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MHC deletion mutants of a B-lymphoblastoid cell line
Abstracts
129 of the "genotype" at individual HLA-D loci rather than the phenotypic results provided by Dw typing.
STUDY OF CLASS II MHC ANTIGENS OF HORSE LYMPHOCYTES USING ANTI-CLASS II HLA MONOCLONAL ANTIBODIES. D. Monos, B. Wolf, S.F. Radka, C.M. Zmijewski, and M. Kamoun; University of Pennsylvania, Philadelphia. PA Evidence for equine class II M H C antigens consists solely of data from mixed lymphocyte cultures. In order to investigate this further, four routine and 11 rat anti-human class II monoclonals were used for the direct detection of class II M H C antigens on the surface of horse lymphocytes. Monoclonals were tested against six unrelated horses using 2-color fluorescence flow cytometry. We found that the mouse monoclonals SG465 (anti-class II HLA), SG157 (DR specific), and XD5 (DR, DP, and some D Q specific) reacted with both Ig + and Ig- cells of all horses tested. However, the Ig ÷ cells were more intensely labeled with red fluorescence indicating either higher affinity for the antibody or higher density o f this antigen on their surface. The fourth mouse monoclonal N297, a D Q specific reagent, reacted primarily with the Ig ÷ cells of all six horses, suggesting that the antigen recognized by N297 is specific for Ig ÷ cells. 5/1l rat anti-class II HLA monoclonals reacted with horse lymphocytes. Eight B9.3 and 11C7.6.7 specific for DR and DP antigens and 10D2.7 specific for DR and D Q reacted with both Ig ÷ and Ig- cells of all horses tested. The fluorescence pattern was similar to that obtained with the first 3 murine monoclonals. I. 13.2 and 1.19.15, both specific for DR, reacted only with 5/6 and 2/6 horses, respectively. The pattern o f reactivity seems to be specific for lg ÷ cells and substantially nonreactive with l g - cells. It is possible that these antibodies detect a polymorphism o f horse class II M H C antigens. Preliminary results from immunoprecipitation and 2DPAGE analysis suggests the presence of a light (28 Kd) and a heavy (32 Kd) chain. It appears that the distribution of class II antigens on horse lymphocytes may be different from that in man since class II M H C antigens are expressed on resting peripheral blood lymphocytes which are Ig-.
MHC DELETION MUTANTS OF A B-LYMPHOBLASTO1D CELL LINE. Bruce Koppelman, Russell Salter, and Peter Cresswell; Duke University Medical Center, Durham, NC Using gamma irradiation followed by immunoselection, a series o f M H C cell surface loss variants has been produced from the B-cell line LICR-LON-HMy2. The initial selection was directed against the HLA A3 allele present on the parent line. The concommitant loss o f linked DP and D Q antigens on the cell surface and the loss of restriction fragments hybridizing to a DR probe lead us to believe this initial variant has lost most or all of an entire M H C haplotype. This line was used for a subsequent round of gamma irradiation and immunoselection directed at the HLA-A2 antigen present on the remaining haplotype. A variant was cloned from the selection which did not express the HLA-A2 antigen on its surface. N o r t h e r n hybridization analysis using a HLA-A locus specific probe indicated that A locus m R N A is being synthesized. We are presently attempting to determine if HLA-A2 protein is being synthesized. A third immunoselection was performed on the initial haplotype loss variant, this time using a monoclonal antibody directed against a monomorphic determinant of the DR locus. The mutant derived from this selection did not bind the selecting antibody but did bind a D Q monomorphic antibody. The latest selection was directed against the
130
Abstracts DQ antigen present on the DR null variant.Clones growing from this selection do not bind the selecting antibody but still bind a monoclonal directed against all three class II loci, suggesting this variant still expresses some class II molecules on its surface. We are presently in the process of determining the nature and extent of the lesions in these variants as well as proceeding with subsequent rounds ofimmunoselection in an effort to elucidate factors involved in the genetic regulation and biosynthesis of MHC molecules. (This work was supported by NIAID grant AI 15575.)
