任意のT細胞エピトープ同定法の開発

1) MHC类分子的定位扫描。利用9聚肽库分析了Kb、Db和Ld的肽结合特异性。文库的基本设计是Oxxxxxxxx，例如，为了评估一个氨基酸在位置上的相对适合度，o.x表示19个氨基酸和Cys相等的位置。通过稳定在TAP缺陷细胞系上表达的空MHC类分子来定量肽的结合。不同氨基酸在MHC结合中的能量优势和劣势在先前通过池测序确定为主要锚点的位置上最为突出。位置4、6、7和8的选择性最低。2) T细胞表位预测。假设每个氨基酸的能量贡献在整个肽的结合中是相加的，根据位置扫描数据设计了一个评分系统。大多数已知的T细胞表位肽在亲本蛋白中得分最高的9-mers中得分为1-5。3)预测与实际结合的相关性。合成任意选择的肽，并将其mhc结合能力与预测的结合分数进行比较。观察到这些值之间存在正相关关系，但也存在较大波动，表明位置扫描的预测能力有限。4)预测不同MHC分子间MHC结合肽的库重叠。利用上述评分系统，预测了三种小鼠MHC异型的MHC结合肽。建议他们的曲目有相对较小的重叠。

1) Positional scanning of MHC calss Imolecules.Peptide binding specificities of Kb, Db and Ld were analyzed with 9-mer peptide libraries. The basic design of the libraries is Oxxxxxxxx, for instance, to evaluate the relative fitness of an amino acid at position, O.xdesignates the positions where 19 am ino acids but Cys are equally represented. Binding of peptides was quantitated by stabilization of empty MHC class Imolecules expressed on TAP deficient cell lines. Energetic advantages and disadvantages among different amino acids in MHC binding are most prominent at the positions that have been previously identified as major anchors by pool sequencing. Positions 4,6,7 and 8 exhibited the least selectivity.2) Prediction of T cell epitopes.A scoring system was devised from the positional scanning data by assuming that the energetic contribution of each amino acid can be additive in binding of the whole peptide. Most of known T cell epitope peptides are scored 1-5th among the highest scoring 9-mers present in parental proteins.3) Correlation of predicted vs actual binding.Arbitorarily chosen peptides were synthesized and their MHCbinding capacities were compared with the predicted binding scores. A positive correlation between these values were observed, but there were also substantial fluctuations, indicating a limit in predicting power by the positional scannning.4) Prediction of repertoire overlap of MHC binding peptides among different MHC molecules.By using the above mentioned scoring system, MHC binding peptides were predicted for thres murine MHC allomorphs. Their repertoiree are suggested to share relatively small overlaps.