共刺激分子配体PD-L1、PD-L2的真核表达及其转基因小鼠的制备
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摘要
PD-L1、PD-L2是新发现的B7家族共刺激分子配体,二者具有共同的受体PD-1,并通过与受体PD-1结合抑制T细胞的增殖和过度活化,在细胞免疫应答过程中起负调控作用,同时通过影响细胞因子的分泌对体液免疫也发挥了调控作用。PD-L1、PD-L2/PD-1路径在机体自身免疫耐受和肿瘤细胞逃逸机体免疫监控过程中都发挥着重要作用。
本研究通过RT-PCR的方法成功地从PHA刺激的小鼠外周血单个核细胞和小鼠胸腺组织中克隆了PD-L1和PD-L2基因,并构建了重组真核表达载体pEF6/PD-L1-V5 His和pEF6/PD-L2-V5 His。两个重组质粒转染CHO细胞,经间接免疫荧光实验证明了构建的重组质粒可以CHO细胞中的成功表达PD-L1和PD-L2基因。稳定筛选后,经western-blot检测得到了稳定表达PD-L1和PD-L2的细胞株,为体外研究PD-L1和PD-L2对免疫调控的作用建立了细胞工具。
重组质粒经酶切,显微注射和PCR筛选分别得到了PD-L1的2只Found小鼠和PD-L2的1只Found小鼠,但只有PD-L2Found小鼠可通过生殖器官稳定遗传了PD-L2基因。并且通过southern-blot得到验证。
本研究通过克隆PD-L1和PD-L2基因来构建稳定表达细胞系和转基因小鼠是成功的。将为进一步研究PD-L1和PD-L2对免疫调控的作用提供有用的工具。
PD-Ll and PD-L2 are two co-stimulate molecule ligands found recent years .which belong to B7 family. They share the same receptor PD-1 .By the interaction with PD-1 they affect activated T cells and diminish their proliferation and cytokine production. The engagement of two ligands to the receptor PD-1 result in a negative regulatory effect in T cell response. On the other hand they can also regulate B cell response though the effect on cytokine production. The PD-Ll and PD-L2/PD-1 pathway is not only take an important role in peripheral self tolerance but also found act on the process of tumor cells evading immune systems.In this research we cloned encoding sequence of PD-L1 and PD-L2 by RT-PCR from the total RNA isolated from PHA stimulated mouse peripheral blood mononuclear cells and the total RNA isolated from mouse thymus. After sequencing we found that the sequence of cloned PD-Ll is exactly the same as Genebanks while the sequence of cloned PD-L2 has one mutation. We consult reference and consider it not important.We successfully construct two recombinant eukaryotic expression vectors pEF6/-PD-Ll-V5 His and pEF6/-PD-Ll-V5 His which contain the target genes. Then the recombinant vectors were transfected into CHO cells separately for transient expression and the expression was identified by immunofluorescence technology. We also obtained the stable expression cell lines through the selection of the transfected cells and was identified using western-blot. These suggest that these cell lines, which stably express PD-Ll or PD-L2, can be strongly useful tools to study the immune response regulated by PD-L2 in vitro.We got the DNA fragments for microinjection by restriction endonuclease digestion. Using microinjection into male nuclei two founder transgenic mice of PD-L1 and one of PD-L2 was established after PCR detection. But only PD-L2 founder mouse can passage the transgene to its offspring and was identified by southern-blot.All these results suggest that the construction of stable cell lines and transgenic mouse are successful and will be strongly useful tools to study the function of PD-Ll and PD-L2 in vitro and vivo.
