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Functional motifs of HLA-DR13 microvariants
Abstracts
41
B-3.3 #69
THE EFFECT OF HLA-DH3 MICROVARIATION ON HUMAN IMMUNE RESPONSES. HA A r a u 4 o , PB P o a c h , AH J o h n s o n a n d C K H u r l e y , D e p a r t m e n t s o f M i c r o b i o l o g y and Pediatrics, Georgetown University, Washington, DC. F o u r D R E I * 0 3 0 2 cDNA w i t h c h a n g e s a t a m i n o a c i d p o s i t i o n s 26, 47, 86 a n d a t b o t h 47 a n d 86 h a v e b e e n c r e a t e d by replacing DREI*0302 residues with DRBI*0301 residues. Murine fibroblast cell lines expressing DR(a,Sl*0301), DR(a,SI*0302) and the four mutant DR(a,$1*0302) molecules were examined for their ability to stimulate DR(~,Sl*0302)-specific a11oproliferative TLC and for their ability to hind peptide antigens. Seven mutant DR(a,SI*0302) recognition patterns were observed with twelve DR(,,Sl*O302)-specific alloproliferativeTLC. These results suggest that the effect that each variant B-chain residue has on TCR recognition is unique to each individual alloproliferative TLC. Interestingly, several a l l o p r o l i f e r a t i v e T L C w e r e a b l e to respond to the single mutants at residues 47 and 86, but w e r e u n a b l e to respond to DR(a,SI*0302) expressed on the surface of the murine fibroblasts. These data suggest that t h e s e a l l o p r o l i f e r a t i v e T L C recognize different DR3-peptide complexes in the murine and human systems. In contrast, none of the mutations introduced had a deleterious effect on the binding of HA 307-319 (DR(u,Sl*0302)-specific). In fact, the mutant DR(a,SI*0302) molecules at residues 47 and 86, alone and in combination, consistently bound HA307-319 better than the wtmolecule. These data suggest that these DR3 variant amino acid residues, which are not thought to be direct T C R c o n t a c t residues, effect DR3 function primarily through the peptide repetoire bound within the antigen binding groove. However, these residues do not affect all peptides that bind in the groove of DR3 in that these mutants were unable to significantly bind HSP 3-13 (DR(u,Sl*0301)-zpecific). In contrast, the mutant at residue 26 had no effect on the binding of either peptide, hut had the most profound effect on allorecognition suggesting that this variant amino acid residue exerts its effect primarily through secondary conformational changes to the DR3-peptide complex.
B-3.3 #70
FUNCTIONAL MOTIFS OF HLA-DR13 MICROVARIANTS. N Steiner, HA Araujo, AH Johnson and CK Hurley, Departments of Microbiology and Pediatrics, Georgetown University, Washington, DC. Two evolutionary pathways may have given rise to two subgroups of alleles encoding molecules that share DR13 serologic determinants yet which possess different structural and, likely, functional motifs. One of these pathways links the DR13 and DR11 allele families. To study the effect of microvariation on function, we have generated DRl3-expressing wild type (DRB1*1301-1305) and mutant murine L cell transfectant8. Mutant DR S chain cDNAs with changes at amino acid positions 32, 37, 57 and 86 were created altering amino acids on the floor and ends of the antigen binding groove. The alterations replaced amino acids found in DR(~,S1"1304) with the amino acid found in DR(a,Sl*1301). Cells expressing DRBl*1303 and DRBI*I304 bound HA 307-319 peptlde at a higher level than cells expressing other wt DR13 molecules, a pattern corresponding to the DR13 subgroups designated by structural studies. When DR(,,S1"1304) was mutated to alter position 57, HA binding was decreased 5 fold. Alterations of positions 32 and 37 reduced binding to the level of DR(a,Sl*1301) binding. The effect of DR13 microvariation on T cell recognition was assessed using alloproliferative T cell clones (TLC) generated using a DRB1*1101 responder and a DRB1*1102 stimulator. These TLC recognize cells (B-LCL, PBL, and DR transfectant8) expressing DRB1*1102 as well as cells expressing DR13 allelic products. For example, TLC 2.22 recognizes cells expressing DRBI*I102, "1301, "1302, "1303, and -1304 as well as S chain mutants. The T cell recognition pattern underscores the close relationship among DR13 and DR11 alleles. These studies illustrate the dichotomy present regarding the function of the DR13 molecules demonstrating the unique and shared functions of this complex family.
