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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

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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 CD4؊CD8؊ 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 CD4؉CD8؉ 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 ؊ ؊ wild-type ZAP-70, inhibits T cell development at the earlier CD4 CD8 stage. The ZAP-70 mutant blocked the generation of Downloaded from CD4؉CD8؉ 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 CD4؉CD8؉ 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 CD4ϪCD8Ϫ 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 CD4ϩCD8ϩ thymocytes. by guest on September 23, 2021 transfer often suffers from technical difficulties, such as low efficiency, 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 pGDЈ (6) or HpaI site of pMSCV (18). Purified and for the sensitive detection of gene-transferred cells. We con- plasmids were transfected into GPϩE-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 measured 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 pGDЈ 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 Ј 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 GPϩE-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 pGDЈ-KDZAP-ires-GFP, virus-producing clones of 106 1 cfu/ml were selected and were used for subsequent experiments. For pGDЈ- 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, pGDЈ-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, ϩ [email protected] BOSC23 cells were sorted for GFP cells to enrich transfected cells. These ϩ 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.

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 GPϩE-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 GPϩE-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 pGDЈ-GFP virus-producing clone (No. ϩ 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 (GFPϩCD45.1Ϫ) cells are displayed. Each dot represents a single cell expressing the indicated intensity of CD4 and CD8. Numbers indicate the frequency of cells within the indicated box. Note that most CD4ϪCD8ϩ cells in fetal thymus cultures represent immature precursor cells for CD4ϩCD8ϩ thymocytes, not CD8ϩ mature T cells expressing TCR at high levels (47–49). Shown are representative results from three individual measurements.

Retrovirus infection to developing thymocytes in suspension polis, MN). Cells were recovered by gentle pipetting, and viable lymphoid cultures cells, identified by small forward scatter intensity and no propidium iodide staining, were sorted for GFPϩ cells using a FACSVantage cell sorter Single-cell suspensions from day 14 fetal thymocytes (0.5–2 ϫ 104/well) equipped with Clone-Cyt hardware and software (Becton Dickinson). In were cultured for 2 to 3 days with virus-producing packaging cells (2–4 ϫ some experiments, cells were also stained for CD45 to identify lymphoid ϩ ϩ 103/well) in the presence of mouse rIL-7 (2–5 ng/well; Genzyme, Cam- cells, and GFP CD45 cells were sorted out from possibly contaminating Ϫ bridge, MA) in 96-well flat-bottom culture plates. IL-7 has been shown to virus-producing cells (CD45 ). Our preliminary experiments indicated that maintain the developmental capability of immature CD4ϪCD8Ϫ thymo- sorting with and without CD45 selection at this process gives essentially cytes in suspension culture (23, 24). In some experiments, the cultures also identical results in the efficiency and profiles of the subsequent T cell included recombinant mouse SCF (2–5 ng/well; R&D Systems, Minnea- differentiation. Equal numbers of sorted GFPϩ cells (CD45.1ϪCD45.2ϩ)

Table I. Virus titers and infection efﬁciencies of cell clones producing GFP-retroviruses a

Efﬁciency to Infect Thymocytes in FTOC

Retrovirus GP ϩ E-86 Clones Virus Titers (cfu/ml) Intact organ Suspension ϩ IL-7

pGDЈ-GFP 48-2-5 107 3 Ϯ 1% (0.1–5) 43 Ϯ 13% (12–86) 6-3-13 107 2 Ϯ 1% (0.2–6) ND pMSCV-GFP 4-1 107 2 Ϯ 1% (0.1–4) 47 Ϯ 13% (11–84) 4-3 107 1 Ϯ 0% (0.2–2) ND

a Titers for virus production were measured using serial dilutions of filtered supernatants. Efficiency to infect thymocytes in FTOC is indicated as the frequency of GFPϩ cells within CD45ϩ thymocytes. Day 14 fetal thymocytes from C57BL/6 (B6) mice were cocultured with virus-producing cells either in intact thymus lobes or in suspension in the presence of IL-7, as described in Materials and Methods, and were cultured for 5 to 10 days in dGuo-treated thymus lobes. Data represent means Ϯ SEs (ranges in parentheses) from five individual measurements. 2890 RETROVIRAL TRANSFER OF ZAP-70 INTO DEVELOPING THYMOCYTES IN CULTURE Downloaded from http://www.jimmunol.org/

