Jacques Y, Le Mauff B, Boeffard F, Godard A, Soulillou J P
Institut National de la Santé et de la Recherche Médicale (INSERM U211), Nantes, France.
J Immunol. 1987 Oct 1;139(7):2308-16.
Several alloreactive human T cell clones derived from a rejected kidney graft were found to produce in their culture supernatants soluble interleukin 2 receptors (IL-2R) upon specific antigenic challenge (irradiated B cell line from the graft's donor). Among them, the 2B11, a high producer clone, was used to purify a soluble IL-2R preparation which was analyzed, in comparison with the high and low affinity cell-surface IL-2R expressed by 2B11 cells, for its parameters of interaction with a set of anti-IL-2R monoclonal antibodies (mAb) and IL-2. This soluble receptor purified by affinity chromatography (anti-IL-2R mAb column) and sodium dodecyl sulfate gel electrophoresis is composed of a single chain of 35,000 to 45,000 Da. Immunoradiometric assays (IRMA) at equilibrium were set up, using pairs of mAb directed against two separate epitopes on the Tac antigen of the human IL-2R, to measure the respective dissociation constant of these mAb for the soluble IL-2R. They were found to be identical to those found on the cell-surface IL-2R. A 1:1 stoichiometry between the two epitopes were found both on the membrane and soluble species. Competition experiments between membrane and soluble IL-2R for binding the mAb allowed the quantitative analysis of the concentration of soluble IL-2R without the need of amino acid analysis on purified material and set up a quantitative IRMA for the human soluble IL-2R (detection limit 5 pM). The affinity of the soluble IL-2R for IL-2 was determined by various techniques including an IRMA using an anti-IL-2R mAb and radiolabeled IL-2. The results obtained led us to conclude that the soluble IL-2R binds IL-2 with a dissociation constant (KD = 30 nM) identical to that found for the binding of IL-2 to low affinity cell-surface IL-2R (Tac antigen). Whereas 2.5% of cell-surface IL-2R expressed 2 days after antigenic stimulation of 2B11 cells were of high affinity for IL-2 (KD = 25 pM), no (less than 0.07%) high affinity binding sites could be detected on the purified soluble IL-2R. This soluble IL-2R therefore likely corresponds to a truncated, extracellular part of the membrane Tac antigen. The amounts of soluble Tac antigen produced by the 2B11 alloreactive human T cell clone did not exceed 1 nM and, as expected from the binding studies, did not affect IL-2-induced T cell proliferation. The physiologic and pathologic implications of our results are discussed.
从一个被排斥的肾移植受者中获得了几个同种异体反应性人类T细胞克隆，发现它们在受到特异性抗原刺激（来自移植供体的经辐照的B细胞系）后，其培养上清液中会产生可溶性白细胞介素2受体（IL-2R）。其中，高产克隆2B11被用于纯化一种可溶性IL-2R制剂，并与2B11细胞表达的高亲和力和低亲和力细胞表面IL-2R进行比较，分析其与一组抗IL-2R单克隆抗体（mAb）和IL-2相互作用的参数。通过亲和层析（抗IL-2R mAb柱）和十二烷基硫酸钠凝胶电泳纯化的这种可溶性受体由一条35,000至45,000 Da的单链组成。建立了平衡免疫放射分析（IRMA），使用针对人IL-2R的Tac抗原上两个不同表位的mAb对，来测量这些mAb与可溶性IL-2R的各自解离常数。发现它们与在细胞表面IL-2R上发现的解离常数相同。在膜结合型和可溶性形式中均发现两个表位之间的化学计量比为1:1。膜结合型和可溶性IL-2R竞争结合mAb的实验使得无需对纯化材料进行氨基酸分析就能对可溶性IL-2R的浓度进行定量分析，并建立了针对人可溶性IL-2R的定量IRMA（检测限为5 pM）。通过包括使用抗IL-2R mAb和放射性标记的IL-2的IRMA在内的各种技术，测定了可溶性IL-2R对IL-2的亲和力。所得结果使我们得出结论，可溶性IL-2R与IL-2结合的解离常数（KD = 30 nM）与IL-2与低亲和力细胞表面IL-2R（Tac抗原）结合的解离常数相同。虽然在2B11细胞抗原刺激后2天表达的细胞表面IL-2R中有2.5%对IL-2具有高亲和力（KD = 25 pM），但在纯化的可溶性IL-2R上未检测到（小于0.07%）高亲和力结合位点。因此，这种可溶性IL-2R可能对应于膜Tac抗原的截短的细胞外部分。2B11同种异体反应性人类T细胞克隆产生的可溶性Tac抗原量不超过1 nM，并且正如结合研究预期的那样，不影响IL-2诱导的T细胞增殖。我们讨论了结果的生理和病理意义。
