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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 conformationally altered class II molecules. Cell surface staining of MHC molecules on mutant 2.7.93. Mutant  2.7.93 (dark line), its progenitor, 3.1.0/DR3 (thin line), control DMA   mutant, 2.2.93 (heavy dashed), and negative control, T2 (thin dashed), were  stained in indirect immunofluorescent flow cytometry using class II conformation-independent mAbs: (a) L-243 (anti-DR); (b) VI-15 (anti-DR);  (c) UK8.1 (anti-DR3, 5, 7); (d) 62.74 (anti-DR1); and class II conformation-sensitive mAbs: (e) 16.23 (anti-DR3); (f) 4AA7 (anti-DQ1); (g)  13.3.B4 (anti-DP4.1); and mAb to class I: (h) W6/32. Only mutant 2.7.93  is labeled to highlight its profile. In b only, T2 is heavy dashed and 2.2.93  is thin dashed.
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Figure 2: Mutant 2.7.93 expresses conformationally altered class II molecules. Cell surface staining of MHC molecules on mutant 2.7.93. Mutant 2.7.93 (dark line), its progenitor, 3.1.0/DR3 (thin line), control DMA mutant, 2.2.93 (heavy dashed), and negative control, T2 (thin dashed), were stained in indirect immunofluorescent flow cytometry using class II conformation-independent mAbs: (a) L-243 (anti-DR); (b) VI-15 (anti-DR); (c) UK8.1 (anti-DR3, 5, 7); (d) 62.74 (anti-DR1); and class II conformation-sensitive mAbs: (e) 16.23 (anti-DR3); (f) 4AA7 (anti-DQ1); (g) 13.3.B4 (anti-DP4.1); and mAb to class I: (h) W6/32. Only mutant 2.7.93 is labeled to highlight its profile. In b only, T2 is heavy dashed and 2.2.93 is thin dashed.

Mentions: For transient expression experiments, cDNA encoding HLA-DMB and cDNA encoding HLA-DMA were cloned into the expression vector pcDNAI/AMP (Invitrogen Corp., Carlsbad, CA) to obtain plasmids pDMB and pDMA, respectively, as described (22). pcDNAI/AMP contains Polyoma and SV40 origins of replication, allowing for episomal replication. For transient transfections, 2.5 × 107 of the indicated cells were resuspended in 0.5 ml RPMI with 15–25-μg circular pDMB or pDMA or both and were electroporated using 2.2 kV. Samples were cultured for 72 h to regain high viability and then were stained and analyzed by flow cytometry as described in Fig. 2.


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 conformationally altered class II molecules. Cell surface staining of MHC molecules on mutant 2.7.93. Mutant  2.7.93 (dark line), its progenitor, 3.1.0/DR3 (thin line), control DMA   mutant, 2.2.93 (heavy dashed), and negative control, T2 (thin dashed), were  stained in indirect immunofluorescent flow cytometry using class II conformation-independent mAbs: (a) L-243 (anti-DR); (b) VI-15 (anti-DR);  (c) UK8.1 (anti-DR3, 5, 7); (d) 62.74 (anti-DR1); and class II conformation-sensitive mAbs: (e) 16.23 (anti-DR3); (f) 4AA7 (anti-DQ1); (g)  13.3.B4 (anti-DP4.1); and mAb to class I: (h) W6/32. Only mutant 2.7.93  is labeled to highlight its profile. In b only, T2 is heavy dashed and 2.2.93  is thin dashed.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199108&req=5

Figure 2: Mutant 2.7.93 expresses conformationally altered class II molecules. Cell surface staining of MHC molecules on mutant 2.7.93. Mutant 2.7.93 (dark line), its progenitor, 3.1.0/DR3 (thin line), control DMA mutant, 2.2.93 (heavy dashed), and negative control, T2 (thin dashed), were stained in indirect immunofluorescent flow cytometry using class II conformation-independent mAbs: (a) L-243 (anti-DR); (b) VI-15 (anti-DR); (c) UK8.1 (anti-DR3, 5, 7); (d) 62.74 (anti-DR1); and class II conformation-sensitive mAbs: (e) 16.23 (anti-DR3); (f) 4AA7 (anti-DQ1); (g) 13.3.B4 (anti-DP4.1); and mAb to class I: (h) W6/32. Only mutant 2.7.93 is labeled to highlight its profile. In b only, T2 is heavy dashed and 2.2.93 is thin dashed.
Mentions: For transient expression experiments, cDNA encoding HLA-DMB and cDNA encoding HLA-DMA were cloned into the expression vector pcDNAI/AMP (Invitrogen Corp., Carlsbad, CA) to obtain plasmids pDMB and pDMA, respectively, as described (22). pcDNAI/AMP contains Polyoma and SV40 origins of replication, allowing for episomal replication. For transient transfections, 2.5 × 107 of the indicated cells were resuspended in 0.5 ml RPMI with 15–25-μg circular pDMB or pDMA or both and were electroporated using 2.2 kV. Samples were cultured for 72 h to regain high viability and then were stained and analyzed by flow cytometry as described in Fig. 2.

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