FIGURE 2. Introduction of ZAP-70 and the catalytically inactive ZAP-70 mutant into developing thymocytes. A, Construct for retrovirus double- expressing ZAP-70 and GFP. Wild-type ZAP-70 (ZAPwt) and the catalytically inactive mutant of human ZAP-70 (KD-ZAP) were cloned into pGDЈ retrovirus vector containing GFP-S65T attached with IRES sequence. B, Expression of ZAP-70 by virus-producing cells. Lysates from GPϩE-86-derived packaging cells producing indicated viruses were electrophoresed and immunoblotted for ZAP-70. C, Expression of GFP by virus-producing cells. Packaging cells producing indicated viruses were analyzed for GFP expression using flow cytometry. Fluorescence intensity by GPϩE-86 control packaging cells indicates the background fluorescence levels. D, Expression of human ZAP-70 by virus-infected thymocytes. RNA transcripts from 5000 GFPϩ fetal thymocytes (FT) infected with indicated viruses were reverse transcribed using random oligonucleotide hexamers and amplified for indicated PCR primers. by guest on September 23, 2021 PCR products were electrophoresed on a polyacrylamide gel and visualized with ethidium bromide staining. Samples prepared without reverse transcriptase (R. T.) were employed to ascertain that the PCR signals were derived from RNA, not from contaminating DNA. RNA from a mouse T cell line 2B4, plasmid DNA, and water alone were also used as controls. were transferred into 2-deoxyguanosine (dGuo)-treated B6-Ly5.1 RT-PCRs ϩ Ϫ (CD45.1 CD45.2 ) fetal thymus lobes in a hanging drop in an inverted ϩ Terasaki well and were organ cultured at the interface between a collagen Total cellular RNA from either virus-producing cells or GFP fetal thymocytes (5000 cells) sorted out of infection cultures were reverse-tran- sponge-supported filter and 5% CO2-humidified air. Details for fetal thy- mal organ culture (FTOC) have been described previously (25, 26). Cells scribed using Superscript II RT (Life Technologies, Gaithersburg, MD) recovered from FTOC were multicolor stained and analyzed on a and random oligonucleotide hexamers and were PCR amplified (55 cycles) Ј Ј Ј FACSVantage as described (27, 28). for either human ZAP-70 (5 -TCTTCTACGGCAGCATCTCG-3 and 5 - AGTAGAACTCGCAGAGCTCTG-3Ј) or mouse ZAP-70 (5Ј-TCTTC Retrovirus infection to developing thymocytes in intact organ TATGGCAGCATCTCG-3Ј and 5Ј-AGTAGAACTGGCAGAGCTCGG- Ј cultures 3 ). PCR products were electrophoresed on a 7% polyacrylamide gel and were visualized with ethidium bromide staining. Day 14 fetal thymus lobes from B6 mice were cocultured with virus-producing cells either in a hanging drop culture or in a high oxygen-supported submersion culture. For the hanging-drop culture, virus-producing cells, Results and Discussion either freshly prepared or precultured for 1 to 2 days, were mixed with Retrovirus gene transfer into developing thymocytes freshly isolated fetal thymus lobes for 1 to 2 days in an inverted Terasaki plate. For high oxygen submersion culture, fetal thymus lobes were placed To evaluate the efficiency of gene transfer into developing thy- onto 1- to 2-day-precultured virus-producing cells in the bottom of 96-well mocytes, we have cloned S65T-GFP into retroviral vectors, ei- flat-bottom culture plates. Cultures were conducted for 2 to 3 days in an ther pGDЈ (6) or pMSCV (18) (Fig. 1A). The recombinant vec- atmosphere containing 70% O2, 25% N2, and 5% CO2 (29). Thymus lobes ϩ were then washed and further cultured under regular FTOC condition. tors were transfected into packaging cell line GP E-86, and stable virus-producer cells at titers more than Ͼ106 cfu/ml were Immunoblot analysis cloned. The mixture of intact mouse fetal thymus lobes with GPϩE-86-derived packaging cells producing either pGDЈ-GFP virus, virus-producing cells for 2 to 3 days resulted in GFP expression pGDЈ-ZAPwt-ires-GFP virus, or pGDЈ-KDZAP-ires-GFP virus were lysed in only 2% of the thymocytes on average (Table I). This low in a buffer containing 1% Nonidet P-40. Cell lysates were electrophoresed efficiency was consistent for day 12 to day 17 fetal thymus lobes in an 8% SDS-polyacrylamide gel, transferred to a nylon membrane, and cocultured with virus-producing cells, either in hanging-drop or detected for ZAP-70 using anti-VSV-G polyclonal Ab (21). Signals were visualized using horseradish peroxidase-conjugated anti-rabbit IgG Ab and in high oxygen submersion cultures. Also, this low efficiency of an enhanced chemiluminescence detection system (ECL; Amersham, virus infection was not improved by 1) the addition of IL-7 Tokyo, Japan). and/or SCF in the cocultures, 2) longer periods of infection The Journal of Immunology 2891 Downloaded from http://www.jimmunol.org/