HLA DR REGION RESTRICTION FRAGMENT LENGTH POLYMORPHISMS DOCUMENTED WITH AN HLA-DR REPEATED ELEMENT PROBE. May Chatila, James McEleney, Lorie Luyrink, Jack Strominger, and David N. Glass; Brigham and Women's Hospital, Boston. MA The restriction fragment length polymorphism (RFLP) technique was utilized to study various homozygous typing cell lines (HTC) in an attempt to delineate haplotypic differences in the number and/or structure of the DR/3 genes and the repeated elements in this region (Spies et al., PNAS, 82:5165, 1985). DNA from HTC was digested with the following restriction endonucleases: BamH I, Sac I, Taq I, KPN I, and EcoR I, and hybridized by Southern blotting with a cDNA DR signal sequence and promoter probe (the repeated element probe). The banding pattern was characteristic for each of the DR haplotypes DRI-w8 except DRw6 that exhibited a heterogenous pattern, and the bands for DR3 and DR5 DNAs were identical. A pattern was also delineated for the HLA DRw53 supertype. As an example, using BamH I, a 22 Kb and a 3.4 Kb band distinguished between DR4 and DR7, respectively. However, a 12.8 Kb band was present in DNA channels from DR4 and DR7 cells which share the DRw53 supertype and absent in all other DNA channels. The general applicability of these findings was tested using DNAs from heterozygous DR4 and DR5 serologically typed individuals; the 22 Kb BamH I DR4-specific band was present in 49/53 DR4 DNAs vs. 0/30 DR5 DNAs. The number of bands per DNA channel was markedly different between the various DR types. The most notable finding was that DR4/ 7/w6 DNAs had more bands regardless of the restriction endonuclease used than all other DNAs. Using Sac I, HLA DR4, w6,7 had 9,10,9 bands, respectively. The remaining cell lines had 5 bands (DR1,3,5), 4 bands (DR2), and 3 bands (DRw8). We conclude that the RFLP technique using the repeated element probe distinguishes between the various HLA-DR haplotypes and the DRw supertypes. These data also indicate that there are differences in the number (known to be six in DNA from one HLA DR4 HTC) and/or structure of the repeated elements and DR3 genes between the various HLA haplotypes.
ISOLATION OF A DQ/3 HOMOLOGOUS GENOMIC CLONE THAT MAY RECOGNIZE A SUBSET OF DQw2 IN SOUTHERN BLOT ANALYSIS. Bruce Barger, H. Wayne Shew, Thomas Hodge, and Ron Acton; University of Alabama at Birmingham, Birmingham, AL A genomic library was constructed using DNA isolated from an individual homozygous for the DR3, DQw2 alleles. The DNA was partially digested with MboI and fractionated by centrifugation through a sucrose density gradient. Fractions containing DNA fragments in the 15-20 kb size range were selected. The DNA was ligated to the bacteriophage vector EMBL3 using the Barn HI site in the multiple cloning region. Recombinant phage were selected based upon the spi phenotype using the E. coli strain P2LE392, amplified, and then screened using
129 of the "genotype" at individual HLA-D loci rather than the phenotypic results provided by Dw typing.
STUDY OF CLASS II MHC ANTIGENS OF HORSE LYMPHOCYTES USING ANTI-CLASS II HLA MONOCLONAL ANTIBODIES. D. Monos, B. Wolf, S.F. Radka, C.M. Zmijewski, and M. Kamoun; University of Pennsylvania, Philadelphia. PA Evidence for equine class II M H C antigens consists solely of data from mixed lymphocyte cultures. In order to investigate this further, four routine and 11 rat anti-human class II monoclonals were used for the direct detection of class II M H C antigens on the surface of horse lymphocytes. Monoclonals were tested against six unrelated horses using 2-color fluorescence flow cytometry. We found that the mouse monoclonals SG465 (anti-class II HLA), SG157 (DR specific), and XD5 (DR, DP, and some D Q specific) reacted with both Ig + and Ig- cells of all horses tested. However, the Ig ÷ cells were more intensely labeled with red fluorescence indicating either higher affinity for the antibody or higher density o f this antigen on their surface. The fourth mouse monoclonal N297, a D Q specific reagent, reacted primarily with the Ig ÷ cells of all six horses, suggesting that the antigen recognized by N297 is specific for Ig ÷ cells. 5/1l rat anti-class II HLA monoclonals reacted with horse lymphocytes. Eight B9.3 and 11C7.6.7 specific for DR and DP antigens and 10D2.7 specific for DR and D Q reacted with both Ig ÷ and Ig- cells of all horses tested. The fluorescence pattern was similar to that obtained with the first 3 murine monoclonals. I. 13.2 and 1.19.15, both specific for DR, reacted only with 5/6 and 2/6 horses, respectively. The pattern o f reactivity seems to be specific for lg ÷ cells and substantially nonreactive with l g - cells. It is possible that these antibodies detect a polymorphism o f horse class II M H C antigens. Preliminary results from immunoprecipitation and 2DPAGE analysis suggests the presence of a light (28 Kd) and a heavy (32 Kd) chain. It appears that the distribution of class II antigens on horse lymphocytes may be different from that in man since class II M H C antigens are expressed on resting peripheral blood lymphocytes which are Ig-.
