Analysis of the Biological Function of CD5 Molecule.

为了确定CD5(T1，Leu1，Tp67)分子的生物学功能，我们在CD5缺陷的Jurkat细胞系和小鼠T细胞杂交瘤中表达了CD5的cDNA克隆。Jurkat亚克隆(Jurkat 9.9)可与固相载体上的抗CD3单抗(MAB)反应产生白介素2(IL-2)。与抗CD5单抗OKT1共培养可促进IL-2的产生。用甲烷磺酸乙酯处理后，用抗CD5单抗加补体筛选，获得CD5缺失突变克隆Jurkat 1.15。在交联型抗CD3单抗存在下，Jurkat 1.15不显示OKT1对IL-2产生的促进作用。将编码CD5的cDNA导入缺陷型逆转录病毒中，用于感染Jurkat 1.15。建立了稳定表达CD5的Jurkat克隆。对okt1的反应中，亲本Jurkat9.9和cd5阳性感染株细胞内钙离子均升高，而cd5阴性突变体和耐g418基因的c-…则没有。更有控制力。此外，CD5的表达恢复了OKT1对交联型抗CD3单抗刺激的IL-2产生的促进作用。用CD5重组逆转录病毒感染小鼠T细胞杂交瘤BY155.16，获得3株稳定的CD5阳性感染株。OKT1刺激这些杂交瘤细胞产生IL-2，并加入不适宜浓度的可溶性抗鼠CD3单抗。这些结果进一步证明CD5为T细胞活化提供了共刺激信号。研究了CD5表面分子在T细胞对白介素1(IL-1)应答中的作用。CD5野生型Jurkat 9.9在抗CD3单抗OKT3的作用下产生白细胞介素2(IL-2)。IL-1或抗CD5单抗联合培养可促进IL-2的产生。CD5突变株和CD5-G418耐药株对抗CD5单抗和IL-1均无反应。CD5感染细胞表面CD5的表达恢复了对IL-1的反应性。CD5野生型Jurkat和CD5感染者的应答均相当于纯化的IL-1、重组IL-1α和重组IL-1β。IL-1和抗CD5单抗的最佳浓度对交联型抗CD3单抗刺激的IL-2产生有相加作用，提示IL-1和CD5通过不同的途径发挥作用。在这些细胞系中检测到重组IL-1β的特异性结合。在CD5感染者和CD5野生型Jurkat细胞中，125I标记的重组IL-1β的特异性结合(在4ﾟC)和随后的内化(在37ﾟC)都是相同的，而在CD5-G418抗性的感染者中，标记的重组IL-1β的特异性结合显著降低。这些观察结果强烈表明，细胞表面CD5的表达调节着IL-1的结合和反应。较少

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