Analysis of the Biological Function of CD5 Molecule.

为了明确CD5 （T1, Leu1, Tp67）分子的生物学功能，我们在CD5缺失的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）产生白细胞介素-2 (IL-2)，以响应抗cd3单克隆抗体（MAB）交联到固体载体。与抗cd5单抗OKT1共培养可增强IL-2的产生。用甲磺酸乙酯处理后，用抗CD5 MAb +补体选择，获得了CD5缺陷突变体Jurkat 1.15。Jurkat 1.15在交联抗cd3单抗存在下没有显示OKT1增强IL-2的产生。将编码CD5的cDNA引入到缺陷逆转录病毒中，用于感染Jurkat 1.15。建立了一个稳定表达CD5的Jurkat克隆。在对OKT1的反应中，在亲本Jurkat 9.9和CD5阳性感染者中观察到细胞内钙的升高，但在CD5阴性突变体或G418抗性c中没有观察到。此外，CD5的表达恢复了OKT1对交联抗cd3单抗的Il-2产生的增加。用CD5重组逆转录病毒感染了对HLA-DR抗原产生IL-2反应的小鼠T细胞杂交瘤By155.16，产生了3种稳定的CD5阳性病毒。这些杂交瘤显示，在可溶性抗小鼠CD3单抗存在的次优浓度下，通过OKT1刺激Il-2的产生增强。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细胞对白细胞介素-1 （IL-1）的反应性中的作用。CD5+野生型Jurkat 9.9产生白细胞介素-2 (IL-2)，响应抗cd3单克隆抗体（MAb） OKT3，交联到固体表面。与IL-1或抗cd5单抗共培养可增强IL-2的产生。CD5突变体和CD5- g418耐药的感染者对抗CD5单抗或IL-1均无应答。CD5+感染者细胞表面表达CD5可恢复对IL-1的反应性。CD5+野生型Jurkat和CD5+感染者对纯化IL-1、重组IL-1 α和重组IL-1 β的反应相同。IL-1和抗CD5单抗的最佳浓度对交联抗cd3单抗刺激IL-2产生的增强具有叠加效应，这表明IL-1和CD5通过不同的途径起作用。在这些细胞系中检测了重组il -1 β的特异性结合。重组il -1 β在CD5+感染细胞和CD5+野生型Jurkat细胞中的特异性结合（4 C）和随后的内化（37 C）都是相同的，而在CD5- g418抗性感染细胞中，重组il -1 β的特异性结合（<125 C）明显降低。这些观察结果强烈表明，细胞表面CD5的表达调节了IL-1的结合和反应性。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