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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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Cell surface staining of MHC class II and MHC class II/CLIP  complexes on 3.4.95 and 3.6.95, and on (2.7.93 × T2) and (3.6.95 ×  T2). (a and b) Cell surface expression of the mAb 16.23 epitope and of  MHC class II–CLIP complexes on 6.3.6/DR3-derived mutants 3.4.95  and 3.6.95. (a) Negative control DMA  mutant 2.2.93, mutants 3.6.95  and 3.4.95, and progenitor 6.3.6/DR3, were stained by indirect immunofluorescence with mAb 16.23. Subclones of 3.4.95 and 3.6.95 manifest  consistent bimodal expression of the mAb 16.23 epitope as seen in mutant  2.7.93 (data not shown). (b) Negative control 6.1.6 (71), progenitor 6.3.6/  DR3, positive control, DMA  mutant 2.2.93, and mutants 3.6.95 and  3.4.95, were stained by indirect immunofluorescence with anti-class II– CLIP mAb I-5, which with HLA class II molecules. (c–f) Cell surface expression of the mAb 16.23 epitope and of MHC class II–CLIP complexes  on somatic cell hybrids. (c) Negative control hybrid (2.2.93 × T2), positive control hybrid (3.1.0/DR3 × T2), and hybrids (2.7.93 × T2) and  (3.6.95 × T2); and (d) control hybrid (3.1.0/DR3 × T2); and hybrids  (2.7.93 × T2) and (3.6.95 × T2) and mutants 2.7.93 and 3.6.95; and (e  and f) negative control (T2), negative control somatic cell hybrids (2.2.93 ×  T2), positive control hybrids (3.1.0/DR3 x T2); and hybrids (2.7.93 ×  T2) were stained in indirect immunofluorescent flow cytometry using the  indicated mAbs.
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Figure 6: Cell surface staining of MHC class II and MHC class II/CLIP complexes on 3.4.95 and 3.6.95, and on (2.7.93 × T2) and (3.6.95 × T2). (a and b) Cell surface expression of the mAb 16.23 epitope and of MHC class II–CLIP complexes on 6.3.6/DR3-derived mutants 3.4.95 and 3.6.95. (a) Negative control DMA mutant 2.2.93, mutants 3.6.95 and 3.4.95, and progenitor 6.3.6/DR3, were stained by indirect immunofluorescence with mAb 16.23. Subclones of 3.4.95 and 3.6.95 manifest consistent bimodal expression of the mAb 16.23 epitope as seen in mutant 2.7.93 (data not shown). (b) Negative control 6.1.6 (71), progenitor 6.3.6/ DR3, positive control, DMA mutant 2.2.93, and mutants 3.6.95 and 3.4.95, were stained by indirect immunofluorescence with anti-class II– CLIP mAb I-5, which with HLA class II molecules. (c–f) Cell surface expression of the mAb 16.23 epitope and of MHC class II–CLIP complexes on somatic cell hybrids. (c) Negative control hybrid (2.2.93 × T2), positive control hybrid (3.1.0/DR3 × T2), and hybrids (2.7.93 × T2) and (3.6.95 × T2); and (d) control hybrid (3.1.0/DR3 × T2); and hybrids (2.7.93 × T2) and (3.6.95 × T2) and mutants 2.7.93 and 3.6.95; and (e and f) negative control (T2), negative control somatic cell hybrids (2.2.93 × T2), positive control hybrids (3.1.0/DR3 x T2); and hybrids (2.7.93 × T2) were stained in indirect immunofluorescent flow cytometry using the indicated mAbs.

Mentions: Mutants 3.6.95 and 3.4.95 have similar phenotypes to mutant 2.7.93, although they were derived from a different progenitor, 6.3.6/DR3 (Fig. 1), and thus are independent of 2.7.93. Mutants 3.6.95 and 3.4.95, like 2.7.93, exhibit substantial loss of the mAb 16.23 epitope (Fig. 6 a), but they also retain levels of MHC HLA-DR and of the DR3 transgene comparable to those of progenitor 6.3.6/DR3 (Table 1).


