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Modulation of the major histocompatibility complex class II-associated peptide repertoire by human histocompatibility leukocyte antigen (HLA)-DO.

van Ham M, van Lith M, Lillemeier B, Tjin E, Grüneberg U, Rahman D, Pastoors L, van Meijgaarden K, Roucard C, Trowsdale J, Ottenhoff T, Pappin D, Neefjes J - J. Exp. Med. (2000)

Bottom Line: DO effectively inhibits antigen presentation of peptides that are loaded onto class II in endosomal compartments that are not very acidic.Thus, DO appears to be a unique, cell type-specific modulator mastering the class II-mediated immune response induced by B cells.DO may serve to increase the threshold for nonspecific B cell activation, restricting class II-peptide binding to late endosomal compartments, thereby affecting the peptide repertoire.

View Article: PubMed Central - PubMed

Affiliation: Division of Tumor Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. vanham@nki.nl

ABSTRACT
Antigen presentation by major histocompatibility complex class II molecules is essential for antibody production and T cell activation. For most class II alleles, peptide binding depends on the catalytic action of human histocompatibility leukocyte antigens (HLA)-DM. HLA-DO is selectively expressed in B cells and impedes the activity of DM, yet its physiological role remains unclear. Cell surface iodination assays and mass spectrometry of major histocompatibility complex class II-eluted peptides show that DO affects the antigenic peptide repertoire of class II. DO generates both quantitative and qualitative differences, and inhibits presentation of large-sized peptides. DO function was investigated under various pH conditions in in vitro peptide exchange assays and in antigen presentation assays using DO(-) and DO(+) transfectant cell lines as antigen-presenting cells, in which effective acidification of the endocytic pathway was prevented with bafilomycin A(1), an inhibitor of vacuolar ATPases. DO effectively inhibits antigen presentation of peptides that are loaded onto class II in endosomal compartments that are not very acidic. Thus, DO appears to be a unique, cell type-specific modulator mastering the class II-mediated immune response induced by B cells. DO may serve to increase the threshold for nonspecific B cell activation, restricting class II-peptide binding to late endosomal compartments, thereby affecting the peptide repertoire.

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DO modulates DM function in a pH-dependent fashion. (A) In vitro association of biotinylated ApoB(2877–2894) to purified DR3–CLIP complexes upon CLIP dissociation was measured at different pH values in the presence of cell lysates containing equal amounts of DM or DM–DOαβ2 GFP complexes (DM/DO) or lysis buffer only (−). Association is depicted as absorbance at 405 nm in arbitrary units (a.U.) upon saturation of the peptide exchange reaction. The data shown were derived from a representative set of experiments (n = 4). (B) Antigen-presenting capacity of p3-13–reactive T cell clone Rp15 1-1 using control (DM) and DOαβGFP-expressing cells (DM/DO) at prevention of acidification of the endosomal/lysosomal pathway. T cell proliferation is expressed as the amount of [3H]thymidine incorporation in cpm and was determined for varying amounts of M. tuberculosis sonicate with 1,500 APCs/assay well. Data shown were derived from a representative experiment performed three times; SEM <10%. T cell proliferation in response to Mel JuSo cells pulsed with HLA-DR3–restricted peptide p56-65 was not significant, and application of Mel JuSo transfected with DO without GFP tag resulted in the same effects as the DOαβGFP transfectants (data not shown). (C) Antigen-presenting capacity of p3-13–reactive T cell clone Rp15 1-1 using varying amounts of control (DM) and DOαβGFP-expressing cells (DM/DO) at prevention of acidification of the endosomal/lysosomal pathway. T cell proliferation (measured by [3H]thymidine incorporation) was determined using 100 μg/ml M. tuberculosis sonicate. Data shown were derived from a representative experiment performed three times; SEM <10%. Application of Mel JuSo transfected with DO without GFP tag yielded the same results as the DOαβGFP transfectants (data not shown). Black bars, DM + M. tuberculosis; gray bars, DM + medium; lined bars, DM-DO + M. tuberculosis; empty bars, DM-DO + medium.
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Figure 4: DO modulates DM function in a pH-dependent fashion. (A) In vitro association of biotinylated ApoB(2877–2894) to purified DR3–CLIP complexes upon CLIP dissociation was measured at different pH values in the presence of cell lysates containing equal amounts of DM or DM–DOαβ2 GFP complexes (DM/DO) or lysis buffer only (−). Association is depicted as absorbance at 405 nm in arbitrary units (a.U.) upon saturation of the peptide exchange reaction. The data shown were derived from a representative set of experiments (n = 4). (B) Antigen-presenting capacity of p3-13–reactive T cell clone Rp15 1-1 using control (DM) and DOαβGFP-expressing cells (DM/DO) at prevention of acidification of the endosomal/lysosomal pathway. T cell proliferation is expressed as the amount of [3H]thymidine incorporation in cpm and was determined for varying amounts of M. tuberculosis sonicate with 1,500 APCs/assay well. Data shown were derived from a representative experiment performed three times; SEM <10%. T cell proliferation in response to Mel JuSo cells pulsed with HLA-DR3–restricted peptide p56-65 was not significant, and application of Mel JuSo transfected with DO without GFP tag resulted in the same effects as the DOαβGFP transfectants (data not shown). (C) Antigen-presenting capacity of p3-13–reactive T cell clone Rp15 1-1 using varying amounts of control (DM) and DOαβGFP-expressing cells (DM/DO) at prevention of acidification of the endosomal/lysosomal pathway. T cell proliferation (measured by [3H]thymidine incorporation) was determined using 100 μg/ml M. tuberculosis sonicate. Data shown were derived from a representative experiment performed three times; SEM <10%. Application of Mel JuSo transfected with DO without GFP tag yielded the same results as the DOαβGFP transfectants (data not shown). Black bars, DM + M. tuberculosis; gray bars, DM + medium; lined bars, DM-DO + M. tuberculosis; empty bars, DM-DO + medium.