本研究通过RT-PCR的方法成功地从PHA刺激的小鼠外周血单个核细胞和小鼠胸腺组织中克隆了PD-L1和PD-L2基因,并构建了重组真核表达载体pEF6/PD-L1-V5 His和pEF6/PD-L2-V5 His。两个重组质粒转染CHO细胞,经间接免疫荧光实验证明了构建的重组质粒可以CHO细胞中的成功表达PD-L1和PD-L2基因。稳定筛选后,经western-blot检测得到了稳定表达PD-L1和PD-L2的细胞株,为体外研究PD-L1和PD-L2对免疫调控的作用建立了细胞工具。
重组质粒经酶切,显微注射和PCR筛选分别得到了PD-L1的2只Found小鼠和PD-L2的1只Found小鼠,但只有PD-L2Found小鼠可通过生殖器官稳定遗传了PD-L2基因。并且通过southern-blot得到验证。
本研究通过克隆PD-L1和PD-L2基因来构建稳定表达细胞系和转基因小鼠是成功的。将为进一步研究PD-L1和PD-L2对免疫调控的作用提供有用的工具。
PD-Ll and PD-L2 are two co-stimulate molecule ligands found recent years .which belong to B7 family. They share the same receptor PD-1 .By the interaction with PD-1 they affect activated T cells and diminish their proliferation and cytokine production. The engagement of two ligands to the receptor PD-1 result in a negative regulatory effect in T cell response. On the other hand they can also regulate B cell response though the effect on cytokine production. The PD-Ll and PD-L2/PD-1 pathway is not only take an important role in peripheral self tolerance but also found act on the process of tumor cells evading immune systems.In this research we cloned encoding sequence of PD-L1 and PD-L2 by RT-PCR from the total RNA isolated from PHA stimulated mouse peripheral blood mononuclear cells and the total RNA isolated from mouse thymus. After sequencing we found that the sequence of cloned PD-Ll is exactly the same as Genebanks while the sequence of cloned PD-L2 has one mutation. We consult reference and consider it not important.We successfully construct two recombinant eukaryotic expression vectors pEF6/-PD-Ll-V5 His and pEF6/-PD-Ll-V5 His which contain the target genes. Then the recombinant vectors were transfected into CHO cells separately for transient expression and the expression was identified by immunofluorescence technology. We also obtained the stable expression cell lines through the selection of the transfected cells and was identified using western-blot. These suggest that these cell lines, which stably express PD-Ll or PD-L2, can be strongly useful tools to study the immune response regulated by PD-L2 in vitro.We got the DNA fragments for microinjection by restriction endonuclease digestion. Using microinjection into male nuclei two founder transgenic mice of PD-L1 and one of PD-L2 was established after PCR detection. But only PD-L2 founder mouse can passage the transgene to its offspring and was identified by southern-blot.All these results suggest that the construction of stable cell lines and transgenic mouse are successful and will be strongly useful tools to study the function of PD-Ll and PD-L2 in vitro and vivo.
引文
[1 ] Bretscher PA,Coln M.A theory of self and nonself discrimination[J].Science , 1970,169:104221049.
[2 ] Hara TF , Hansen JA. Human T cell activation IIA new activation pathway used by a major T cell population via adisulfide2bonded dimer of a 44 kilo-dation polypeptide (9.3 antigen) [J]J Exp Med, 1985,161:15132
[3 ] Ishida Y, Agata Y, Shibahara K, et al.Induced expression of PD-1 , anovel member of the immunoglobulin gene superfamily,upon programmed cell death[J ] . EMBOJ, 1992 ,11: 388723895.
[4 ] Hiroyuki Nishimura and Tasuku Honjo PD-1: an inhibitory immunoreceptor involved in peripheral tolerance.TRENDS in Immunology Vol.22 No. 5 May 2001
[5 ] Finger LR, Pu JY, Wasserman R, et al. The human PD-1 gene : complete cDNA , genomic organization , and developmentally regulated expression in B cell progenitors[J ] . Gene , 1997, 197: 1772187.
[6 ] Ravetch JV , Lanier LL. Immune inhibitory receptors[J ] . Science , 2000 ,290 : 84289.
[ 7 ] Latchman Y, Wood CR, Tatyana C , et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation[J ]. Nat Immunol, 2001 , 2 (3) : 2612-267.
[8] Sathish JG.Johnson KG .Fuller KJ .et al.Constitutive association of SHP-1 with leukocyte-associated Ig-like receptor-1 in human T cells [J ].J Immunol,2001,166:1763
[ 9 ] Okazaki T,Maeda,Nishimura H, et al. PD-1 immunoreceptor inhibit B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine [J ].Proc Natl Acad Sci USA.2001.98:13866
[ 10 ] Hiroyuki Nishimura, 1 Taku Okazaki, et al. Autoimmune: Dilated Cardiomyopathy in PD-1 Receptor-Deficient Mice [J ]. SCIENCE VOL 291 12 JANUARY 2001
[11] Nishimura H,Nose M,Hiai H, et al.Development of lupusi-like autoimmune diseases by disruption of PD-1 gene encoding an ITIM motif-carrying immunoreceptor [J ]. immunity. 1999,11:141
[12] Dong HD , Zhu GF , Tamada K, et al. B7-H1 , a third member of the B7 family , co-stimulates T-cell proliferation and interleukin210 secretion[J ] .Nat Med, 1999 ,5(12): 136521369.
