Lamont A G, Powell M F, Colón S M, Miles C, Grey H M, Sette A
Cytel, La Jolla, CA 92037.
J Immunol. 1990 Apr 1;144(7):2493-8.
The identification of a core region for OVA 323-339, which is critical in determining binding to IAd, has enabled us to generate a series of analog peptides in which this core region was extended at both the N and C termini with different amino acid residues. When assessed for binding capacity, several peptides were shown to have increased affinity for IAd compared with the parent sequence, and in addition, some peptides had acquired binding specificities for class II MHC haplotypes not present for OVA 323-339. These peptides were next examined for their ability to inhibit T cell responses in vitro and in vivo. The correlation between binding and the ability to inhibit T cell activation in vitro was good. However, when assessed in vivo, it was clear that high Ia binding was not sufficient in itself to define the inhibitory capacity of a given peptide. That this discrepancy was due to differences in degradation of the core-extended peptides was suggested by 1) results from an inhibition of Ag presentation assay, in which the pulse period with Ag and inhibitor was extended to 20 h; and 2) direct analysis of peptide stability by using reverse phase HPLC. Finally, by protecting the peptide from degradation with N- and C-terminal substitutions of D-amino acids, the inhibitory capacity of an unstable core-extended peptide in vitro could be greatly enhanced. These data indicate that the core extension approach may be one method by which antagonists for MHC class II molecules may be generated.
确定OVA 323 - 339的核心区域（这对于确定与IAd的结合至关重要），使我们能够生成一系列类似肽，其中该核心区域在N端和C端均用不同氨基酸残基进行了延伸。在评估结合能力时，与亲本序列相比，一些肽显示出对IAd的亲和力增加，此外，一些肽获得了对OVA 323 - 339不存在的II类MHC单倍型的结合特异性。接下来检查这些肽在体外和体内抑制T细胞反应的能力。体外结合与抑制T细胞活化能力之间的相关性良好。然而，在体内评估时，很明显高Ia结合本身不足以确定给定肽的抑制能力。1）抗原呈递测定抑制实验结果表明这种差异是由于核心延伸肽降解的差异，在该实验中抗原和抑制剂的脉冲期延长至20小时；2）通过反相HPLC直接分析肽的稳定性。最后，通过用D - 氨基酸进行N端和C端取代来保护肽不被降解，不稳定的核心延伸肽在体外的抑制能力可大大增强。这些数据表明核心延伸方法可能是一种产生II类MHC分子拮抗剂的方法。
