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Molecular variants of diabetic DR3 and DR4 haplotypes
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Abstracts (22) F u n c t / o n a l A n a l y s i s o f D R 3 - A s s o c i a t e d Class I I Molecules U s i n g g p 3 5 0 Specific H u m a n T-Cell C l o n e s (TCC.s) B- Carreno, C.K_ Hurley, G.R. Pearson, 1LJ. Hartzman
Georgetown University, Washington, D.C. The major envelope glycoproteln of Epstein-Burr virus, gp350, has been postulated as a candidate subviral vaccine against EBV-assocLated diseases. H,lman TCC-spec/fic for gp350 have been generated in order to understand T-cell responses to this g]ycoproteio including its MHC-restricted recognltion. Using these gp350-specific TCCs, we have analyzed struccarai/functional relationship of DR and DQ molecules using fatuities and o panel of unrelated Caucasian and American bla~k donors as ant/gee-presenting ceils (APCs). Two TCCs derived from a Caucasian donor are described. TCC T3 recognized gp350 only when presented by cells expressing DR3,DQw2; gp350-pulsed APCs expressing other haplotypes, including DR3,DQw4 and other DQw2-assoclated haploq/pes, were unable to stimulate this clone. The two DR3 haplotypes express identical DR#III chains and DR#! chains thus differ at amino acids positions 26,28,47, and 86. The fact that the DR3,DQw4 haplotTpe fails m present antigen to T3 implicates these four amino acids in antigen and/or T-cell-receptor binding. TCC W4 showed the same pattern of restriction as T3 with DR3 APes but, in DR9 APCs. Moreover, this pattern of restriction segregated within a family expressing an unusual DR-,DQw2 haplotype. Based on DNA sequence, DR3- and DRT-assedared DQw2 beterodimers present identical ~ chains paired with different DQ~ chains. In addition, APCs expressing the same or a similar DQw2a chain as DR3,DQw2 are unable to present gp350 to W4. These data suggest that the DQw2~ chain may not only define the serniogic specificity of the molecule hut also control its MHC-restricting capacity.
(23) M o l e c u l a r V a r i a n t s o f D i a b e t i c D R 5 a n d D R 4 H a p l o t y p e s C. Carrier, S. Rodriguez de Cordoba, and P. Rubinstein
lmmunogenetics Laboratory, New York Blood Center, New York, N Y Genetic variants of the DQ~ and fl and of the DXcz genes, detectable as RFI.Ps, have been suggested re independently improve the definition of high relative risks for type I diabetes (IDDM). We have tested this hypothesis by analyzing the DNA phenotypes of all members of 32 multiplex families, by digestion with 6 restriction enzymes and Southern blotting with eDNA probes for those 3 genes. Assignment of fragments to serologically and biochemically (IEF) characterized HLA class I1 haptotypes was based on the/r segregation and performed with a computerized algorithm. The results indicate that the DNA (RF) alleles of DX~, DQa, and DOff maintain linkage disequifibrium (p < 0.001) so that the preponderant (26 of 30 examples) diabetic DR4 haplotype carries both the DX~,. Taql-2.2kb and DQ~3.2 high-risk alleles. Rare, affected and unaffected, DQw3 haplotvpes that canV eit,berthe DQ or the DX portions of the diabetic DR4 haplotype were encountered. In the same families, affected but not unaffected, DR5 and DR7 haplotypes were shown m be in significant linkage disequilibrium with the IEF-defined DP¢ allele. The molecular veriants thus defined are now being used as markers to pinpoint the location of the susceptibifity gene in the respective haplot~es.
