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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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DOαβGFP affects peptide presentation by HLA-DR3. (A) FACS® analysis of 5,000 Mel JuSo cells transfected either with DOαβ2 GFP (GFP+ population) or vector only (GFP− population) showing staining with secondary antibody only (−), or staining that is specific for class II molecules (L243), CLIP-bound HLA-DR3 (CerCLIP.1), or HLA-DR3 complexed to stably bound peptides (16.23). (B) FACS® analysis of 5,000 Mel JuSo cells expressing various levels of DOαβGFP, showing specific staining of HLA-DR3–CLIP complexes by CerCLIP.1 antibody. The vertical axis represents GFP and the horizontal axis PE-fluorescence derived from the secondary antibody, each in arbitrary units on a logarithmic scale. Values next to the right axis indicate which DOαβGFP populations were FACS® sorted for analysis of relative DO/DM expression levels.
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Figure 1: DOαβGFP affects peptide presentation by HLA-DR3. (A) FACS® analysis of 5,000 Mel JuSo cells transfected either with DOαβ2 GFP (GFP+ population) or vector only (GFP− population) showing staining with secondary antibody only (−), or staining that is specific for class II molecules (L243), CLIP-bound HLA-DR3 (CerCLIP.1), or HLA-DR3 complexed to stably bound peptides (16.23). (B) FACS® analysis of 5,000 Mel JuSo cells expressing various levels of DOαβGFP, showing specific staining of HLA-DR3–CLIP complexes by CerCLIP.1 antibody. The vertical axis represents GFP and the horizontal axis PE-fluorescence derived from the secondary antibody, each in arbitrary units on a logarithmic scale. Values next to the right axis indicate which DOαβGFP populations were FACS® sorted for analysis of relative DO/DM expression levels.

Mentions: The function of DO was studied using the human cell line Mel JuSo (DO−, DM+, and HLA–DR3+; 23) and stable transfectants expressing DOαβGFP with GFP 37 linked to the cytoplasmic tail of DOβ without affecting DO function 23. Although DO hampers the release of CLIP from MHC class II molecules, it affects presentation of some particular antigenic peptides, but not of others 22 23 24. To get insight in the effect of DO on the overall efficiency of antigenic peptide presentation by class II, FACS® analysis was performed on both DO− and DO+ cells. The amount of DOαβGFP in the DO+ cells was such that the whole DM pool was quantitatively associated to DO 23. DOαβGFP expression caused an ∼50% reduction in staining with the antibody 16.23 that recognizes HLA-DR3–antigenic peptide complexes 28 29, concomitant with an increase in the relative amount of class II/CLIP ( Fig. 1 A). The total level of cell-surface expressed class II remained unchanged ( Fig. 1 A), nor did the level of DM vary significantly 23. Thus, in cells with DM quantitatively associated to DO, the pool of class II presenting antigenic peptides is reduced, but not eliminated. Since it was recently suggested 38 that the inhibitory effect of DO on CLIP release only occurred at high DO expression levels, FACS® analysis on a population of transfectants with various DOαβGFP levels was performed. This demonstrated an almost linear correlation between DOαβGFP expression and cell surface–expressed class II/CLIP, ranging from a low, but detectable, amount of class II/CLIP in DO− cells to an almost two-log increase in cells with relatively high DOαβGFP levels ( Fig. 1 B). Additional augmentation of DOαβGFP expression did not increase the relative CLIP expression any further, suggesting that from this point on DO expression was saturating for quantitative association of DM to DO ( Fig. 1 B). Identical results were obtained when GFP and DOβ were expressed as separate proteins from one bicistronic transcript (data not shown). FACS® sorting of different DOαβGFP transfectant populations (populations 2–5, Fig. 1 B) was performed to quantify DM and DO expression (Table ). Comparison of the relative DO/DM levels with that observed in primary B cells showed that the relative DO/DM level in primary B cells falls within the range of values obtained from the transfectants studied (Table ). Thus, DO invariably and quantitatively impedes CLIP removal from newly synthesised class II molecules, also when expressed at levels that are comparable to the physiological B cell levels.