[ 13] Engin O" zkaynak,2 Liqing Wang, et al. Programmed Death-1 Targeting Can Promote Allograft Survival [J ].The Journal of Immunology 0022-1767/02
[ 14 ] Ishida M, Iwai Y, Tanaka Y, et al. Differential expression of PD-L1 and PD-L2 , ligands for an inhibitory receptor PD-1 , in the cells of lymphohe matopoietic tissues[J ] . Immunol Lett, 2002 , 84 : 57262.
[ 15 ] Eppihimer MJ , Gunn J , Freeman GJ ,et al. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells [J ] . Microcirculation , 2002,9(2): 1332145.
[16] Dong HD , Strome SE , Salomao DR , et al. Tumor2associated B7-H1 promotes T-cell apoptosis : A potential mechanism of immune evasion [J ] .Nat Med, 2002 ,8(8): 7932800.
[17] Yamazaki T, Akiba H, IwaiH, et al. Expression of programmed death I ligands by murine T cels and APC[J]. J Immunol, 2002169(7):5538-5545
[18] Tseng SY, Otsuji M, Gorsaki K, et al. B7-DC , a new dendritic cell molecule with potent costimulatory properties for Tcells[J ] .J Exp Med, 2001 , 193 (7) : 8392845.
[19 ] Petroff MG, Chen L , Phillips TA ,et al. B7 family molecules : novel immunomodulators at the matemal2fetal interface [J ] . Placenta , 2002 , 16 :952101.
[20 ] Chambers CA. The expanding world of co-stimulation : the two-signal model revisited [J ] . Trends Immunol, 2001 , 22 (4): 2172223.
[21] Lanzaveccjia A,Sallusto F,Dynamics of T lymphocyte responses:intermediates effectors,and memory cells [J ] .Science,2000,290:92
[22] Sharpe AH , Freeman GJ . The B7-CD28 Superfamily[J ]. Nat Rev Immunol, 2002 , 2 (2): 1162126.
[23] Freeman GJ , Long AJ , Iwai Y, et al. Engagement of the PD-1 immunoin-hibitory receptor by a novel B7 family member leads to negative regulationof lymphcyte activation[J ] . J Exp Med, 2000 , 192 (7) : 102721034.
[24] Tamura H , Dong HD , Zhu GF ,et al. B7-H1 costimulation preferentiallyenhances CD28-independent T-helper cell function [J ] . Blood, 2001 , 97(6) : 180921816.
[25 ] Fagarasan S , Honjo T. T-indepent immune response : New aspects of B cell biology [J ] . Science , 2001 ,290 : 89292.
[26] 徐宽枫综述 张学光审阅 B7家族新成员PD-L1、PD-L2的研究进展ISSN1007-8738细胞与分子免疫学杂志(Chin J Cell Mol Immunol)2003,19(5)
[27 ] Xingluo Liu, Jian Xin Gao, Jing Wen, Lijie Yin, B7DC/PDL2 Promotes Tumor Immunity by a PD-1-independent Mechanism J. Exp. Med. 0022-1007/2003/06/1721/10 Volume 197, Number 12, June 16, 2003 1721-1730
[28 ] Yvette E. Latchman, Spencer C. Liang, et al. PD-L1-deficient mice show that PD-L1 on T cells,antigen-presenting cells, and host tissues negatively regulates T cells [J ] .PNAS July 20, 2004 vol. 101 no. 29 10691-10696
[29 ] P'ng Loke and James P. Allison PD-L1 and PD-L2 are differentially regulated by Th 1 and Th2 cells [J ] PNAS April 29, 2003 vol. 100 no. 9 5336-5341
[30 ]Shengdian Wang,Jurgen, et al .Molecular Modeling and Functional Mapping of B7-H1 and B7-DC Uncouple Costimulatory Function from PD-1 Interaction. The Journal of Experimental MedicineVolume 197, Number 9, May 5, 2003 1083-1091
[31] Freeman GJ,Long AJ,Iwai Y,et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphcyte activation,[J ] J Exp Med,2000,192:1027.