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)

Cell surface staining of MHC class II and MHC class II/CLIP  complexes on 3.4.95 and 3.6.95, and on (2.7.93 × T2) and (3.6.95 ×  T2). (a and b) Cell surface expression of the mAb 16.23 epitope and of  MHC class II–CLIP complexes on 6.3.6/DR3-derived mutants 3.4.95  and 3.6.95. (a) Negative control DMA  mutant 2.2.93, mutants 3.6.95  and 3.4.95, and progenitor 6.3.6/DR3, were stained by indirect immunofluorescence with mAb 16.23. Subclones of 3.4.95 and 3.6.95 manifest  consistent bimodal expression of the mAb 16.23 epitope as seen in mutant  2.7.93 (data not shown). (b) Negative control 6.1.6 (71), progenitor 6.3.6/  DR3, positive control, DMA  mutant 2.2.93, and mutants 3.6.95 and  3.4.95, were stained by indirect immunofluorescence with anti-class II– CLIP mAb I-5, which with HLA class II molecules. (c–f) Cell surface expression of the mAb 16.23 epitope and of MHC class II–CLIP complexes  on somatic cell hybrids. (c) Negative control hybrid (2.2.93 × T2), positive control hybrid (3.1.0/DR3 × T2), and hybrids (2.7.93 × T2) and  (3.6.95 × T2); and (d) control hybrid (3.1.0/DR3 × T2); and hybrids  (2.7.93 × T2) and (3.6.95 × T2) and mutants 2.7.93 and 3.6.95; and (e  and f) negative control (T2), negative control somatic cell hybrids (2.2.93 ×  T2), positive control hybrids (3.1.0/DR3 x T2); and hybrids (2.7.93 ×  T2) were stained in indirect immunofluorescent flow cytometry using the  indicated mAbs.
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Figure 6: Cell surface staining of MHC class II and MHC class II/CLIP complexes on 3.4.95 and 3.6.95, and on (2.7.93 × T2) and (3.6.95 × T2). (a and b) Cell surface expression of the mAb 16.23 epitope and of MHC class II–CLIP complexes on 6.3.6/DR3-derived mutants 3.4.95 and 3.6.95. (a) Negative control DMA mutant 2.2.93, mutants 3.6.95 and 3.4.95, and progenitor 6.3.6/DR3, were stained by indirect immunofluorescence with mAb 16.23. Subclones of 3.4.95 and 3.6.95 manifest consistent bimodal expression of the mAb 16.23 epitope as seen in mutant 2.7.93 (data not shown). (b) Negative control 6.1.6 (71), progenitor 6.3.6/ DR3, positive control, DMA mutant 2.2.93, and mutants 3.6.95 and 3.4.95, were stained by indirect immunofluorescence with anti-class II– CLIP mAb I-5, which with HLA class II molecules. (c–f) Cell surface expression of the mAb 16.23 epitope and of MHC class II–CLIP complexes on somatic cell hybrids. (c) Negative control hybrid (2.2.93 × T2), positive control hybrid (3.1.0/DR3 × T2), and hybrids (2.7.93 × T2) and (3.6.95 × T2); and (d) control hybrid (3.1.0/DR3 × T2); and hybrids (2.7.93 × T2) and (3.6.95 × T2) and mutants 2.7.93 and 3.6.95; and (e and f) negative control (T2), negative control somatic cell hybrids (2.2.93 × T2), positive control hybrids (3.1.0/DR3 x T2); and hybrids (2.7.93 × T2) were stained in indirect immunofluorescent flow cytometry using the indicated mAbs.
Mentions: Mutants 3.6.95 and 3.4.95 have similar phenotypes to mutant 2.7.93, although they were derived from a different progenitor, 6.3.6/DR3 (Fig. 1), and thus are independent of 2.7.93. Mutants 3.6.95 and 3.4.95, like 2.7.93, exhibit substantial loss of the mAb 16.23 epitope (Fig. 6 a), but they also retain levels of MHC HLA-DR and of the DR3 transgene comparable to those of progenitor 6.3.6/DR3 (Table 1).

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