Baixeras E, Huard B, Miossec C, Jitsukawa S, Martin M, Hercend T, Auffray C, Triebel F, Piatier-Tonneau D
Laboratoire d'Hémato-Immunologie, INSERM U333, Institut Gustave-Roussy, Villejuif, France.
J Exp Med. 1992 Aug 1;176(2):327-37. doi: 10.1084/jem.176.2.327.
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.
淋巴细胞激活基因3（LAG-3）在人类活化的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LAG-3编码蛋白的初步特征。在用对应于LAG-3免疫球蛋白样第一结构域中一个暴露的额外环区域的30个氨基酸肽免疫小鼠后，我们产生了两种单克隆抗体。这些试剂的反应性针对LAG-3，因为它们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。在对表达LAG-3的活化淋巴细胞进行的竞争实验中评估，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。细胞分布分析表明，LAG-3.1表位在活化的T细胞（CD4 +和CD8 +亚群）和NK细胞上表达，而不在活化的B细胞或单核细胞上表达。在对活化的T细胞和NK细胞裂解物进行的免疫沉淀实验中，SDS-PAGE分析后检测到一种70-kD的蛋白。还免疫沉淀了45-kD的蛋白种类。70-kD和45-kD的蛋白均显示为N-糖基化。在蛋白质印迹分析中，只有前一种分子被抗LAG-3抗体识别，表明它是LAG-3编码的。这些抗LAG-3抗体用于研究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和定量细胞粘附测定，我们证明转染LAG-3的COS-7细胞与人白细胞抗原（HLA）II类B淋巴细胞之间的玫瑰花结形成特别依赖于LAG-3/HLA II类相互作用。与CD4相反，LAG-3不结合人类免疫缺陷病毒gp120。这一初步特征将指导对该分子功能的进一步研究，该分子可能在由T和NK淋巴细胞介导的免疫反应中起重要作用。
