Díaz G, Catálfamo M, Coiras M T, Alvarez A M, Jaraquemada D, Nombela C, Sánchez-Pérez M, Arroyo J
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Tissue Antigens. 1998 Jul;52(1):27-36. doi: 10.1111/j.1399-0039.1998.tb03020.x.
To investigate the contribution to allorecognition of the individual polymorphic positions Glu 69 and Val 36 from the DPB102012 allele, DPB102012 cDNA was subjected to site-directed mutagenesis and alleles expressing Lys at 69 and Ala at 36 were generated. The lymphoblastoid cell line (LCL) 45.EM1, a previously generated mutant B-LCL which expresses normal levels of DPA mRNA but is not able to transcribe DPB, was transfected with wild-type or mutant DPB102012 cDNAs. The ability of two HLA-DPw2 alloreactive CD4+ cytotoxic T-lymphocyte (CTL) clones to lyse the panel of DPB102012 wild-type and site-directed mutant B-cell lines was tested. Both CTL clones (8.3 and 8.9) lysed the B-LCL 45.1, which is haploid for HLA and expresses wild-type DPB1*02012, and transfectants expressing Ala at 36 instead of Val, indicating that this polymorphic residue is not critical for T-cell recognition. However, the change of Glu to Lys at 69 prevented recognition by clones 8.3 and 8.9. These data demonstrate that the residue at peptide-binding position 69 is crucial for T-cell receptor recognition and suggest the requirement for a negatively charged residue at this position for allostimulation of these T-cell clones. The side chain of DPbeta-69 is predicted to point into the peptide-binding groove, and the existence of positive(Lys) or negative (Glu) residues probably leads to substantial differences in the allo- or auto-DP-bound peptides or to differences in the conformation of the peptide-MHC complex, which would therefore be responsible for specific DPw2 allorecognition. The binding of a panel of monomorphic and polymorphic anti-HLA-DP monoclonal antibodies (mAbs) to these transfectants was also tested by flow cytometry. The changes at Glu 69 and Val 36 did not affect recognition by any of the monomorphic antibodies tested. However, the binding pattern of some of the polymorphic mAbs was clearly modified. Therefore, even though it is not crucial for T-cell allorecognition, polymorphic residue 36 must be involved in epitopes recognized by some polymorphic anti-DP antibodies, while residue 69 of the DPB molecule is crucial both for T-cell allorecognition and recognition by some mAbs.
为研究DPB102012等位基因中单个多态性位点Glu 69和Val 36对同种异体识别的贡献，对DPB102012 cDNA进行了定点诱变，生成了在69位表达赖氨酸且在36位表达丙氨酸的等位基因。淋巴母细胞系（LCL）45.EM1是先前生成的突变B-LCL，其表达正常水平的DPA mRNA但无法转录DPB，用野生型或突变型DPB102012 cDNA转染。测试了两个HLA-DPw2同种异体反应性CD4+细胞毒性T淋巴细胞（CTL）克隆裂解DPB102012野生型和定点突变B细胞系的能力。两个CTL克隆（8.3和8.9）均裂解了HLA单倍型且表达野生型DPB1*02012的B-LCL 45.1以及在36位表达丙氨酸而非缬氨酸的转染子，这表明该多态性残基对T细胞识别并不关键。然而，69位的谷氨酸突变为赖氨酸则阻止了克隆8.3和8.9的识别。这些数据表明，肽结合位置69处的残基对T细胞受体识别至关重要，并提示这些T细胞克隆的同种异体刺激需要该位置带有负电荷的残基。预测DPβ-69的侧链指向肽结合槽，正（赖氨酸）或负（谷氨酸）残基的存在可能导致与同种异体或自身DP结合的肽存在实质性差异，或者导致肽-MHC复合物构象的差异，这因此可能是特异性DPw2同种异体识别的原因。还通过流式细胞术测试了一组单态性和多态性抗HLA-DP单克隆抗体（mAb）与这些转染子的结合。Glu 69和Val 36处的变化不影响所测试的任何单态性抗体的识别。然而，一些多态性mAb的结合模式明显改变。因此，尽管多态性残基36对T细胞同种异体识别并不关键，但它必定参与了一些多态性抗DP抗体所识别的表位，而DPB分子的残基69对T细胞同种异体识别和一些mAb的识别均至关重要。
