Analysis of the Biological Function of CD5 Molecule.

为了确定CD 5（T1，Leu 1，Tp 67）分子的生物学功能，在CD 5缺陷型Jurkat细胞系和鼠T细胞杂交瘤中表达CD 5的cDNA克隆。Jurkat亚克隆（Jurkat 9.9）响应于与固体支持物交联的抗CD 3单克隆抗体（MAB）产生白细胞介素-2（IL-2）。IL-2的产生通过与抗CD 5单克隆抗体OKT 1共培养而增强。通过用甲磺酸乙酯处理，然后用抗CD 5 MAb加补体选择，产生CD 5缺陷型突变克隆Jurkat 1.15。Jurkat 1.15未显示在交联抗CD 3 MAb存在下OKT 1对IL-2产生的增强作用。将编码CD 5的cDNA引入用于感染Jurkat 1.15的缺陷型逆转录病毒中。建立稳定表达CD 5的Jurkat克隆。在对OKT 1的应答中，在亲本Jurkat 9.9和CD 5阳性感染物中均观察到细胞内钙升高，但在CD 5阴性突变体或G418抗性突变体中未观察到。 关于我们 控制。此外，CD 5的表达恢复了OKT 1对交联抗CD 3单抗的IL-2产生的增强作用。用CD 5重组逆转录病毒感染鼠T细胞杂交瘤By155.16（其响应于HLA-DR抗原而产生IL-2），并产生三种稳定的CD 5阳性感染物。这些杂交瘤表现出增强IL-2生产的刺激与OKT 1的存在下，次优浓度的可溶性抗鼠CD 3单克隆抗体。这些结果进一步证明了CD 5为T细胞活化提供了共刺激信号，研究了CD 5表面分子在T细胞对白细胞介素-1（IL-1）的反应性中的作用。CD 5+野生型Jurkat 9.9响应于与固体表面交联的抗CD 3单克隆抗体（MAb）OKT 3产生白细胞介素-2（IL-2）。与IL-1或抗CD 5单抗共培养可增强IL-2的产生。无论是CD 5-突变体还是CD 5- G418-抗性感染者都对抗CD 5单克隆抗体或IL-1没有应答。通过CD 5+感染物中CD 5的细胞表面表达恢复对IL-1的应答。CD 5+野生型Jurkat和CD 5+感染物对纯化的IL-1、重组IL-1 α和重组IL-1 β的应答等同。最佳浓度的IL-1和抗CD 5单克隆抗体对交联抗CD 3单克隆抗体刺激的IL-2产生有相加作用，表明IL-1和CD 5通过不同的途径起作用。在这些细胞系中检测重组IL-1 β的特异性结合。125 I标记的重组IL-1 β的特异性结合（4 ℃）和随后的内化（37 ℃）在CD 5+感染物和CD 5+野生型Jurkat细胞中是相等的，而125 I标记的重组IL-1 β的特异性结合<125>在CD 5- G418抗性感染物中显著降低。这些观察结果强烈表明，细胞表面表达的CD 5调节结合和响应IL-1。少

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