41
B-3.3 #69
THE EFFECT OF HLA-DH3 MICROVARIATION ON HUMAN IMMUNE RESPONSES. HA A r a u 4 o , PB P o a c h , AH J o h n s o n a n d C K H u r l e y , D e p a r t m e n t s o f M i c r o b i o l o g y and Pediatrics, Georgetown University, Washington, DC. F o u r D R E I * 0 3 0 2 cDNA w i t h c h a n g e s a t a m i n o a c i d p o s i t i o n s 26, 47, 86 a n d a t b o t h 47 a n d 86 h a v e b e e n c r e a t e d by replacing DREI*0302 residues with DRBI*0301 residues. Murine fibroblast cell lines expressing DR(a,Sl*0301), DR(a,SI*0302) and the four mutant DR(a,$1*0302) molecules were examined for their ability to stimulate DR(~,Sl*0302)-specific a11oproliferative TLC and for their ability to hind peptide antigens. Seven mutant DR(a,SI*0302) recognition patterns were observed with twelve DR(,,Sl*O302)-specific alloproliferativeTLC. These results suggest that the effect that each variant B-chain residue has on TCR recognition is unique to each individual alloproliferative TLC. Interestingly, several a l l o p r o l i f e r a t i v e T L C w e r e a b l e to respond to the single mutants at residues 47 and 86, but w e r e u n a b l e to respond to DR(a,SI*0302) expressed on the surface of the murine fibroblasts. These data suggest that t h e s e a l l o p r o l i f e r a t i v e T L C recognize different DR3-peptide complexes in the murine and human systems. In contrast, none of the mutations introduced had a deleterious effect on the binding of HA 307-319 (DR(u,Sl*0302)-specific). In fact, the mutant DR(a,SI*0302) molecules at residues 47 and 86, alone and in combination, consistently bound HA307-319 better than the wtmolecule. These data suggest that these DR3 variant amino acid residues, which are not thought to be direct T C R c o n t a c t residues, effect DR3 function primarily through the peptide repetoire bound within the antigen binding groove. However, these residues do not affect all peptides that bind in the groove of DR3 in that these mutants were unable to significantly bind HSP 3-13 (DR(u,Sl*0301)-zpecific). In contrast, the mutant at residue 26 had no effect on the binding of either peptide, hut had the most profound effect on allorecognition suggesting that this variant amino acid residue exerts its effect primarily through secondary conformational changes to the DR3-peptide complex.
B-3.3 #70
FUNCTIONAL MOTIFS OF HLA-DR13 MICROVARIANTS. N Steiner, HA Araujo, AH Johnson and CK Hurley, Departments of Microbiology and Pediatrics, Georgetown University, Washington, DC. Two evolutionary pathways may have given rise to two subgroups of alleles encoding molecules that share DR13 serologic determinants yet which possess different structural and, likely, functional motifs. One of these pathways links the DR13 and DR11 allele families. To study the effect of microvariation on function, we have generated DRl3-expressing wild type (DRB1*1301-1305) and mutant murine L cell transfectant8. Mutant DR S chain cDNAs with changes at amino acid positions 32, 37, 57 and 86 were created altering amino acids on the floor and ends of the antigen binding groove. The alterations replaced amino acids found in DR(~,S1"1304) with the amino acid found in DR(a,Sl*1301). Cells expressing DRBl*1303 and DRBI*I304 bound HA 307-319 peptlde at a higher level than cells expressing other wt DR13 molecules, a pattern corresponding to the DR13 subgroups designated by structural studies. When DR(,,S1"1304) was mutated to alter position 57, HA binding was decreased 5 fold. Alterations of positions 32 and 37 reduced binding to the level of DR(a,Sl*1301) binding. The effect of DR13 microvariation on T cell recognition was assessed using alloproliferative T cell clones (TLC) generated using a DRB1*1101 responder and a DRB1*1102 stimulator. These TLC recognize cells (B-LCL, PBL, and DR transfectant8) expressing DRB1*1102 as well as cells expressing DR13 allelic products. For example, TLC 2.22 recognizes cells expressing DRBI*I102, "1301, "1302, "1303, and -1304 as well as S chain mutants. The T cell recognition pattern underscores the close relationship among DR13 and DR11 alleles. These studies illustrate the dichotomy present regarding the function of the DR13 molecules demonstrating the unique and shared functions of this complex family.