MHC DELETION MUTANTS OF A B-LYMPHOBLASTO1D CELL LINE. Bruce Koppelman, Russell Salter, and Peter Cresswell; Duke University Medical Center, Durham, NC Using gamma irradiation followed by immunoselection, a series o f M H C cell surface loss variants has been produced from the B-cell line LICR-LON-HMy2. The initial selection was directed against the HLA A3 allele present on the parent line. The concommitant loss o f linked DP and D Q antigens on the cell surface and the loss of restriction fragments hybridizing to a DR probe lead us to believe this initial variant has lost most or all of an entire M H C haplotype. This line was used for a subsequent round of gamma irradiation and immunoselection directed at the HLA-A2 antigen present on the remaining haplotype. A variant was cloned from the selection which did not express the HLA-A2 antigen on its surface. N o r t h e r n hybridization analysis using a HLA-A locus specific probe indicated that A locus m R N A is being synthesized. We are presently attempting to determine if HLA-A2 protein is being synthesized. A third immunoselection was performed on the initial haplotype loss variant, this time using a monoclonal antibody directed against a monomorphic determinant of the DR locus. The mutant derived from this selection did not bind the selecting antibody but did bind a D Q monomorphic antibody. The latest selection was directed against the
130
Abstracts DQ antigen present on the DR null variant.Clones growing from this selection do not bind the selecting antibody but still bind a monoclonal directed against all three class II loci, suggesting this variant still expresses some class II molecules on its surface. We are presently in the process of determining the nature and extent of the lesions in these variants as well as proceeding with subsequent rounds ofimmunoselection in an effort to elucidate factors involved in the genetic regulation and biosynthesis of MHC molecules. (This work was supported by NIAID grant AI 15575.)
HLA DR REGION RESTRICTION FRAGMENT LENGTH POLYMORPHISMS DOCUMENTED WITH AN HLA-DR REPEATED ELEMENT PROBE. May Chatila, James McEleney, Lorie Luyrink, Jack Strominger, and David N. Glass; Brigham and Women's Hospital, Boston. MA The restriction fragment length polymorphism (RFLP) technique was utilized to study various homozygous typing cell lines (HTC) in an attempt to delineate haplotypic differences in the number and/or structure of the DR/3 genes and the repeated elements in this region (Spies et al., PNAS, 82:5165, 1985). DNA from HTC was digested with the following restriction endonucleases: BamH I, Sac I, Taq I, KPN I, and EcoR I, and hybridized by Southern blotting with a cDNA DR signal sequence and promoter probe (the repeated element probe). The banding pattern was characteristic for each of the DR haplotypes DRI-w8 except DRw6 that exhibited a heterogenous pattern, and the bands for DR3 and DR5 DNAs were identical. A pattern was also delineated for the HLA DRw53 supertype. As an example, using BamH I, a 22 Kb and a 3.4 Kb band distinguished between DR4 and DR7, respectively. However, a 12.8 Kb band was present in DNA channels from DR4 and DR7 cells which share the DRw53 supertype and absent in all other DNA channels. The general applicability of these findings was tested using DNAs from heterozygous DR4 and DR5 serologically typed individuals; the 22 Kb BamH I DR4-specific band was present in 49/53 DR4 DNAs vs. 0/30 DR5 DNAs. The number of bands per DNA channel was markedly different between the various DR types. The most notable finding was that DR4/ 7/w6 DNAs had more bands regardless of the restriction endonuclease used than all other DNAs. Using Sac I, HLA DR4, w6,7 had 9,10,9 bands, respectively. The remaining cell lines had 5 bands (DR1,3,5), 4 bands (DR2), and 3 bands (DRw8). We conclude that the RFLP technique using the repeated element probe distinguishes between the various HLA-DR haplotypes and the DRw supertypes. These data also indicate that there are differences in the number (known to be six in DNA from one HLA DR4 HTC) and/or structure of the repeated elements and DR3 genes between the various HLA haplotypes.
ISOLATION OF A DQ/3 HOMOLOGOUS GENOMIC CLONE THAT MAY RECOGNIZE A SUBSET OF DQw2 IN SOUTHERN BLOT ANALYSIS. Bruce Barger, H. Wayne Shew, Thomas Hodge, and Ron Acton; University of Alabama at Birmingham, Birmingham, AL A genomic library was constructed using DNA isolated from an individual homozygous for the DR3, DQw2 alleles. The DNA was partially digested with MboI and fractionated by centrifugation through a sucrose density gradient. Fractions containing DNA fragments in the 15-20 kb size range were selected. The DNA was ligated to the bacteriophage vector EMBL3 using the Barn HI site in the multiple cloning region. Recombinant phage were selected based upon the spi phenotype using the E. coli strain P2LE392, amplified, and then screened using