Mentions: By which mechanism does DO generate qualitative differences in the class II–associated peptide repertoire? Different class II–peptide complexes can be generated in distinct compartments of the endosomal/lysosomal pathway in different pH environments 40 41 42 43 44 45. The DM–DO complex constitutively recycles between the MIICs and the plasma membrane (data not shown), implying that en route to the MIIC, both DM and DO traverse all endocytic compartments where they may support and modulate peptide loading. Recently, recombinant DO was reported to affect DM function in a pH-sensitive manner in vitro 24. If true, this may illustrate how DO modulates the peptide repertoire. To determine if DO modulates DM activity in a pH-dependent fashion in our transfectants, in vitro peptide loading experiments were performed using DM and DM–DO complexes obtained from either DO− or DO+ cells in which the whole DM pool was quantitatively associated to DO 23 30 35. Equal amounts of DM and DM–DO were applied in the experiments as quantified using the FluorChem™ imaging system and AlphaEase™ FC software (data not shown). Exchange of CLIP from purified HLA-DR3–CLIP for the antigenic peptide ApoB(2877–2894) was indeed increasingly catalyzed by DM on acidification with an optimum pH ∼5 ( Fig. 4 A). DO-mediated inhibition of DM was strongest at the pH normally observed in early lysosomes (pH ∼6), conditions in which the catalytic capacity of DM is almost abrogated. DO still inhibited DM at the pH described for the MIICs (pH 4.5–5), but only by half ( Fig. 4 A). Thus, these in vitro data suggests that DO inhibits DM preferentially at the pH of earlier endosomal compartments, while allowing substantial DM-mediated peptide loading at the pH present in MIICs.


Modulation of the major histocompatibility complex class II-associated peptide repertoire by human histocompatibility leukocyte antigen (HLA)-DO.

van Ham M, van Lith M, Lillemeier B, Tjin E, Grüneberg U, Rahman D, Pastoors L, van Meijgaarden K, Roucard C, Trowsdale J, Ottenhoff T, Pappin D, Neefjes J - J. Exp. Med. (2000)

