Analysis of autoantigens in aplastic anemia : identification of an epitope recognized by CD4^+ T cells specific to hematopoietic progenitor cells

为了鉴定再生障碍性贫血（plastic anemia，AA）患者血清IgG所识别的白血病细胞系UT-7中的一个80 kD蛋白质，我们采用免疫沉淀法纯化了该蛋白质，并用质谱指纹法测定了其氨基酸序列。该蛋白质被证明是膜突蛋白。用ELISA和重组膜突蛋白筛选患者血清，43例AA患者中有20例（46.5%）抗膜突蛋白抗体阳性。在阵发性睡眠性血红蛋白尿（PNH）型细胞增多的22例患者中有14例（64%）检测到该抗体，而在18例PNH型细胞未增多的患者中仅3例（17%）检测到该抗体。通过对UT-7 cDNA文库的免疫筛选，确定其为AA的候选自身抗原。在84例（38.1%）AA患者中有32例（38.1%）PNH型细胞增加，可检测到该蛋白的抗体。使用衍生自DRS-1 cDNA的GST融合蛋白片段的表位作图显示，26 mer肽（氨基酸173-198）包含抗体表位的热点，其被显示抗DRS-1抗体的近一半AA患者识别。当使用ELISPOT检测外周血淋巴细胞中是否存在对HLA-DR 15具有高亲和力的DRS-1肽具有特异性的T细胞前体时，两名携带HLA-DR 15和抗DRS-1抗体的AA患者显示出高频率的DRS-1特异性CD 4 ^+ T细胞。通过用DRS-1肽刺激T细胞，从两名AA患者之一产生的DRS-1特异性T细胞显示出对表达HLA-DR 15 ^+和DRS-1的白血病细胞系KH 88的细胞毒性，且具有剂量依赖性。这些发现表明，在具有HLA-DR 15和抗DRS-1抗体的AA患者中，DRS-1特异性T细胞可能有助于骨髓衰竭的发展。

To identify an 80-kD protein derived from a leukemia cell line, UT-7, that was recognized by scrum IgG of a plastic anemia (AA) patients, we purified this protein using immunoprecipitation and determined amino acid sequence with mass fingerprinting. The protein proved to be moesin. When patients' sera were screened using ELISA and recombinant moesin, 20 of 43 (46.5%) AA patients were positive for anti-moesin antibodies. The antibody was detected in 14 of 22 (64%) patients possessing increased paroxysmal nocturnal hemoglobinuria (PNH)-type cells which are known to be a marker for lmmunopathophysiology of AA while it was detected in only 3 of 18 (17%) patients without increased PNH-type cells.We also identified diazepam-binding inhibitor-related sequence-1 (DRS-1), as a candidate autoantigen of AA using immunoscreening of cDNA library derived from UT-7. Antibodies to this protein were detectable in 32 of 84 (38.1%) of AA patients possessing increased PNH-type cells. Epitope mapping using GST-fusion protein fragments derived from DRS-1 cDNA showed that a 26 mer peptide (amino acid 173-198) contained a hot spot of antibody epitopes which was recognized by nearly half of AA patients showing anti-DRS-1 antibodies. When peripheral blood lymphocytes were examined using ELISPOT assay for the presence of T-cell precursors specific to a DRS-1 peptide which has high affinity to HLA-DR15, two AA patients carrying HLA-DR15 and anti-DRS-1 antibodies showed high frequency of DRS-1-specific CD4^+ T cells. DRS-1-specific T cells generated from one of the two AA patients by stimulating T cells with the DRS-1 peptide showed cytotoxicity against an HLA-DR15^+ and DRS-1-expressing leukemia cell line KH88 in a dose-dependent manner. These findings indicate that in AA patients possessing HLA-DR15 and anti-DRS-1 antibodies, DRS-1-specific T cells may contribute to development of bone marrow failure.