Analysis of the Biological Function of CD5 Molecule.

为了确定 CD5（T1、Leu1、Tp67）分子的生物学功能，在 CD5 缺陷型 Jurkat 细胞系和鼠 T 细胞杂交瘤中表达了 CD5 的 cDNA 克隆。 Jurkat 亚克隆 (Jurkat 9.9) 响应与固体支持物交联的抗 CD3 单克隆抗体 (MAB)，产生白细胞介素 2 (IL-2)。通过与抗 CD5 MAb OKT1 共培养增强了 IL-2 的产生。 CD5 缺陷突变体克隆 Jurkat 1.15 通过用甲磺酸乙酯处理然后用抗 CD5 MAb 加补体进行选择而产生。 Jurkat 1.15 没有表现出在交联抗 CD3 MAb 存在的情况下 OKT1 增强 IL-2 的产生。将编码 CD5 的 cDNA 引入有缺陷的逆转录病毒中，该逆转录病毒用于感染 Jurkat 1.15。建立了稳定表达CD5的Jurkat克隆。作为对 OKT1 的反应，在亲本 Jurkat 9.9 和 CD5 阳性感染体中观察到细胞内钙增加，但在 CD5 阴性突变体或 G418 抗性对照中没有观察到。此外，CD5 的表达恢复了 OKT1 响应交联抗 CD3 MAb 产生的 IL-2 的增加。响应HLA-DR抗原产生IL-2的鼠T细胞杂交瘤By155.16也用CD5重组逆转录病毒感染，并产生了三个稳定的CD5阳性感染者。在可溶性抗鼠 CD3 MAb 浓度次佳的情况下，这些杂交瘤显示出通过 OKT1 刺激可增强 IL-2 的产生。这些结果进一步证明CD5为T细胞激活提供了共刺激信号。研究了CD5表面分子在T细胞对白介素-1 (IL-1)反应中的作用。 CD5 野生型 Jurkat 9.9 响应与固体表面交联的抗 CD3 单克隆抗体 (MAb) OKT3，产生白介素 2 (IL-2)。通过与 IL-1 或抗 CD5 MAb 共培养可增强 IL-2 的产生。 CD5-突变体和CD5-G418抗性感染者均不响应抗CD5 MAb或IL-1。 CD5 感染者的细胞表面 CD5 表达恢复了对 IL-1 的反应性。 CD5 野生型 Jurkat 和 CD5 感染者的反应与纯化的 IL-1、重组 IL-1α 和重组 IL-1β 相当。 IL-1 和抗 CD5 MAb 的最佳浓度对交联抗 CD3 MAb 刺激的 IL-2 产生的增强具有累加效应，表明 IL-1 和 CD5 通过不同的途径发挥作用。在这些细胞系中检查了重组 IL-1β 的特异性结合。 125I 标记的重组 IL-1β 的特异性结合（4°C）和随后的内化（37°C）在 CD5 感染者和 CD5 野生型 Jurkat 细胞中是相同的，而 125I 标记的重组 I1-1β 的特异性结合在 CD5-G418 抗性感染者中显着降低。这些观察结果强烈表明 CD5 的细胞表面表达调节 IL-1 的结合和反应。较少的

In order to define biological function of the CD5 (T1, Leu1, Tp67) molecule, a cDNA clone of CD5 was expressed in a CD5 deficient Jurkat cell line and in a murine T cell hybridoma. A Jurkat subclone (Jurkat 9.9) produced interleukin-2 (IL-2) in response to anti-CD3 monoclonal antibody (MAB) crosslinked to solid support. IL-2 production was enhanced by co-culture with the anti-CD5 MAb OKT1. A CD5 deficient mutant clone Jurkat 1.15 was generated by treatment with ethyl methanesulfonate followed by selection with anti-CD5 MAb plus complement. Jurkat 1.15 did not demonstrate enhancement of IL-2 production by OKT1 in the presence of crosslinked anti-CD3 MAb. A cDNA encoding CD5 was introduced into a defective retrovirus which was used to infect Jurkat 1.15. A Jurkat clone stably expressing CD5 was established. In response to OKT1, a rise in intracellular calcium was observed in both the parent Jurkat 9.9 and the CD5 positive infectant but not in the CD5 negative mutant or a G418 resistant c … More ontrol. Furthermore, expression of CD5 restored the augmentation of Il-2 production by OKT1 in response to crosslinked anti-CD3 MAb. A murine T cell hybridoma By155.16 which produces IL-2 in response to HLA-DR antigens was also infected with the CD5 recombinant retrovirus and three stable CD5 positive infectants were generated. These hybridomas showed enhancement of Il-2 production by stimulation with OKT1 in the presence of suboptimal concetrations of soluble anti-murine CD3 MAb. These results provide further evidence that CD5 provides a costimulatory signal for T cell activation.The role of the CD5 surface molecule in T cell responsiveness to interleukin-1 (IL-1) was examined. The CD5+ wild type Jurkat 9.9 produced interleukin-2 (IL-2) in response to anti-CD3 monoclonal antibocy (MAb), OKT3, crosslinked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 MAb. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 MAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalent to purified IL-1, recomvinant IL-1alpha and recobinant IL-1beta. Optimal concentrations of IL-1and anti-CD5 MAb had an additive effect upon the enhancement of IL-2 production stimulated with crosslinked anti-CD3 MAb suggesting that IL-1 and CD5 act through distinct pathways. The specific binding of recombinant IL-1beta was examined in these cell lines. Both the specific binding (at 4ﾟC) and subsequent internalization (at 37ﾟC) of 125I labeled recombinant IL-1beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of ^<125>I labeled recombinant I1-1beta was markedly decreased in the CD5- G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1. Less