同种T细胞前体频率与MHC-抗原肽种类呈正相关

目的用种类数目不同的肽段加载T2（表达空载HLA-A2分子）细胞与HLA-A2阴性个体的PBL混合培养，探讨同种反应性CIL前体的频率与同种抗原表位种类多少的关系。方法通过人工合成HLA-A2限制性单一病毒抗原肽、10种细胞正常成分的肽段以及冻融酸处理法制备细胞混合肽。加载T2细胞，经丝裂霉素灭活后作为刺激细胞，与PKH67预染HLA-A2阴性个体PBL进行混合淋巴细胞培养，PKH67荧光强度随细胞增殖而递减，通过流式细胞仪增殖软件（ModFit）分析同种T细胞前体频率。结果单独PBL增殖不明显；空载T2细胞刺激同种反应性CTL前体的频率为0．052819；加载单一表位肽的T2细胞刺激时，前体的频率为0．030429；10种HLA-A2自身限制性的混合抗原肽负载时，前体的频率为0．144942；混合多肽负载时，前体的频率为0．203649。结论本研究结果显示了同种T细胞前体的频率随着同种抗原表位种类的增多而增高，与同种抗原的密度不相关；支持了强烈的同种反应的主要原因是同种细胞表面表达极其繁多的T细胞识别表位（即pMHC）。

The proliferation magnitude and precursor frequency of alloreactive CTL are proportional to the number of T cell epitope specificities

Objective To find the experimental evidence to show the precursor frequency of alloreactive CTL are proportional to the number of the T-cell epitope specificities. Methods Ten HLA-A2 restricted peptides (all are normal cell components) were synthesized commercially, and cell peptide extract was prepared by freezing, thawing, and acid treating T2 cell. The number of T-cell epitope specificities is manipulated by pulsing different number of HLA-A2 restricted peptide(s) onto the T2 cell. T2 cells loaded with different number of peptide(s) were co-cultured with peripheral blood lymphocytes (PBL) of HLA-A2 negative individual which were stained with PKH67 in advance. The amount of dye in a cell was then partitioned equally between daughter cells during mitosis and therefore decreases by half at each cell division. Then the proliferation was monitored with flow cytometry, and the precursor frequency of the effector cells was analyzed by the ModFit Software. Results After 6 days of culture, no proliferation was observed in the bulk culture of PBL alone, and obvious proliferation took place when PBL of the HLA-A2 negative were co-cultured with T2 cells loaded with or without peptide(s). The precursor frequency of the co-cultured alloreactive CTL was 0.052 819 for T2 cells loaded with nothing; the precursor frequency was 0.030 429 for T2 cells loaded with single peptide, and increased up to 0.144 942 for T2 cells loaded with 10 pep-tides; the precursor frequency was 0.203 649 when co-cultured with T2 cells loaded with peptides extracted from the cytoplasm of T2 cells. Conclusion This study reveals that the proliferation magnitude and precursor frequency of alloreactive CTL are proportional to the number of T-cell epitope specificities, and independent from the density of the allogeneic HLA class I molecule. Our finding supports the hypothesis that the alloreactive T cell populations comprise miscellaneous T cell clones, which are specific to co-responding pMHC. The novel constellation of peptides presented by allogeneic MHC molecules makes thousands of different epitopes, which account for the exceptional high precursor frequency of alloreactive T cells.