2F1 DCs were pulsed with OVA or BSA (1 mg/ml) for 2h in 24 well-plates where the cells were originally plated, washed carefully to avoid disruption of cell clusters (by adding media slowly along the wall of each well and then aspirating gently media at the same spot, repeatedly), and chased for 7 h with or without stimulation before fixation and culture with OT.1 T cells. Right panel: OVA-pulsed cells and T cells were cultured in the presence of OVA peptide. T cell responses were monitored at 24 h by measuring IL-2 release. (B) After the pulse-chase, MHC I H-2Kb surface expression was evaluated by flow cytometry on the CD11c-positive population of “LPS-treated and cluster disrupted” (solid black line) or unstimulated OVA-pulsed cells (dashed black line). The solid and dashed gray lines depict staining with an isotype control on stimulated and unstimulated cells, respectively. On the x-axis, the fluorescence intensity is given, whereas the y-axis depicts the relative cell number. (C) DCs were pulsed with OVA (5 mg/ml) for 30 min, washed, and chased for 30 min. CD11c-positive cells purified using magnetic beads conjugated to anti-CD11c mAb. After homogenization, cytosolic and membrane/vesicle fractions were separated, and probed for OVA and Cat L by Western blot. (D) After 7 h in culture, maturation state of “unstimulated” 2 h OVA-pulsed DCs (i.e., not treated with added LPS and not subjected to cluster disruption) and “mock-treated” DCs was examined by measuring surface expression of MHC II (x-axis) and CD86 (y-axis) by flow cytometry on the CD11c-positive population. (E) The capacity of the stimuli LPS and “cluster disruption” to trigger DC maturation was monitored on the CD11c-positive population of unpulsed cultures activated for 7 h as described above. One representative experiment out of three is shown.
Mentions: Because peptide–MHC I complexes could form from endogenous OVA in immature DCs, we next asked if cross presentation by DCs is similarly independent of DC maturation. Cultures were pulsed with OVA for 2 h and washed carefully to avoid disruption of cell clusters, itself a potential maturation stimulus (14). After a 7 h chase, the cells were then fixed to prevent maturation and cultured with OT.1 CD8+ T cells. As shown in Fig. 4 A (left panel), the unstimulated DCs were unable to cross present OVA, suggesting that DC maturation was in fact required to activate cross presentation. This was in marked contrast to DCs subjected to dispersal of the cell clusters by repeated pipetting and addition of LPS before the 7 h culture period. These “LPS-treated and cluster disrupted” DCs were efficient at cross presentation despite expressing no more MHC I than unstimulated cells (Fig. 4 B); presentation of preprocessed OVA peptide was similar in all cases (Fig. 4 A, right panel). In fact, cluster disruption alone was found to be a potent stimulus of cross presentation while LPS alone (in the absence of cluster disruption) was almost totally ineffective (Fig. 4 A, left panel).

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