DO modulates DM function in a pH-dependent fashion. (A) In vitro association of biotinylated ApoB(2877–2894) to purified DR3–CLIP complexes upon CLIP dissociation was measured at different pH values in the presence of cell lysates containing equal amounts of DM or DM–DOαβ2 GFP complexes (DM/DO) or lysis buffer only (−). Association is depicted as absorbance at 405 nm in arbitrary units (a.U.) upon saturation of the peptide exchange reaction. The data shown were derived from a representative set of experiments (n = 4). (B) Antigen-presenting capacity of p3-13–reactive T cell clone Rp15 1-1 using control (DM) and DOαβGFP-expressing cells (DM/DO) at prevention of acidification of the endosomal/lysosomal pathway. T cell proliferation is expressed as the amount of [3H]thymidine incorporation in cpm and was determined for varying amounts of M. tuberculosis sonicate with 1,500 APCs/assay well. Data shown were derived from a representative experiment performed three times; SEM <10%. T cell proliferation in response to Mel JuSo cells pulsed with HLA-DR3–restricted peptide p56-65 was not significant, and application of Mel JuSo transfected with DO without GFP tag resulted in the same effects as the DOαβGFP transfectants (data not shown). (C) Antigen-presenting capacity of p3-13–reactive T cell clone Rp15 1-1 using varying amounts of control (DM) and DOαβGFP-expressing cells (DM/DO) at prevention of acidification of the endosomal/lysosomal pathway. T cell proliferation (measured by [3H]thymidine incorporation) was determined using 100 μg/ml M. tuberculosis sonicate. Data shown were derived from a representative experiment performed three times; SEM <10%. Application of Mel JuSo transfected with DO without GFP tag yielded the same results as the DOαβGFP transfectants (data not shown). Black bars, DM + M. tuberculosis; gray bars, DM + medium; lined bars, DM-DO + M. tuberculosis; empty bars, DM-DO + medium.
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Figure 4: DO modulates DM function in a pH-dependent fashion. (A) In vitro association of biotinylated ApoB(2877–2894) to purified DR3–CLIP complexes upon CLIP dissociation was measured at different pH values in the presence of cell lysates containing equal amounts of DM or DM–DOαβ2 GFP complexes (DM/DO) or lysis buffer only (−). Association is depicted as absorbance at 405 nm in arbitrary units (a.U.) upon saturation of the peptide exchange reaction. The data shown were derived from a representative set of experiments (n = 4). (B) Antigen-presenting capacity of p3-13–reactive T cell clone Rp15 1-1 using control (DM) and DOαβGFP-expressing cells (DM/DO) at prevention of acidification of the endosomal/lysosomal pathway. T cell proliferation is expressed as the amount of [3H]thymidine incorporation in cpm and was determined for varying amounts of M. tuberculosis sonicate with 1,500 APCs/assay well. Data shown were derived from a representative experiment performed three times; SEM <10%. T cell proliferation in response to Mel JuSo cells pulsed with HLA-DR3–restricted peptide p56-65 was not significant, and application of Mel JuSo transfected with DO without GFP tag resulted in the same effects as the DOαβGFP transfectants (data not shown). (C) Antigen-presenting capacity of p3-13–reactive T cell clone Rp15 1-1 using varying amounts of control (DM) and DOαβGFP-expressing cells (DM/DO) at prevention of acidification of the endosomal/lysosomal pathway. T cell proliferation (measured by [3H]thymidine incorporation) was determined using 100 μg/ml M. tuberculosis sonicate. Data shown were derived from a representative experiment performed three times; SEM <10%. Application of Mel JuSo transfected with DO without GFP tag yielded the same results as the DOαβGFP transfectants (data not shown). Black bars, DM + M. tuberculosis; gray bars, DM + medium; lined bars, DM-DO + M. tuberculosis; empty bars, DM-DO + medium.
Mentions: By which mechanism does DO generate qualitative differences in the class II–associated peptide repertoire? Different class II–peptide complexes can be generated in distinct compartments of the endosomal/lysosomal pathway in different pH environments 40 41 42 43 44 45. The DM–DO complex constitutively recycles between the MIICs and the plasma membrane (data not shown), implying that en route to the MIIC, both DM and DO traverse all endocytic compartments where they may support and modulate peptide loading. Recently, recombinant DO was reported to affect DM function in a pH-sensitive manner in vitro 24. If true, this may illustrate how DO modulates the peptide repertoire. To determine if DO modulates DM activity in a pH-dependent fashion in our transfectants, in vitro peptide loading experiments were performed using DM and DM–DO complexes obtained from either DO− or DO+ cells in which the whole DM pool was quantitatively associated to DO 23 30 35. Equal amounts of DM and DM–DO were applied in the experiments as quantified using the FluorChem™ imaging system and AlphaEase™ FC software (data not shown). Exchange of CLIP from purified HLA-DR3–CLIP for the antigenic peptide ApoB(2877–2894) was indeed increasingly catalyzed by DM on acidification with an optimum pH ∼5 ( Fig. 4 A). DO-mediated inhibition of DM was strongest at the pH normally observed in early lysosomes (pH ∼6), conditions in which the catalytic capacity of DM is almost abrogated. DO still inhibited DM at the pH described for the MIICs (pH 4.5–5), but only by half ( Fig. 4 A). Thus, these in vitro data suggests that DO inhibits DM preferentially at the pH of earlier endosomal compartments, while allowing substantial DM-mediated peptide loading at the pH present in MIICs.

Bottom Line: DO effectively inhibits antigen presentation of peptides that are loaded onto class II in endosomal compartments that are not very acidic.Thus, DO appears to be a unique, cell type-specific modulator mastering the class II-mediated immune response induced by B cells.DO may serve to increase the threshold for nonspecific B cell activation, restricting class II-peptide binding to late endosomal compartments, thereby affecting the peptide repertoire.

View Article: PubMed Central - PubMed

Affiliation: Division of Tumor Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. vanham@nki.nl

ABSTRACT
Antigen presentation by major histocompatibility complex class II molecules is essential for antibody production and T cell activation. For most class II alleles, peptide binding depends on the catalytic action of human histocompatibility leukocyte antigens (HLA)-DM. HLA-DO is selectively expressed in B cells and impedes the activity of DM, yet its physiological role remains unclear. Cell surface iodination assays and mass spectrometry of major histocompatibility complex class II-eluted peptides show that DO affects the antigenic peptide repertoire of class II. DO generates both quantitative and qualitative differences, and inhibits presentation of large-sized peptides. DO function was investigated under various pH conditions in in vitro peptide exchange assays and in antigen presentation assays using DO(-) and DO(+) transfectant cell lines as antigen-presenting cells, in which effective acidification of the endocytic pathway was prevented with bafilomycin A(1), an inhibitor of vacuolar ATPases. DO effectively inhibits antigen presentation of peptides that are loaded onto class II in endosomal compartments that are not very acidic. Thus, DO appears to be a unique, cell type-specific modulator mastering the class II-mediated immune response induced by B cells. DO may serve to increase the threshold for nonspecific B cell activation, restricting class II-peptide binding to late endosomal compartments, thereby affecting the peptide repertoire.

Show MeSH
Related in: MedlinePlus