[32] Tahiro Shin.Gene Kennedy,Kevin Gorski.Haruo Tsuchiya Cooperative B7-l/2(CD80/CD86) and B7-DC Costimulation of CD4~+T Cells Independent of the PD-1 Receptor J. Exp. Med.The Rockefeller University Press-0022-1007/2003/07/31/8 Volume 198, Number 1, July 7, 2003 31-38
[33] Tseng SY.Dustin M. T-cell activation: a multidimensional signaling network [J ] .Curr Biol.2002,14:575
[34] Gabriel L. Sica, In-Hak Choi,et al. B7-H4, a Molecule of the B7 Family ,Negatively Regulates T Cell Immunity [J ] .Immunity, Vol. 18, 849-861, June, 2003.
[35] Durbaka V.R. Chen Dong,et al. B7S1, a Novel B7 Family Member that Negatively Regulates T Cell Activation[J ] .Immunity, Vol. 18, 863-873, June, 2003,
[36] Iwai Y,Okazaki T,Nishimura H, et al.Microanatomical location of PD-1 in human tonsils[J ] .Immunol Lett,2002,83:215.
[2 ] Hara TF , Hansen JA. Human T cell activation IIA new activation pathway used by a major T cell population via adisulfide2bonded dimer of a 44 kilo-dation polypeptide (9.3 antigen) [J]J Exp Med, 1985,161:15132
[3 ] Ishida Y, Agata Y, Shibahara K, et al.Induced expression of PD-1 , anovel member of the immunoglobulin gene superfamily,upon programmed cell death[J ] . EMBOJ, 1992 ,11: 388723895.
[4 ] Hiroyuki Nishimura and Tasuku Honjo PD-1: an inhibitory immunoreceptor involved in peripheral tolerance.TRENDS in Immunology Vol.22 No. 5 May 2001
[5 ] Finger LR, Pu JY, Wasserman R, et al. The human PD-1 gene : complete cDNA , genomic organization , and developmentally regulated expression in B cell progenitors[J ] . Gene , 1997, 197: 1772187.
[6 ] Ravetch JV , Lanier LL. Immune inhibitory receptors[J ] . Science , 2000 ,290 : 84289.
[ 7 ] Latchman Y, Wood CR, Tatyana C , et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation[J ]. Nat Immunol, 2001 , 2 (3) : 2612-267.
[8] Sathish JG.Johnson KG .Fuller KJ .et al.Constitutive association of SHP-1 with leukocyte-associated Ig-like receptor-1 in human T cells [J ].J Immunol,2001,166:1763
[ 9 ] Okazaki T,Maeda,Nishimura H, et al. PD-1 immunoreceptor inhibit B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine [J ].Proc Natl Acad Sci USA.2001.98:13866
[ 10 ] Hiroyuki Nishimura, 1 Taku Okazaki, et al. Autoimmune: Dilated Cardiomyopathy in PD-1 Receptor-Deficient Mice [J ]. SCIENCE VOL 291 12 JANUARY 2001
[11] Nishimura H,Nose M,Hiai H, et al.Development of lupusi-like autoimmune diseases by disruption of PD-1 gene encoding an ITIM motif-carrying immunoreceptor [J ]. immunity. 1999,11:141
[12] Dong HD , Zhu GF , Tamada K, et al. B7-H1 , a third member of the B7 family , co-stimulates T-cell proliferation and interleukin210 secretion[J ] .Nat Med, 1999 ,5(12): 136521369.
[ 13] Engin O" zkaynak,2 Liqing Wang, et al. Programmed Death-1 Targeting Can Promote Allograft Survival [J ].The Journal of Immunology 0022-1767/02
[ 14 ] Ishida M, Iwai Y, Tanaka Y, et al. Differential expression of PD-L1 and PD-L2 , ligands for an inhibitory receptor PD-1 , in the cells of lymphohe matopoietic tissues[J ] . Immunol Lett, 2002 , 84 : 57262.
[ 15 ] Eppihimer MJ , Gunn J , Freeman GJ ,et al. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells [J ] . Microcirculation , 2002,9(2): 1332145.
[16] Dong HD , Strome SE , Salomao DR , et al. Tumor2associated B7-H1 promotes T-cell apoptosis : A potential mechanism of immune evasion [J ] .Nat Med, 2002 ,8(8): 7932800.
