Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.
Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.
复制标题
DOI：
10.1084/jem.176.2.327
复制
发表时间：
1992-08-01
期刊：
影响因子：
--
通讯作者：
Piatier-Tonneau D
中科院分区：
文献类型：
--
作者：
Baixeras E;Huard B;Miossec C;Jitsukawa S;Martin M;Hercend T;Auffray C;Triebel F;Piatier-Tonneau D
淋巴细胞激活基因3（LAG - 3）在人类激活的T细胞和自然杀伤（NK）细胞中表达，在基因和蛋白质水平上与CD4密切相关。我们在此报告对LAG - 3编码蛋白的初步特性描述。我们用一个30个氨基酸的肽段免疫小鼠后制备了两种单克隆抗体，该肽段对应于LAG - 3免疫球蛋白样第一结构域中一个暴露的额外环区。这些试剂的反应性针对LAG - 3，因为它们能识别在杆状病毒表达系统中产生的膜表达和可溶性重组LAG - 3分子。这两种抗体可能与LAG - 3第一结构域额外环上暴露的相同或密切相关的表位（称为LAG - 3.1）发生反应，这在对表达LAG - 3的激活淋巴细胞进行的竞争实验中得到了验证。细胞分布分析表明，LAG - 3.1表位在激活的T细胞（包括CD4⁺和CD8⁺亚群）和NK细胞上表达，而不在激活的B细胞或单核细胞上表达。在对激活的T细胞和NK细胞裂解物进行的免疫沉淀实验中，经SDS - PAGE分析检测到一种70 - kD的蛋白质。还免疫沉淀出了45 - kD的蛋白质种类。70 - kD和45 - kD蛋白质都被证明是N - 糖基化的。在蛋白质印迹分析中，只有前者能被抗LAG - 3抗体识别，表明它是由LAG - 3编码的。这些抗LAG - 3抗体被用于研究LAG - 3蛋白是否与CD4配体相互作用。通过使用基于用重组CDM8载体转染COS - 7细胞的高水平表达细胞系统以及一种定量细胞黏附测定法，我们证明了转染LAG - 3的COS - 7细胞与人白细胞抗原（HLA）II类阳性B淋巴细胞之间形成花环特异性地依赖于LAG - 3/HLA II类相互作用。与CD4不同，LAG - 3不与人免疫缺陷病毒gp120结合。这一初步特性描述将指导对该分子功能的进一步研究，该分子可能在T细胞和NK淋巴细胞介导的免疫应答中发挥重要作用。
The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3- expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.