FIGURE 3. The effects of KD-ZAP expression in thymocyte development. Day 14 fetal thymocytes from normal B6 mice were cocultured in suspension with packaging cells producing indicated retroviruses. Equal numbers (ranging between 1000 and 2500 cells) of GFP-expressing lymphoid cells were sorted and transferred into a dGuo-treated B6-Ly5.1 fetal thymus lobe. A, T cell development of CD45.1Ϫ thymocytes in FTOC were measured on day 6 (Expt. 1) or day 9 (Expt. 2) in FTOC, as described in the legend to Figure 1. In parallel staining analyses, Ͼ98% of CD45.1Ϫ cells expressed CD45.2ϩ, indicating that CD45.1Ϫ cells were indeed derived from B6 thymocytes. CD4ϪCD8ϩ cells found in KD-ZAP-introduced FTOC (Expt. 2) were mostly TCRϪ/low, representing immature precursor cells (47–49) rather than mature T cells. B, FTOC cells were four-color analyzed for GFP, CD45.1, CD4, and CD8 on day 7, as described in the legend to Figure 1. Expression profiles of GFP and CD45.1 in all cells recovered from FTOC are indicated in the left-hand panels. by guest on September 23, 2021 CD4/CD8 profiles of CD45.1Ϫ B6-derived thymocytes further gated by the expression levels of GFP are indicated in the right-hand panels. C, FTOC cells were four-color analyzed for GFP CD45.1, CD4, and CD8; for GFP CD45.1 TCR-␤ (APC-H57, PharMingen) and TCR-␦ (PE-GL3, PharMingen); and three-color analyzed for GFP CD45.1 and CD44 (PE-IM7, PharMingen). Shown are fluorescence profiles of indicated molecules by CD45.1Ϫ-gated B6-derived thymocytes. Data shown in B and C are representative of three individual experiments.

cocultures up to 10 days, and 3) graded numbers of virus-pro- the infection culture in suspension, as GFPϩ cells infected only ducing cells in cocultures (data not shown), despite the fact that in the presence of IL-7, but not SCF, retained the capability to a previous study had reported a very efficient (Ͼ80%) infection enter the CD4/CD8 developmental pathway. The capacity of to intact day 13 fetal thymus lobes (17). Nonetheless, we found IL-7-treated thymocytes to undergo T cell development was that a much higher efficiency of gene transfer, ϳ40% on aver- further ascertained in FTOC by the acquisition of TCR and CD5 age, was consistently obtained by 2- to 3-day cocultures of vi- and by the down-regulation of CD44 and heat-stable antigen rus-producing cells with fetal thymocytes in single-cell suspen- (data not shown). Thus, we have established an efficient method sion cultures in the presence of IL-7 (Table I). Neither virus- of retroviral gene transfer and gene detection in developing containing supernatants nor virus-producing cells separated by thymocytes. filter membrane resulted in successful gene transfer to developing thymocytes. Two retroviral vectors, pGDЈ and pMSCV, Retroviral introduction of ZAP-70 and GFP into developing gave a similar efficiency of gene transfer (Table I). Expression thymocytes of GFP upon retroviral transfer is useful, as cells can be readily Using the method described above, we began introducing genes purified by FACS cell sorting, and their subsequent develop- encoding intracellular signaling molecules into immature thymo- ment in the thymic environment can then be easily analyzed by cytes. To examine the role of ZAP-70 in thymocyte development, FTOC (Fig. 1B). The sorting of GFPϩ thymocytes and transfer we constructed a retrovirus that is capable of producing ZAP-70 into retrovirus-free FTOC also rules out any unwanted side ef- and GFP (Fig. 2A). The IRES sequence allows cap-independent fects by possible gene transfer into other cell types such as translation (30, 31), so that GFP-encoding RNA transcripts can be thymic epithelial cells, a possibility that could not be ruled out translated even though the IRES-GFP sequence is located down- by previous studies (17). Indeed, in most experiments, stream of the translation termination sequence of ZAP-70. The CD45ϩGFPϩ-infected thymocytes were sorted out to distin- recombinant virus indeed produced both ZAP-70 (Fig. 2B) and guish thymocytes from CD45Ϫ virus-producing cells and thy- GFP (Fig. 2C) in the cells, thereby enabling ZAP-70-expressing mic stromal cells. Figure 1C shows that IL-7, a growth factor cells to be identified and sorted out by virtue of their GFP expres- for immature lymphocytes (23, 24), was essential in supporting sion. The lower expression of GFP by ZAP-70-expressing virus 2892 RETROVIRAL TRANSFER OF ZAP-70 INTO DEVELOPING THYMOCYTES IN CULTURE Downloaded from