[17] Yamazaki T, Akiba H, IwaiH, et al. Expression of programmed death I ligands by murine T cels and APC[J]. J Immunol, 2002169(7):5538-5545
[18] Tseng SY, Otsuji M, Gorsaki K, et al. B7-DC , a new dendritic cell molecule with potent costimulatory properties for Tcells[J ] .J Exp Med, 2001 , 193 (7) : 8392845.
[19 ] Petroff MG, Chen L , Phillips TA ,et al. B7 family molecules : novel immunomodulators at the matemal2fetal interface [J ] . Placenta , 2002 , 16 :952101.
[20 ] Chambers CA. The expanding world of co-stimulation : the two-signal model revisited [J ] . Trends Immunol, 2001 , 22 (4): 2172223.
[21] Lanzaveccjia A,Sallusto F,Dynamics of T lymphocyte responses:intermediates effectors,and memory cells [J ] .Science,2000,290:92
[22] Sharpe AH , Freeman GJ . The B7-CD28 Superfamily[J ]. Nat Rev Immunol, 2002 , 2 (2): 1162126.
[23] Freeman GJ , Long AJ , Iwai Y, et al. Engagement of the PD-1 immunoin-hibitory receptor by a novel B7 family member leads to negative regulationof lymphcyte activation[J ] . J Exp Med, 2000 , 192 (7) : 102721034.
[24] Tamura H , Dong HD , Zhu GF ,et al. B7-H1 costimulation preferentiallyenhances CD28-independent T-helper cell function [J ] . Blood, 2001 , 97(6) : 180921816.
[25 ] Fagarasan S , Honjo T. T-indepent immune response : New aspects of B cell biology [J ] . Science , 2001 ,290 : 89292.
[26] 徐宽枫综述 张学光审阅 B7家族新成员PD-L1、PD-L2的研究进展ISSN1007-8738细胞与分子免疫学杂志(Chin J Cell Mol Immunol)2003,19(5)
[27 ] Xingluo Liu, Jian Xin Gao, Jing Wen, Lijie Yin, B7DC/PDL2 Promotes Tumor Immunity by a PD-1-independent Mechanism J. Exp. Med. 0022-1007/2003/06/1721/10 Volume 197, Number 12, June 16, 2003 1721-1730
[28 ] Yvette E. Latchman, Spencer C. Liang, et al. PD-L1-deficient mice show that PD-L1 on T cells,antigen-presenting cells, and host tissues negatively regulates T cells [J ] .PNAS July 20, 2004 vol. 101 no. 29 10691-10696
[29 ] P'ng Loke and James P. Allison PD-L1 and PD-L2 are differentially regulated by Th 1 and Th2 cells [J ] PNAS April 29, 2003 vol. 100 no. 9 5336-5341
[30 ]Shengdian Wang,Jurgen, et al .Molecular Modeling and Functional Mapping of B7-H1 and B7-DC Uncouple Costimulatory Function from PD-1 Interaction. The Journal of Experimental MedicineVolume 197, Number 9, May 5, 2003 1083-1091
[31] Freeman GJ,Long AJ,Iwai Y,et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphcyte activation,[J ] J Exp Med,2000,192:1027.
[32] Tahiro Shin.Gene Kennedy,Kevin Gorski.Haruo Tsuchiya Cooperative B7-l/2(CD80/CD86) and B7-DC Costimulation of CD4~+T Cells Independent of the PD-1 Receptor J. Exp. Med.The Rockefeller University Press-0022-1007/2003/07/31/8 Volume 198, Number 1, July 7, 2003 31-38
[33] Tseng SY.Dustin M. T-cell activation: a multidimensional signaling network [J ] .Curr Biol.2002,14:575
[34] Gabriel L. Sica, In-Hak Choi,et al. B7-H4, a Molecule of the B7 Family ,Negatively Regulates T Cell Immunity [J ] .Immunity, Vol. 18, 849-861, June, 2003.
[35] Durbaka V.R. Chen Dong,et al. B7S1, a Novel B7 Family Member that Negatively Regulates T Cell Activation[J ] .Immunity, Vol. 18, 863-873, June, 2003,
[36] Iwai Y,Okazaki T,Nishimura H, et al.Microanatomical location of PD-1 in human tonsils[J ] .Immunol Lett,2002,83:215.