Isolation of T-cell clones cytotoxic agcirst nemotopoietic progenitor cells from a patienl with aplastic aremia

虽然免疫介导的造血抑制一直被认为是特发性再生障碍性贫血发生的最重要机制，但尚未获得这种免疫攻击的直接证据。为了确定可能导致骨髓衰竭发展的T淋巴细胞，我们从一名未融合的再生障碍性贫血患者的骨髓中建立了T细胞克隆，该患者拥有HLA-DRB1^&lt；**&gt；1501和DRB1^&lt；**&gt；0405。在分离的CD4^+T细胞克隆中，一个Vbeta21 T细胞克隆NT4.2不仅能对照射的自体CD34^+细胞或EBV转化的淋巴母细胞系(LCL)产生干扰素-γ，而且还能裂解具有人类白细胞抗原-DRB1^和同种异体LCL的自体LCL和异体LCL。Vbeta21基因扩增产物的单链构象多态性分析表明，NT4.2是患者骨髓中Vbeta21^+T细胞中最具优势的克隆。加入抗人类白细胞抗原-DR单抗或抗CD3单抗可阻断NT4.2对LCL细胞的杀伤作用。当CD34^+细胞在各种集落刺激因子存在下培养一周后，NT4.2细胞也能裂解自体CD34^+细胞和拥有HLADRB1；**&gt；0405的正常人的同种异体CD34^+细胞。当NT4.2细胞与培养的CD34^+细胞共同孵育时，NT4.2细胞强烈抑制自体造血祖细胞或带有HLA-DRB1^&lt；**&gt；0405的同种异体祖细胞的集落形成。这些发现提示，这种T细胞克隆可能通过识别与人类白细胞抗原DR4结合的造血祖细胞上的一种自体多肽而参与再生障碍性贫血的造血抑制。

Although immune-mediated suppression of hematopoiesis has been considered to be the most important mechanism for the development of idiopathic aplastic anemia, direct evidence for such immunologic attack has not been obtained. To identify T lymphocytes that may contribute to the development of bone marrow failure, we established T-cell clones from the bone marrow of an untrasnfused patient with aplastic anemia, who owned HLA-DRB1^<**>1501 and DRB1^<**>0405. Among CD4^+ T-cell clones isolated, a Vbeta21 T-cell clone, NT4.2, not only secreted interferon-gamma in response to irradiated autologous CD34^+ cells or EBV-transformed lymphoblastoid cell line (LCL) but also lysed the autologous LCL and allogneic LCL owning HLA-DRB1^<**>0405. Single strand conformation polymorphism analysis of amplified Vbeta21 cDNA products revealed that NT4.2 was the most dominant clone among Vbeta21^+ T cells in the bone marrow of the patient. The cytotoxicity against LCL cells by NT4.2 was blocked by the addition of anti HLA-DR monoclonal antibodies (mAb) or anti CD3 mAb into the culture. NT4.2 cells also lysed autologous CD34^+ cells as well as allogeneic CD34^+ cells of a normal individual owining HLA-DRB1^<**>0405 when the CD34^+ cells were cultured in the presence of various colony-stimulaing factors for one week prior to the ^<51>Cr release assay. The NT4.2 cells strongly inhibited colony formation by autologous hematopoietic progenitor cells or by the allogeneic progenitor cells with HLA-DRB1^<**>0405 when they were incubated with the cultured CD34^+ cells prior to plating with the methylcellulose medium. These findings suggest that this T-cell clone may contribute to the suppression of hematopoiesis in aplastic anemia by recognizing a self-peptide on hematopoietic progenitor cells bound to HLA-DR4.