FIGURE 4. The effects of KD-ZAP expression in thymocyte development of ZAP-70-knockout mice. Day 14 fetal thymocytes from ZAP-70Ϫ/Ϫ mice were cocultured in suspension with packaging cells producing indicated retroviruses. Equal numbers (ranging between 1500 and 2000 cells) of GFP- expressing lymphoid cells were sorted and transferred into a dGuo-treated B6-Ly5.1 fetal thymus lobe. T cell development of CD45.1Ϫ thymocytes were measured on day 10 in FTOC, as described in the legend to Figure 1. Expression proﬁles of GFP and CD45.1 in all cells recovered from FTOC are indicated in the left-hand panels. CD4/CD8 proﬁles of CD45.1Ϫ ZAP-70Ϫ/Ϫ-derived thymocytes further gated by the expression levels of GFP are indicated in the Ϫ ϩ Ϫ/low right-hand panels. CD4 CD8 cells found in the KD-ZAP-introduced culture were mostly TCR , representing immature precursor cells rather than http://www.jimmunol.org/ mature T cells (47–49). Shown are representative results of four individual experiments.

than the virus expressing GFP alone (Fig. 2C) appeared to be con- almost complete arrest at CD4ϪCD8Ϫ stage, than in GFPlow cells, sistent for the IRES-GFP virus construction used in the present i.e., arrest mostly at CD4ϪCD8ϩ intermediate precursor stage study, since we observed a similar decrease of GFP expression in (Fig. 3B and the legend for Fig. 3A), and GFPhigh cells indeed the bicistronic viruses producing MKK1, MKK6, and calcineurin expressed higher levels of KD-ZAP transcripts than GFPlow cells instead of ZAP-70 (T.S. and Y.T., unpublished observation). (data not shown), suggesting that the kinase-inactive ZAP-70 mu- Nonetheless, GFPϩ cells sorted out from the infection culture tant inhibits T cell development in a dominant negative manner by guest on September 23, 2021 abundantly expressed virus-transferred human ZAP-70 in addition and that the variation in the inhibition by KD-ZAP (Fig. 3A, Expt. to endogenous mouse ZAP-70 (Fig. 2D), although the limited 1 vs 2) is due to the variation in the levels of KD-ZAP expression. numbers of fetal thymocytes (5 ϫ 103 to 1 ϫ 104 GFPϩ cells in a Nonetheless, T cell development was impaired even in GFPlow typical experiment) did not allow us to detect ZAP-70 proteins in cells derived from KD-ZAP-introduced GFPϩ-sorted cells (Fig. gene-transferred thymocytes by immunoblot analysis. The diffi- 3B), suggesting that most cells have expressed KD-ZAP during culty in obtaining large numbers of cells should be noted as a FTOC. KD-ZAP introduction inhibited T cell development at the limitation of the FTOC-based gene transfer approach. A similar immature CD44high stage within the CD4ϪCD8Ϫ compartment, gene transfer approach for human CD34ϩ T/NK-progenitor cells resulting in the developmental arrest of TCR-␤ϩ TCR-␣␤ lineage has been recently reported (32). cells as well as TCR-␦ϩ TCR-␥␦ lineage cells (Fig. 3C). Thus, the introduction of KD-ZAP in immature thymocytes severely inhibits Introduction into immature thymocytes of kinase-inactive ZAP-70 Ϫ Ϫ Ϫ Ϫ T cell development beyond the CD4 CD8 stage, both for mutant inhibits T cell development beyond the CD4 CD8 stage TCR-␣␤ and TCR-␥␦ lineages. Immature CD4ϪCD8Ϫ thymocytes from day 14 fetal mice were infected with retroviruses producing GFP and either wild-type Ϫ Ϫ ZAP-70 or a catalytically inactive mutant of ZAP-70 (KD-ZAP), Kinase-inactive ZAP-70 Inhibits CD4 CD8 thymocyte which can antagonize ZAP-70 signals in mature T cells (21). As development even in the absence of endogenous ZAP-70 shown in Figure 3, the development of CD4ϪCD8Ϫ thymocytes ZAP-70-knockout mice (36) and mutant mice expressing unstable was severely impaired by the introduction of KD-ZAP. Since the ZAP-70 (37) have been shown to exhibit defects in both the pos- developmental arrest appeared to be mapped to the early stage of itive and negative selection of CD4ϩCD8ϩ thymocytes. Immuno- T cell development before the generation of CD4ϩCD8ϩ thymo- deficient patients lacking the expression of ZAP-70 also show a cytes, gene-introduced cells were cultured in FTOC for Ͻ10 days defect in the generation of CD8ϩ T cells beyond the CD4ϩCD8ϩ so that we could focus our analysis on the effect of KD-ZAP on stage (38–40). The generation of CD4ϩCD8ϩ cells in these ZAP- early T cell development. 