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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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Derivation of mutant cell lines. (a) Regions of  chromosome 6p hemizygously  deleted in progenitors 3.1.0  (∼40 Mb) and 6.3.6 (∼30 Mb)  and their positions relative to the  HLA complex. (b) The HLA class  II region haplotypes of B-LCL  3.1.0, the progenitor of mutant  2.7.93 and mutant 2.2.93 (DMA  ) (22). (c) The HLA class II  region haplotypes of B-LCL  6.3.6, the progenitor of mutants  3.6.95 and 3.4.95. Deletions are  indicated by horizontal hatched  bars on the deleted haplotype  and have been previously described for 3.1.0 and 6.3.6 (71).  Vertical bars indicate approximate locations of genetic loci  and details of the HLA class II  region (72). (d) The 1-Mb class  II region HLA homozygous deletion in T2 (T × B hybrid line:  721.174 × CEM.T2) (40). The  centromeres are to the left. Not  to scale.
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Figure 1: Derivation of mutant cell lines. (a) Regions of chromosome 6p hemizygously deleted in progenitors 3.1.0 (∼40 Mb) and 6.3.6 (∼30 Mb) and their positions relative to the HLA complex. (b) The HLA class II region haplotypes of B-LCL 3.1.0, the progenitor of mutant 2.7.93 and mutant 2.2.93 (DMA ) (22). (c) The HLA class II region haplotypes of B-LCL 6.3.6, the progenitor of mutants 3.6.95 and 3.4.95. Deletions are indicated by horizontal hatched bars on the deleted haplotype and have been previously described for 3.1.0 and 6.3.6 (71). Vertical bars indicate approximate locations of genetic loci and details of the HLA class II region (72). (d) The 1-Mb class II region HLA homozygous deletion in T2 (T × B hybrid line: 721.174 × CEM.T2) (40). The centromeres are to the left. Not to scale.

Mentions: EBV transformed human B-lymphoblastoid cell lines (B-LCL) with hemizygous MHC deletions have been useful as progenitors for immunoselecting recessive mutants which are defective in MHC class II/peptide assembly, including DM mutants previously isolated (21, 22, 26, 27). Using this approach, we derived mutant 2.7.93 from progenitor 3.1.0, which contains an ∼40-Mb hemizygous deletion in chromosome 6p, removing one HLA haplotype (59) (Fig. 1). Mutants 3.6.95 and 3.4.95 were similarly derived from progenitor 6.3.6, which contains a smaller, ∼30 Mb hemizygous deletion in chromosome 6p, which is overlapped by the 3.1.0 deletion (59) (Fig. 1). As a basis for immunoselection of these mutants, progenitors 3.1.0 (DR1+/DR3-) and 6.3.6 (DR1+/DR3-) were first transduced with DRB1.0301. HLA-DR3 molecules exhibit reduced binding of certain mAbs, e.g., mAb 16.23 and mAb 7.3.19.1, as a result of changes in occupancy of the peptide binding groove (22, 26). The 3.1.0/DR3 and 6.3.6/DR3 progenitors were mutagenized with EMS and were immunoselected with mAb 7.3.19.1. After immunoselection, surviving clones were screened for loss of mAb 16.23 binding and further analyzed for class II alterations suggestive of defects in MHC class II/ peptide assembly. Mutants 2.7.93, 3.4.95, and 3.6.95 were isolated from mutagenized progenitors at frequencies of ∼10−4, consistent with observed frequencies for EMS-induced recessive mutations in other hemizygous loci using equivalent EMS concentrations and standard protocols (55, 60).


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)

Derivation of mutant cell lines. (a) Regions of  chromosome 6p hemizygously  deleted in progenitors 3.1.0  (∼40 Mb) and 6.3.6 (∼30 Mb)  and their positions relative to the  HLA complex. (b) The HLA class  II region haplotypes of B-LCL  3.1.0, the progenitor of mutant  2.7.93 and mutant 2.2.93 (DMA  ) (22). (c) The HLA class II  region haplotypes of B-LCL  6.3.6, the progenitor of mutants  3.6.95 and 3.4.95. Deletions are  indicated by horizontal hatched  bars on the deleted haplotype  and have been previously described for 3.1.0 and 6.3.6 (71).  Vertical bars indicate approximate locations of genetic loci  and details of the HLA class II  region (72). (d) The 1-Mb class  II region HLA homozygous deletion in T2 (T × B hybrid line:  721.174 × CEM.T2) (40). The  centromeres are to the left. Not  to scale.
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Related In: Results  -  Collection

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Figure 1: Derivation of mutant cell lines. (a) Regions of chromosome 6p hemizygously deleted in progenitors 3.1.0 (∼40 Mb) and 6.3.6 (∼30 Mb) and their positions relative to the HLA complex. (b) The HLA class II region haplotypes of B-LCL 3.1.0, the progenitor of mutant 2.7.93 and mutant 2.2.93 (DMA ) (22). (c) The HLA class II region haplotypes of B-LCL 6.3.6, the progenitor of mutants 3.6.95 and 3.4.95. Deletions are indicated by horizontal hatched bars on the deleted haplotype and have been previously described for 3.1.0 and 6.3.6 (71). Vertical bars indicate approximate locations of genetic loci and details of the HLA class II region (72). (d) The 1-Mb class II region HLA homozygous deletion in T2 (T × B hybrid line: 721.174 × CEM.T2) (40). The centromeres are to the left. Not to scale.
Mentions: EBV transformed human B-lymphoblastoid cell lines (B-LCL) with hemizygous MHC deletions have been useful as progenitors for immunoselecting recessive mutants which are defective in MHC class II/peptide assembly, including DM mutants previously isolated (21, 22, 26, 27). Using this approach, we derived mutant 2.7.93 from progenitor 3.1.0, which contains an ∼40-Mb hemizygous deletion in chromosome 6p, removing one HLA haplotype (59) (Fig. 1). Mutants 3.6.95 and 3.4.95 were similarly derived from progenitor 6.3.6, which contains a smaller, ∼30 Mb hemizygous deletion in chromosome 6p, which is overlapped by the 3.1.0 deletion (59) (Fig. 1). As a basis for immunoselection of these mutants, progenitors 3.1.0 (DR1+/DR3-) and 6.3.6 (DR1+/DR3-) were first transduced with DRB1.0301. HLA-DR3 molecules exhibit reduced binding of certain mAbs, e.g., mAb 16.23 and mAb 7.3.19.1, as a result of changes in occupancy of the peptide binding groove (22, 26). The 3.1.0/DR3 and 6.3.6/DR3 progenitors were mutagenized with EMS and were immunoselected with mAb 7.3.19.1. After immunoselection, surviving clones were screened for loss of mAb 16.23 binding and further analyzed for class II alterations suggestive of defects in MHC class II/ peptide assembly. Mutants 2.7.93, 3.4.95, and 3.6.95 were isolated from mutagenized progenitors at frequencies of ∼10−4, consistent with observed frequencies for EMS-induced recessive mutations in other hemizygous loci using equivalent EMS concentrations and standard protocols (55, 60).

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