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

为了确定CD4^+ t细胞克隆ZN1的表位，它似乎在再生障碍性贫血的发展中起重要作用，我们试图建立一个系统来筛选能够刺激这种t细胞克隆的肽，使用CLIP-replacement ii链基因表达载体。用猴疱疹病毒感染使ZN1永生化。白细胞介素-3和GM-CSF刺激培养的骨髓单核细胞（BMMCs）诱导ZN1合成DNA。在培养中加入抗HLA-DR、HLA-DR2或HLA-DQ的单克隆抗体（mab），抗dr和抗dr2单克隆抗体均抑制ZN1对培养BMMCs的DNA合成，提示HLA-DR2对**抗原识别有限制。然后，我们用dr2b基因质粒和dr2a链质粒转染COS7细胞。流式细胞术证实了COS7转染后DR2的表达。我们现在正在测试用CLIP-replacement Ii链基因载体转染的HLA-DR2-COS7刺激ZN1对干扰素γ的排泄。本项目利用重组表达克隆技术对患者骨髓单个核细胞的抗原进行血清学鉴定，筛选cDNA文库，鉴定AA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^<1-101>）的IgG抗体。25例AA患者中有21例（84.0%）采用重组α珠蛋白^<1-101>进行免疫印迹检测。当患者的淋巴细胞受到α珠蛋白^<1-11>的刺激时，产生了低百分比的CD8* T细胞对该肽的反应。培养的T细胞对用该肽脉冲的HLA-A*0201^+JY细胞表现出细胞毒性。α珠蛋白^<1-11>刺激的T细胞加入自体CD34^+细胞后，BFU-E和CFU-GM的菌落数量减少到对照组的近一半，α珠蛋白可能是HLA-A-*0201 AA患者免疫系统攻击的靶标。

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.