O'Brien R M, Cram D S, Coppel R L, Harrison L C
Burnet Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research, Australia.
J Autoimmun. 1990 Dec;3(6):747-57. doi: 10.1016/s0896-8411(05)80041-1.
High-titre IgG antibodies against the immunodominant 70-kDa protein of the (U1)ribonucleoprotein (RNP) complex are present in virtually 100% of patients with mixed connective tissue disease (MCTD), and less commonly in a variety of other autoimmune rheumatic diseases. As T-cell 'help' is assumed to be required for this potentially pathogenic form of immune response, investigations to define T-cell epitopes on the 70-kDa protein were undertaken. In prior studies we expressed the 70-kDa protein and a number of its fragments, spanning most of the molecule, as recombinant fusion proteins using the pGEX expression-vector system. These fusion proteins were used as antigens in the epitope mapping studies reported here. PBMC were isolated from patients with (U1)RNP-positive rheumatic diseases and from both normal controls and rheumatologic patients with other autoantibody reactivities, including those to Ro, La and dsDNA. Reactivity to the purified 70-kDa protein was assayed by thymidine incorporation and was evident only in anti-(U1)RNP positive patients but was not restricted to MCTD patients, being present also in patients with SLE and rheumatoid arthritis. The stimulation indices (SIs) observed were in the two- to five-fold range. Using the 70-kDa protein fragments, a T-cell stimulatory epitope was localized to the C-terminal 63 amino acids of the autoantigen. A T-cell line, derived from PBMC of a (U1)RNP positive patient with MCTD, also reacted predominantly with this C-terminal fragment but with an SI of approximately 15-fold. Thus, we have demonstrated the presence and specificity of autoreactive T lymphocytes to a defined peptide epitope in systemic rheumatic disease.
针对（U1）核糖核蛋白（RNP）复合物中免疫显性70kDa蛋白的高滴度IgG抗体几乎在100%的混合性结缔组织病（MCTD）患者中存在，而在其他多种自身免疫性风湿性疾病中较少见。由于这种潜在致病性免疫反应形式被认为需要T细胞“辅助”，因此开展了确定70kDa蛋白上T细胞表位的研究。在先前的研究中，我们使用pGEX表达载体系统表达了70kDa蛋白及其许多片段，这些片段覆盖了该分子的大部分区域，作为重组融合蛋白。这些融合蛋白在此处报道的表位作图研究中用作抗原。从（U1）RNP阳性风湿性疾病患者以及正常对照和具有其他自身抗体反应性的风湿性患者（包括那些针对Ro、La和双链DNA的患者）中分离外周血单个核细胞（PBMC）。通过掺入胸苷来检测对纯化的70kDa蛋白的反应性，仅在抗（U1）RNP阳性患者中明显，但不限于MCTD患者，也存在于系统性红斑狼疮（SLE）和类风湿关节炎患者中。观察到的刺激指数（SI）在2至5倍范围内。使用70kDa蛋白片段，一个T细胞刺激表位定位于自身抗原的C末端63个氨基酸。从一名MCTD的（U1）RNP阳性患者的PBMC中获得的一个T细胞系也主要与这个C末端片段反应，但刺激指数约为15倍。因此，我们已经证明了系统性风湿性疾病中自身反应性T淋巴细胞对一个确定的肽表位的存在和特异性。
