An Improved Retroviral Gene Transfer Technique Demonstrates Inhibition of CD4− CD8− Thymocyte Development by Kinase-Inactive ZAP-70 This information is current as of September 23, 2021. Takehiko Sugawara, Vincenzo Di Bartolo, Tadaaki Miyazaki, Hiromitsu Nakauchi, Oreste Acuto and Yousuke Takahama J Immunol 1998; 161:2888-2894; ; http://www.jimmunol.org/content/161/6/2888 Downloaded from References This article cites 49 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/161/6/2888.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. An Improved Retroviral Gene Transfer Technique Demonstrates Inhibition of CD42CD82 Thymocyte Development by Kinase-Inactive ZAP-701 Takehiko Sugawara,* Vincenzo Di Bartolo,§ Tadaaki Miyazaki,‡ Hiromitsu Nakauchi,* Oreste Acuto,§ and Yousuke Takahama2*† ZAP-70 is a Syk family tyrosine kinase that plays an essential role in initiating TCR signals. Deficiency in ZAP-70 causes a defect in the development at CD41CD81 thymocytes due to defective TCR-mediated positive and negative selection. Using a newly devised retrovirus gene transfer and an efficient green fluorescence protein detection technique in fetal thymus organ cultures, the present study shows that forced expression in developing thymocytes of a catalytically inactive mutant of ZAP-70, but not 2 2 wild-type ZAP-70, inhibits T cell development at the earlier CD4 CD8 stage. The ZAP-70 mutant blocked the generation of Downloaded from CD41CD81 thymocytes even in the absence of endogenous ZAP-70. Thus, the present results demonstrate a novel technique for gene transfer into developing T cells and suggest that ZAP-70/Syk family tyrosine kinases are involved in the signals inducing the generation of CD41CD81 thymocytes. The Journal of Immunology, 1998, 161: 2888–2894. ntroducing a given gene into developing thymocytes is a was useful, as gene-transferred cells could be readily detected and powerful technique for analyzing molecular mechanisms reg- sorted using flow cytometry. Immature thymocytes were success- http://www.jimmunol.org/ I ulating T cell differentiation. Transgenic expression of a gene fully infected with these retroviruses in suspension culture in the under control of a T cell-specific promoter/enhancer has been presence of IL-7 and were examined for their developmental ca- widely used for gene manipulation of thymocytes (1, 2). Recently, pability by transferring to the thymus organ culture. Using this retroviral gene transfer has been used successfully for a wide va- retroviral gene transfer technique, the present study shows that riety of cells including hemopoietic cells (3–8) and developing B forced expression in developing thymocytes of a kinase-inactive lymphocytes (9, 10). Retrovirus-mediated gene transfer has several mutant of ZAP-70 inhibits T cell development at the immature advantages over the transgenic techniques, including rapid and CD42CD82 thymocytes, suggesting that ZAP-70/Syk family ty- close analysis of specific cellular events in vitro and its potential rosine kinases are involved in the signals that induce the genera- application for gene therapy. However, retrovirus-mediated gene tion of CD41CD81 thymocytes. by guest on September 23, 2021 transfer often suffers from technical difficulties, such as low effi- ciency, which hamper applications in various cell types. Conse- quently, attempts to introduce exogenous genes using retroviruses Materials and Methods have had limited success in developing T lymphocytes (11–17). Retrovirus constructs and virus-producing cells The present study reports an effective technique for retroviral gene transfer into developing T cells in fetal thymus organ cultures The S65T mutant of GFP (Clontech, Palo Alto, CA) was cloned into either the BclI site of pGD9 (6) or HpaI site of pMSCV (18). Purified and for the sensitive detection of gene-transferred cells. We con- plasmids were transfected into GP1E-86-packaging cells (19). G418- structed recombinant retroviruses expressing green fluorescence resistant cells were clone sorted for GFPhigh clones using a protein (GFP)3 along with a protein of interest, using the internal FACSVantage cell sorter (Becton Dickinson, San Jose, CA). Graded ribosomal entry site (IRES) sequence. The coexpression of GFP dilutions of filtered supernatants from the selected clones were