原发性胆汁性肝硬化PDC-E2特异性T细胞拮抗性模拟肽的筛选

Screening of analogue peptides antagonizing PDC-E2 specific T cells in patients with primary biliary cirrhosis

目的筛选可拮抗原发性胆汁性肝硬化(PBC)中PDC-E2_(165-174)特异性CTL功能的模拟肽。方法以自身抗原表位PDC-E2_(165-174)(LLAEIETDKA)为原型肽,采用丙氨酸突变扫描法,通过亲和力分析及其对PDC-E2_(165-174)特异性CTL细胞毒性和分泌细胞因子等功能的抑制效应,从中筛选拮抗性模拟肽。结果8条模拟肽中,5号位突变的模拟肽(I5A)显示与HLA-A*0201分子高亲和力,5名PBC患者PDc-E2 特异性CTL对其负载的T2细胞的杀伤率均小于10％,并且I5A也未能诱导PBC患者PDC-E2特异性CTL 产生IFN-γ。在原型肽的存在下,I5A可抑制PDC-E2_(165-174)CTL细胞毒性和分泌IFN-γ,随着模拟肽浓度增加,抑制效应越强,在10 μmol／L浓度时可以明显抑制PDC-E2_(165-174)CTL的细胞毒活性和分泌IFN-γ的能力。结论模拟肽I5A(LLAEAETDKA)可能是PDC-E2_(165-174)特异性CTL的一个拮抗性多肽,其抑制效应并不是其能竞争结合靶细胞上的HLA-A*0201分子,而是对TCR的一种拮抗作用,为将来设计以TCR为基础的特异性免疫治疗提供实验依据。

Objective To screen for analogue peiptie antagonizing functions of PDC-E2165-174 specific CTL in primary biliary cirrhosis（PBC）. Methods Based on the prototype peptide PDC-E2165-174 （LLAEIETDKA）, analogue peptides were designed using alanine scan mutation. HLA affinity and inhibition effect to the functions of PDC-E2165-174 specific CTL were analyzed. Results Among the 8 analogue peptides, the peptide I5A alanine substituted at position 5 showed a high affinity to HLA-A ＊ 0201 and cytotoxicity of PDC-E2165-174 specific CTL induced from 5 PBC patients against ISA loaded T2 cells were less than 10%. The ISA peptide also failed to induce detectable levels of IFN-γsynthesis in the PDC-E2165-174 specific CTL derived from any of the 5 patients. Inhibition effects of I5A on the cytotoxicity and IFN-γ production of PDC-E2165-174 specific CTL were observed when incubated with the prototype peptide. Conclusion Our data indicate that analogue peptide I5A （LLAEAETDKA） can be utilized to manipulate the CD8^＋ T cell responses against PDC-E2165-174 and to provide important clues for TCR-based specific immunotherapy of PBC.