Analysis of autoantigens in immune-mediated aplastic anemia-identification of an epitope of a CD4^+ T-cell clone

为了确定CD 4 ^+ T细胞克隆ZN 1的表位，该表位似乎在再生障碍性贫血的发展中具有重要作用，我们试图建立一个系统，使用CLIP替代II链基因表达载体筛选能够刺激该T细胞克隆的肽。ZN 1是永生化使用疱疹病毒saimiri感染。在IL-3和GM-CSF存在下用培养的骨髓单个核细胞（BMMC）刺激ZN 1诱导DNA合成。当将抗HLA-DR、HLA-DR 2或HLA-DQ的单克隆抗体（mAb）加入培养物中时，抗DR和抗DR 2 mAb均抑制ZN 1响应于培养的BMMC的DNA合成，表明HLA-DR 2限制 ** 抗原识别。然后，我们用DR 2 B基因质粒和DR a链质粒转染COS 7细胞。流式细胞术证实C 0 S7转染子表达DR 2。本研究利用CLIP置换Ii链基因载体转染HLA-DR 2-COS 7，通过筛选AA患者骨髓单个核细胞的cDNA文库，利用重组表达克隆进行抗原血清学鉴定，以鉴定AA患者的自身抗原。在患者血清中检测到识别α珠蛋白（残基1-101，α珠蛋白^）的IgG抗体<1-101>。用重组α珠蛋白免疫印迹法<1-101>检测25例AA患者中21例（84.0%）检出特异性抗体。当患者的淋巴细胞用α珠蛋白刺激时<1-11>，产生了低百分比的对该肽反应的CD 8 * T细胞。培养的T细胞显示出对用该肽脉冲的HLA-A*0201^+JY细胞的细胞毒性。将α珠蛋白刺激的T细胞加入<1-11>自体CD 34 ^+细胞中，可使BFU-E和CFU-GM的集落数减少到对照的近一半。α珠蛋白可能是HLA-A-*0201型再障患者免疫系统攻击的靶点。

To determine an epitope of a CD4^+ T-cell clone, ZN1, that appears to have an essential role in the development of aplastic anemia, we attempted to establish a system to screen a peptide capable of stimulating this T-cell clone using CLIP-replacement Ii-chain gene expression vectors. ZN1 was immortalized using infection with Herpesvirus saimiri. Stimulation with cultured bone marrow mononuclear cells (BMMCs) in the presence of IL-3 and GM-CSF induced DNA synthesis by ZN1. When monoclonal antibodies (mAbs) against HLA-DR, HLA-DR2 or HLA-DQ were added to the culture, both anti-DR and anti-DR2 mAbs inhibited DNA synthesis by ZN1 in response to cultured BMMCs, suggesting restriction ** antigen recognition by HLA-DR2. Then, we transfected COS7 cells with a DR2 b gene plasmid and a DR a chain plasmid. Flow cytometry confirmed expression of DR2 by the COS7 transfectant. We are now testing interferon-γ excretion by ZN1 stimulated by the HLA-DR2-COS7 that was transfected with CLIP-replacement Ii chain gene vectors.In relation to this project, we screened cDNA library derived from the patient's bone marrow mononuclear cells using serological identification of antigens by recombinant expression cloning to identify autoantigens in AA. IgG antibodies recognizing α globin (residue 1-101, α globin^<1-101>) was detected in the patient's serum. Immunoblotting using recombinant α globin^<1-101> detected the specific antibodies in 21 of 25 (84.0%) AA patients. When the patient's lymphocytes were stimulated with α globin^<1-11>, a low percentage of CD8* T cells reactive to this peptide were generated. The cultured T cells showed cytotoxicity against HLA-A*0201^+JY cells that were pulsed with this peptide. Addition of T cells stimulated by α globin^<1-11> to autologous CD34^+ cells reduced the number of colonies derived from BFU-E and CFU-GM to nearly a half of the control, α globin may serve as a target of immune system attack in AA patients possessing HLA-A-*0201.