mea- sured for virus titers, using G418-resistance of NIH-3T3 cells. NIH-3T3 cells were cultured with the supernatants for 1 day, then were assayed for G418 resistance. Clones producing more than 106 CFU/ml were *Department of Immunology and †PRESTO Research Project, Institute of Basic Med- selected for subsequent experiments. ical Sciences, University of Tsukuba, Tsukuba, Japan; ‡Department of Immunology, GFP-S65T attached downstream of the IRES sequence from encepha- Faculty of Medicine, University of Tokyo, Tokyo, Japan; and §Laboratory of Molec- lomyocarditis virus (20) was cloned into the pGD9 vector. Either wild-type ular Immunology, Department of Immunology, Institut Pasteur, Paris, France human ZAP-70 or its catalytically inactive (kinase-dead) mutant (KD- Received for publication April 3, 1998. Accepted for publication May 8, 1998. ZAP; D461N), tagged with the VSV-G sequence (21), was cloned into the 9 The costs of publication of this article were defrayed in part by the payment of page XhoI site of pGD -ires-GFP vector. Resulting retrovirus vectors were trans- charges. This article must therefore be hereby marked advertisement in accordance fected into GP1E-86 cells, and virus producing cells were cloned as de- with 18 U.S.C. Section 1734 solely to indicate this fact. scribed above. For pGD9-KDZAP-ires-GFP, virus-producing clones of 106 1 cfu/ml were selected and were used for subsequent experiments. For pGD9- This work was supported by the University of Tsukuba Research Projects, PRESTO 5 Research Project “Unit Process and Combined Circuit,” and the Ministry of Educa- ZAPwt-ires-GFP, virus-producing cells of more than 10 CFU/ml were not tion, Science, Sports, and Culture of Japan. generated. Consequently, to compare the effects of ZAPwt-producing virus and KD-ZAP-producing virus, pGD9-based plasmids were transiently 2 Address correspondence and reprint requests to Dr. Y. Takahama, Department of Immunology and PRESTO Research Project, Institute of Basic Medical Sciences, transfected into BOSC23 packaging cells (22) obtained from American University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8577, Japan. E-mail address: Type Culture Collection (Manassas, VA). Two days after the transfection, 1 [email protected] BOSC23 cells were sorted for GFP cells to enrich transfected cells. These 1 3 Abbreviations used in this paper: GFP, green fluorescence protein; IRES, internal GFP BOSC23 cells were used for virus-producing cells to infect fetal ribosomal entry site; KD-ZAP, kinase-dead mutant of ZAP-70; FTOC, fetal thymus thymocytes. organ culture; dGuo, 2-deoxyguanosine; ITAM, immunoreceptor tyrosine-based ac- All experiments using retroviruses were conducted in accordance with tivation motif. the guidelines of the University of Tsukuba. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 2889 Downloaded from http://www.jimmunol.org/ FIGURE 1. Retrovirus gene transfer into developing thymocytes in FTOC. A, Constructs for retroviruses producing S65T-mutant of GFP. Genes encoding gag, pol, and env were deleted from the sequences to prevent virus production by gene transferred cells. Instead, these viral proteins were supplied by the packaging cells GP1E-86. B, Experimental design for retrovirus gene transfer and GFP-detection in developing thymocytes in FTOC. Forward light scatter intensity (FSC) representing cell size distinguishes small lymphoid cells from GP1E-86-based virus-producing cells. C, Requirement for cytokines during infection culture. Fetal thymocytes from normal C57BL/6 (B6) mice were cultured in suspension with the pGD9-GFP virus-producing clone (No. 1 48-2-5) for 2 days in the absence or presence of indicated cytokines (4 ng/culture). Equal numbers of GFP FSCsmall thymocytes sorted out of the infection by guest on September 23, 2021 culture were cultured for indicated number of days in dGuo-treated B6-Ly5.1 fetal thymus lobes. FTOC cells were stained with allophycocyanin-labeled anti-CD4 Ab, phycoerythrin-labeled anti-CD8 Ab, and biotinylated anti-CD45.1 Ab, followed by Texas Red-streptavidin. Stained cells were analyzedby four-color flow cytometry; CD4/CD8 staining profiles of cells within electronically gated gene-transferred (GFP1CD45.12) cells are displayed. Each dot represents a single cell expressing the indicated intensity