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Novel mutants define genes required for the expression of human histocompatibility leukocyte antigen DM: evidence for loci on human chromosome 6p.

Fling SP, Rak J, Muczynski KA, Arp B, Pious D - J. Exp. Med. (1997)

Bottom Line: However, we show that the defects in two of these new mutants do not map to the DM locus.These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules.The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

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mAb 16.23 and  mAb 16.23low sorted subpopulations of 2.7.93-type mutants express differential levels of stable  HLA-DR dimers. (a) SDS stability of HLA-DR molecules from  cell-sorted mAb16.23 and  mAb 16.23low subpopulations of  mutant 2.7.93. Western blots  were stained with a combination of mAbs DA6.147 and  HB10.A. (b) Bimodal reexpression of the mAb 16.23 epitope in  subclones of the 2.7.93 mAb  16.23 sorted subpopulations.  2.7.93 cells were stained in indirect immunofluorescence with  mAb 16.23. Each panel represents mAb 16.23 staining of an individual subclone of 2.7.93 (mAb 16.23); subclones of the mAb 16.23low subpopulation show similar bimodal reexpression of the mAb 16.23 epitope (data not shown).
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Figure 5: mAb 16.23 and mAb 16.23low sorted subpopulations of 2.7.93-type mutants express differential levels of stable HLA-DR dimers. (a) SDS stability of HLA-DR molecules from cell-sorted mAb16.23 and mAb 16.23low subpopulations of mutant 2.7.93. Western blots were stained with a combination of mAbs DA6.147 and HB10.A. (b) Bimodal reexpression of the mAb 16.23 epitope in subclones of the 2.7.93 mAb 16.23 sorted subpopulations. 2.7.93 cells were stained in indirect immunofluorescence with mAb 16.23. Each panel represents mAb 16.23 staining of an individual subclone of 2.7.93 (mAb 16.23); subclones of the mAb 16.23low subpopulation show similar bimodal reexpression of the mAb 16.23 epitope (data not shown).

Mentions: Although these changes resemble those of DM mutants, there are features of the mAb binding profile that distinguish the new mutants from DMA mutant 2.2.93 and all other DM mutants. In particular, whereas DM mutants are essentially mAb 16.23, mutant 2.7.93 has the mAb 16.23 peak but also a reproducible mAb 16.23low shoulder (Figs. 2 e and 5). Mutants 3.4.95 and 3.6.95 also exhibit this unusual, bimodal distribution of mAb 16.23 staining (Fig. 6 a), although relative to 2.7.93, mutant 3.4.95 consistently displays a greater proportion of mAb 16.23 cells and 3.6.95 consistently displays a greater proportion of mAb 16.23low cells in their respective populations. Thus, these mutants have related, yet distinguishable phenotypes. The bimodal mAb 16.23 staining pattern in these mutants persists despite repeated subcloning (see below and Fig. 5).


Novel mutants define genes required for the expression of human histocompatibility leukocyte antigen DM: evidence for loci on human chromosome 6p.

Fling SP, Rak J, Muczynski KA, Arp B, Pious D - J. Exp. Med. (1997)

mAb 16.23 and  mAb 16.23low sorted subpopulations of 2.7.93-type mutants express differential levels of stable  HLA-DR dimers. (a) SDS stability of HLA-DR molecules from  cell-sorted mAb16.23 and  mAb 16.23low subpopulations of  mutant 2.7.93. Western blots  were stained with a combination of mAbs DA6.147 and  HB10.A. (b) Bimodal reexpression of the mAb 16.23 epitope in  subclones of the 2.7.93 mAb  16.23 sorted subpopulations.  2.7.93 cells were stained in indirect immunofluorescence with  mAb 16.23. Each panel represents mAb 16.23 staining of an individual subclone of 2.7.93 (mAb 16.23); subclones of the mAb 16.23low subpopulation show similar bimodal reexpression of the mAb 16.23 epitope (data not shown).
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Related In: Results  -  Collection

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Figure 5: mAb 16.23 and mAb 16.23low sorted subpopulations of 2.7.93-type mutants express differential levels of stable HLA-DR dimers. (a) SDS stability of HLA-DR molecules from cell-sorted mAb16.23 and mAb 16.23low subpopulations of mutant 2.7.93. Western blots were stained with a combination of mAbs DA6.147 and HB10.A. (b) Bimodal reexpression of the mAb 16.23 epitope in subclones of the 2.7.93 mAb 16.23 sorted subpopulations. 2.7.93 cells were stained in indirect immunofluorescence with mAb 16.23. Each panel represents mAb 16.23 staining of an individual subclone of 2.7.93 (mAb 16.23); subclones of the mAb 16.23low subpopulation show similar bimodal reexpression of the mAb 16.23 epitope (data not shown).
Mentions: Although these changes resemble those of DM mutants, there are features of the mAb binding profile that distinguish the new mutants from DMA mutant 2.2.93 and all other DM mutants. In particular, whereas DM mutants are essentially mAb 16.23, mutant 2.7.93 has the mAb 16.23 peak but also a reproducible mAb 16.23low shoulder (Figs. 2 e and 5). Mutants 3.4.95 and 3.6.95 also exhibit this unusual, bimodal distribution of mAb 16.23 staining (Fig. 6 a), although relative to 2.7.93, mutant 3.4.95 consistently displays a greater proportion of mAb 16.23 cells and 3.6.95 consistently displays a greater proportion of mAb 16.23low cells in their respective populations. Thus, these mutants have related, yet distinguishable phenotypes. The bimodal mAb 16.23 staining pattern in these mutants persists despite repeated subcloning (see below and Fig. 5).

Bottom Line: However, we show that the defects in two of these new mutants do not map to the DM locus.These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules.The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

Show MeSH
Related in: MedlinePlus