(24) R e g u l a t i o n o f H L A Clsss I I G e n e E x p r e s s i o n b y P h o r b o l 1 2 , 1 3 - D i b u t y r a t e M.N. Carrington, O. King, and ]~.]L Ward
Department of Microbiology and luwt~unology,Duke University Medical Center, Durham, NC In order to test the effect of protein kinase C (PKC) on regulation of HLA class II gene expression, a class II-positive melanomacell line, DU-Mel 17, was treated with phorbo112, I3-dihutyt~e (PdBU). Cells were treated with 24aM PdBU for 1, 24, and 48 hours, ~ d class II mRNA and protein levels were then measured. After 1 hour, D I ~ mRNA levels were increased relative to the untreated control At 24 hours, DRa mRNA levels were similar to those of the untreated group, and, by 48 hours, levels of the message were continuing to decrease. Cycloheximide had no effect on the ability of PdBU to decrease DRa mR.NA, suggesting that preformed mediato~ are responsible for altering these R ~ A levels. Further, 4a-phorbol did not alter DR~ mRNA levels, in accordance with its inability to bind PKC. Fluorescein Label/ngof the DU-Mel
Abstracts (22) F u n c t / o n a l A n a l y s i s o f D R 3 - A s s o c i a t e d Class I I Molecules U s i n g g p 3 5 0 Specific H u m a n T-Cell C l o n e s (TCC.s) B- Carreno, C.K_ Hurley, G.R. Pearson, 1LJ. Hartzman
Georgetown University, Washington, D.C. The major envelope glycoproteln of Epstein-Burr virus, gp350, has been postulated as a candidate subviral vaccine against EBV-assocLated diseases. H,lman TCC-spec/fic for gp350 have been generated in order to understand T-cell responses to this g]ycoproteio including its MHC-restricted recognltion. Using these gp350-specific TCCs, we have analyzed struccarai/functional relationship of DR and DQ molecules using fatuities and o panel of unrelated Caucasian and American bla~k donors as ant/gee-presenting ceils (APCs). Two TCCs derived from a Caucasian donor are described. TCC T3 recognized gp350 only when presented by cells expressing DR3,DQw2; gp350-pulsed APCs expressing other haplotypes, including DR3,DQw4 and other DQw2-assoclated haploq/pes, were unable to stimulate this clone. The two DR3 haplotypes express identical DR#III chains and DR#! chains thus differ at amino acids positions 26,28,47, and 86. The fact that the DR3,DQw4 haplotTpe fails m present antigen to T3 implicates these four amino acids in antigen and/or T-cell-receptor binding. TCC W4 showed the same pattern of restriction as T3 with DR3 APes but, in DR9 APCs. Moreover, this pattern of restriction segregated within a family expressing an unusual DR-,DQw2 haplotype. Based on DNA sequence, DR3- and DRT-assedared DQw2 beterodimers present identical ~ chains paired with different DQ~ chains. In addition, APCs expressing the same or a similar DQw2a chain as DR3,DQw2 are unable to present gp350 to W4. These data suggest that the DQw2~ chain may not only define the serniogic specificity of the molecule hut also control its MHC-restricting capacity.
(23) M o l e c u l a r V a r i a n t s o f D i a b e t i c D R 5 a n d D R 4 H a p l o t y p e s C. Carrier, S. Rodriguez de Cordoba, and P. Rubinstein
lmmunogenetics Laboratory, New York Blood Center, New York, N Y Genetic variants of the DQ~ and fl and of the DXcz genes, detectable as RFI.Ps, have been suggested re independently improve the definition of high relative risks for type I diabetes (IDDM). We have tested this hypothesis by analyzing the DNA phenotypes of all members of 32 multiplex families, by digestion with 6 restriction enzymes and Southern blotting with eDNA probes for those 3 genes. Assignment of fragments to serologically and biochemically (IEF) characterized HLA class I1 haptotypes was based on the/r segregation and performed with a computerized algorithm. The results indicate that the DNA (RF) alleles of DX~, DQa, and DOff maintain linkage disequifibrium (p < 0.001) so that the preponderant (26 of 30 examples) diabetic DR4 haplotype carries both the DX~,. Taql-2.2kb and DQ~3.2 high-risk alleles. Rare, affected and unaffected, DQw3 haplotvpes that canV eit,berthe DQ or the DX portions of the diabetic DR4 haplotype were encountered. In the same families, affected but not unaffected, DR5 and DR7 haplotypes were shown m be in significant linkage disequilibrium with the IEF-defined DP¢ allele. The molecular veriants thus defined are now being used as markers to pinpoint the location of the susceptibifity gene in the respective haplot~es.
(24) R e g u l a t i o n o f H L A Clsss I I G e n e E x p r e s s i o n b y P h o r b o l 1 2 , 1 3 - D i b u t y r a t e M.N. Carrington, O. King, and ]~.]L Ward
Department of Microbiology and luwt~unology,Duke University Medical Center, Durham, NC In order to test the effect of protein kinase C (PKC) on regulation of HLA class II gene expression, a class II-positive melanomacell line, DU-Mel 17, was treated with phorbo112, I3-dihutyt~e (PdBU). Cells were treated with 24aM PdBU for 1, 24, and 48 hours, ~ d class II mRNA and protein levels were then measured. After 1 hour, D I ~ mRNA levels were increased relative to the untreated control At 24 hours, DRa mRNA levels were similar to those of the untreated group, and, by 48 hours, levels of the message were continuing to decrease. Cycloheximide had no effect on the ability of PdBU to decrease DRa mR.NA, suggesting that preformed mediato~ are responsible for altering these R ~ A levels. Further, 4a-phorbol did not alter DR~ mRNA levels, in accordance with its inability to bind PKC. Fluorescein Label/ngof the DU-Mel