| Project/Area Number |
63480170
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Immunology
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| Research Institution | Kyushu University |
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Principal Investigator |
NISHIMURA Yasuharu Department of Genetics, Medical Institute of Bioregulation, Kyushu University, Associate Professor, 生体防御医学研究所, 助教授 (10156119)
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| Co-Investigator(Kenkyū-buntansha) |
KIMURA Akinori Department of Genetics, Medical Institute of Bioregulation, Kyushu University, R, 生体防御医学研究所, 助手 (60161551)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1990: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1988: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | CD5 molecule / T cell activation / Mutant cell / Retroviral vector / Gene transfection / Interleukin 1 / 突然変異細胞 / トランスフェクション / CD5 / CD5欠損突然変異株 / ILー1 / ILー1受容体 / IL-1 / IL-1受容体 / T細胞レセプター / CD3複合体 / Jurkat細胞株 / 欠損変異株 / 組み換えレトロウィルス |
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| Research Abstract |
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
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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
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