Brief Definitive Report Identification of a T3/t Cell Receptor Complex in Chickens-猫眼课题宝

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Chen-lo H. Chen-Chen-lo-H.-Chen-2109482134;L. Lanier Ager;G. Larry Gartland;M D Cooper

与人 T 细胞表面抗原受体分子密切相关的是由 Mr 为 25,000（3' 链）和 20,000（~ 链）的两种糖蛋白和 Mr 20,000（E 链）的非糖基化蛋白组成的 T3 复合物 (1, 2)。鼠类单克隆抗体可以识别人类 T3 分子。抗 T3 抗体共沉淀 TCR 的 a 和/3 链的能力表明了 T3 分子与 T 细胞受体 (TCR) 的物理关联 (3, 4)。针对 T3 或 TCR 的 mAb 会诱导相同的 T 细胞激活信号 (5, 6)。由于 T3/TCR 复合物在人类 T 细胞功能中的重要性，我们预计 T3/TCR 复合物将被证明在系统发育上是保守的。最近 (7-9)，通过与 TCR 和/3 链的共沉淀，鉴定了一种鼠 T3 候选物。在这里，我们报告了表明鸟类中存在功能性 T3/TCR 复合物的证据。材料和方法淋巴细胞分离和免疫荧光。近亲繁殖的白来亨鸡、鸡蛋和年轻的印第安纳巨型鹌鹑均来自当地。从血液、胸腺、法氏囊、脾脏和骨髓中分离单核细胞以及免疫荧光染色如所述(10, 11)。通过荧光显微镜或自动流式细胞术分析染色细胞的频率。将染色细胞在 37°C 下孵育 30 小时后，评估免疫荧光染色后淋巴细胞表面分子的加帽情况。 mAb CT-3的制备。 BALB/C 小鼠通过皮下和足垫注射鸡胸腺细胞和 Ig 血单核细胞进行免疫，其淋巴结细胞与 P3-X63Ag8.653 骨髓瘤细胞融合 (11)。由此产生的杂交瘤产生了 IgG1 抗体 CT-3，显示出与血液淋巴细胞亚群的免疫荧光反应性。将杂交瘤亚克隆，在腹水中生长，并在 Protein A-Sepharose 4 B 柱上纯化 CT-3 抗体。对于有丝分裂研究，将纯化的 CT-3 抗体与 Sepharose 4 B 珠缀合，并计算与珠结合的 CT-3 的准确量。免疫沉淀和凝胶电泳。使用乳过氧化物酶方法用 NalZSl 对血液单核细胞进行表面标记，并在 1% NP-40 或 1% 洋地黄皂苷加 0.2% NP-40 的 0.05 Tris（pH 7.5）溶液（含有蛋白酶抑制剂）中于 4°C 裂解 30 分钟（9, 10）。免疫分离是在微量滴定板中通过固体

Closely associated with the antigen receptor molecule on the surface of human T cells is the T3 complex composed of two glycoproteins with Mr of 25,000 (3' chain) and 20,000 (~ chain), and a nonglycosylated protein of Mr 20,000 (E chain) (1, 2). Murine mAb have been made that recognize the human T3 molecule. Physical association of the T3 molecule with the T cell receptor (TCR) has been suggested by the ability of anti-T3 antibodies to coprecipitate a and/3 chains of the TCR (3, 4). mAb directed against either T3 or the TCR induce the same T cell activation signal (5, 6). Because of its importance in human T cell function, we anticipated that the T3/TCR complex would prove to be phylogenetically conserved. Recently (7-9), a murine T3 candidate has been identified by its coprecipitation with TCR and/3 chains. Here we report evidence indicating the existence of a functional T3/TCR complex in birds. Materials and Methods Lymphocyte Isolation and Immunofluorescence. Outbred white leghorn chickens, eggs, and young Indiana giant quails were obtained from local sources. Isolation of mononuclear cells from blood, thymus, bursa, spleen, and bone marrow, and immunofluorescent staining were as described (10, 11). The frequency of stained cells was analyzed by fluorescence microscopy or automated flow cytometry. Capping of lymphocyte surface molecules after immunofluorescent staining was evaluated after incubation of stained cells for 30 rain at 37°C. Preparation ofmAb CT-3. BALB/C mice were immunized by subcutaneous and footpad injections with chicken thymocytes and Ig-blood mononuclear cells, and their lymph node ceils were fused with P3-X63Ag8.653 myeloma cells (11). One resultant hybridoma produced an IgG1, antibody, CT-3, showing immunofluorescence reactivity with a sub-population of blood lymphocytes. The hybridoma was subcloned, grown in ascites, and the CT-3 antibody was purified on a protein A-Sepharose 4 B column. For mitogenicity studies, the purified CT-3 antibody was conjugated to Sepharose 4 B beads, and the exact amount of CT-3 bound to the beads calculated. Immunoprecipitation and Gel Electrophoresis. Blood mononuclear ceils were surface-labeled with NalZSl using the lactoperoxidase method, and lysed in either 1% NP-40 or 1% digitonin plus 0.2% NP-40 in 0.05 Tris, pH 7.5, containing protease inhibitors, at 4°C for 30 min (9, 10). Immunoisolation was performed in a microtiter plate by a solid

Brief Definitive Report Identification of a T3/t Cell Receptor Complex in Chickens

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