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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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Mutant 2.7.93 expresses increased levels of class II which are  associated with CLIP. (a) Control cells, DMA  mutant 2.2.93 and  DMB  9.5.3, their progenitors 3.1.0/DR3 and 8.1.6, and negative  control T2, were stained in indirect immunofluorescent flow cytometry  with mAb CER.CLIP.1, which recognizes CLIP associated with HLA-DR molecules. (b) Mutant 2.7.93, control cell DMA mutant 2.2.93, their  progenitor, 3.1.0/DR3, and negative control T2, were stained in indirect  immunofluoresence with mAb CER.CLIP.
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Figure 4: Mutant 2.7.93 expresses increased levels of class II which are associated with CLIP. (a) Control cells, DMA mutant 2.2.93 and DMB 9.5.3, their progenitors 3.1.0/DR3 and 8.1.6, and negative control T2, were stained in indirect immunofluorescent flow cytometry with mAb CER.CLIP.1, which recognizes CLIP associated with HLA-DR molecules. (b) Mutant 2.7.93, control cell DMA mutant 2.2.93, their progenitor, 3.1.0/DR3, and negative control T2, were stained in indirect immunofluoresence with mAb CER.CLIP.

Mentions: The altered conformation of class II dimers expressed by these new mutants is similar to that of DM mutants, in which the alterations in class II conformation result from the presence of CLIP and the absence of cognate peptides in the binding groove. To determine if the new mutants express aberrantly high levels of CLIP–class II complexes, we measured cell surface levels of CLIP using mAbs CER.CLIP1 and mAb I-5, both of which recognize CLIP associated with MHC class II molecules. Mutants 2.7.93, 3.4.95, and 3.6.95, like DM mutants, exhibit marked increases in cell surface levels of class II molecules associated with CLIP peptides (Figs. 4 b and 6 b). However, the levels of anti-CLIP staining on these mutants are not as high as those of DM mutants 2.2.93 or 9.5.3 (Figs. 4 and 6).


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)

Mutant 2.7.93 expresses increased levels of class II which are  associated with CLIP. (a) Control cells, DMA  mutant 2.2.93 and  DMB  9.5.3, their progenitors 3.1.0/DR3 and 8.1.6, and negative  control T2, were stained in indirect immunofluorescent flow cytometry  with mAb CER.CLIP.1, which recognizes CLIP associated with HLA-DR molecules. (b) Mutant 2.7.93, control cell DMA mutant 2.2.93, their  progenitor, 3.1.0/DR3, and negative control T2, were stained in indirect  immunofluoresence with mAb CER.CLIP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199108&req=5

Figure 4: Mutant 2.7.93 expresses increased levels of class II which are associated with CLIP. (a) Control cells, DMA mutant 2.2.93 and DMB 9.5.3, their progenitors 3.1.0/DR3 and 8.1.6, and negative control T2, were stained in indirect immunofluorescent flow cytometry with mAb CER.CLIP.1, which recognizes CLIP associated with HLA-DR molecules. (b) Mutant 2.7.93, control cell DMA mutant 2.2.93, their progenitor, 3.1.0/DR3, and negative control T2, were stained in indirect immunofluoresence with mAb CER.CLIP.
Mentions: The altered conformation of class II dimers expressed by these new mutants is similar to that of DM mutants, in which the alterations in class II conformation result from the presence of CLIP and the absence of cognate peptides in the binding groove. To determine if the new mutants express aberrantly high levels of CLIP–class II complexes, we measured cell surface levels of CLIP using mAbs CER.CLIP1 and mAb I-5, both of which recognize CLIP associated with MHC class II molecules. Mutants 2.7.93, 3.4.95, and 3.6.95, like DM mutants, exhibit marked increases in cell surface levels of class II molecules associated with CLIP peptides (Figs. 4 b and 6 b). However, the levels of anti-CLIP staining on these mutants are not as high as those of DM mutants 2.2.93 or 9.5.3 (Figs. 4 and 6).

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