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)

DOαβGFP affects peptide presentation by HLA-DR3. (A) FACS® analysis of 5,000 Mel JuSo cells transfected either with DOαβ2 GFP (GFP+ population) or vector only (GFP− population) showing staining with secondary antibody only (−), or staining that is specific for class II molecules (L243), CLIP-bound HLA-DR3 (CerCLIP.1), or HLA-DR3 complexed to stably bound peptides (16.23). (B) FACS® analysis of 5,000 Mel JuSo cells expressing various levels of DOαβGFP, showing specific staining of HLA-DR3–CLIP complexes by CerCLIP.1 antibody. The vertical axis represents GFP and the horizontal axis PE-fluorescence derived from the secondary antibody, each in arbitrary units on a logarithmic scale. Values next to the right axis indicate which DOαβGFP populations were FACS® sorted for analysis of relative DO/DM expression levels.
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Related In: Results  -  Collection

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Figure 1: DOαβGFP affects peptide presentation by HLA-DR3. (A) FACS® analysis of 5,000 Mel JuSo cells transfected either with DOαβ2 GFP (GFP+ population) or vector only (GFP− population) showing staining with secondary antibody only (−), or staining that is specific for class II molecules (L243), CLIP-bound HLA-DR3 (CerCLIP.1), or HLA-DR3 complexed to stably bound peptides (16.23). (B) FACS® analysis of 5,000 Mel JuSo cells expressing various levels of DOαβGFP, showing specific staining of HLA-DR3–CLIP complexes by CerCLIP.1 antibody. The vertical axis represents GFP and the horizontal axis PE-fluorescence derived from the secondary antibody, each in arbitrary units on a logarithmic scale. Values next to the right axis indicate which DOαβGFP populations were FACS® sorted for analysis of relative DO/DM expression levels.
Mentions: The function of DO was studied using the human cell line Mel JuSo (DO−, DM+, and HLA–DR3+; 23) and stable transfectants expressing DOαβGFP with GFP 37 linked to the cytoplasmic tail of DOβ without affecting DO function 23. Although DO hampers the release of CLIP from MHC class II molecules, it affects presentation of some particular antigenic peptides, but not of others 22 23 24. To get insight in the effect of DO on the overall efficiency of antigenic peptide presentation by class II, FACS® analysis was performed on both DO− and DO+ cells. The amount of DOαβGFP in the DO+ cells was such that the whole DM pool was quantitatively associated to DO 23. DOαβGFP expression caused an ∼50% reduction in staining with the antibody 16.23 that recognizes HLA-DR3–antigenic peptide complexes 28 29, concomitant with an increase in the relative amount of class II/CLIP ( Fig. 1 A). The total level of cell-surface expressed class II remained unchanged ( Fig. 1 A), nor did the level of DM vary significantly 23. Thus, in cells with DM quantitatively associated to DO, the pool of class II presenting antigenic peptides is reduced, but not eliminated. Since it was recently suggested 38 that the inhibitory effect of DO on CLIP release only occurred at high DO expression levels, FACS® analysis on a population of transfectants with various DOαβGFP levels was performed. This demonstrated an almost linear correlation between DOαβGFP expression and cell surface–expressed class II/CLIP, ranging from a low, but detectable, amount of class II/CLIP in DO− cells to an almost two-log increase in cells with relatively high DOαβGFP levels ( Fig. 1 B). Additional augmentation of DOαβGFP expression did not increase the relative CLIP expression any further, suggesting that from this point on DO expression was saturating for quantitative association of DM to DO ( Fig. 1 B). Identical results were obtained when GFP and DOβ were expressed as separate proteins from one bicistronic transcript (data not shown). FACS® sorting of different DOαβGFP transfectant populations (populations 2–5, Fig. 1 B) was performed to quantify DM and DO expression (Table ). Comparison of the relative DO/DM levels with that observed in primary B cells showed that the relative DO/DM level in primary B cells falls within the range of values obtained from the transfectants studied (Table ). Thus, DO invariably and quantitatively impedes CLIP removal from newly synthesised class II molecules, also when expressed at levels that are comparable to the physiological B cell levels.

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