70-deficient thymocytes appears inconsistent with our results in- The introduction of either wild-type ZAP-70 or GFP alone did dicating an earlier arrest at the CD4ϪCD8Ϫ stage of thymic de- not affect the generation of CD4ϩCD8ϩ thymocytes (Fig. 3A), velopment following the introduction of catalytically inactive suggesting that the inhibition by KD-ZAP is caused by the lack of ZAP-70. Transient introduction of KD-ZAP in Jurkat cells specif- ZAP-70 kinase activity and is not solely due to overexpression of ically inhibited TCR-induced NFAT activation without exhibiting ZAP-70, which is capable of interacting with Lck (33)-, Shc (34)-, a generalized suppressive effect on transcription (21), suggesting and ITAM-containing receptors (35). The block of thymocyte de- that KD-ZAP did not inactivate immature thymocytes in a non- velopment by KD-ZAP was more severe in GFPhigh cells, i.e., specific manner. It is possible, however, that ZAP-70 signals are The Journal of Immunology 2893 involved in, but not essential for, the signals inducing the gener- 11. Blaese, R. M., K. W. Culver, A. D. Miler, C. S. Carter, T. Fleisher, M. Clerici, ation of CD4ϩCD8ϩ thymocytes, triggered by the pre-TCR com- G. M. Shearer, L. Chang, Y. Chiang, P. Tolstoshev, J. J. Greenblatt, S. A. Rosenberg, H. Klein, M. Berger, C. A. Mullen, W. J. Ramsey, L. Muul, plex containing ITAM motifs (41). In addition to ZAP-70, another R. A. Morgan, and W. F. Anderson. 1995. T lymphocyte-directed gene therapy member of the Syk/ZAP-70 tyrosine kinase family, Syk, is ex- for ADA-SCID: initial trial results after 4 years. Science 270:475. pressed by CD4ϪCD8Ϫ thymocytes (42). In the absence of ZAP- 12. DeMatteo, R. P., S. E. Raper, M. Ahn, K. J. Fisher, C. Burke, A. Radu, ϩ ϩ G. Widera, B. R. Claytor, C. F. Barker, and J. F. Markmann. 1995. Gene transfer 70, Syk may compensate for the signals inducing CD4 CD8 to the thymus: a means of abrogating the immune response to recombinant ad- thymocytes, whereas KD-ZAP may inhibit those signals by com- enovirus. Ann. Surg. 222:229. 13. Plavec, I., A. Voytovich, K. Moss, D. Webster, M. B. Hanley, S. Escaich, peting for Syk binding sites. This possibility is indeed supported K. E. Ho, E. Bohnlein, and D. L. GiGiusto. 1996. Sustained retroviral gene by our results showing that KD-ZAP inhibited the generation of marking and expression in lymphoid and myeloid cells derived from transduced CD4ϩCD8ϩ thymocytes even in ZAP-70-knock-out mice (Fig. 4), hematopoietic progenitor cells. Gene Ther. 3:717. 14. Gu, J., M. L. Kuo, A. Rivera, N. Sutkowski, Y. Ron, and J. P. Dougherty. 1996. indicating that KD-ZAP can interfere with signaling events not A murine model for genetic manipulation of the T cell compartment. Exp. He- necessarily mediated by ZAP-70. The interchangeable but essen- matol. 24:1432. tial involvement by ZAP-70 and Syk in the signals inducing 15. Kohn, D. B. 1996. Gene therapy for hematopoietic and immune disorders. Bone CD4ϩCD8ϩ thymocytes has also been supported by the recent Marrow Transplant. 18S:55. Ϫ Ϫ 16. Sharma, S., M. Cantwell, T. J. Kipps, and T. Friedmann. 1996. Efficient infection finding that T cell development is arrested at the CD4 CD8 stage of a human T-cell line and of human primary peripheral blood leukocytes with a of thymocytes in ZAP-70Ϫ/SykϪ double knock-out mice (43). pseudotyped retrovirus vector. Proc. Acad. Natl. Sci. USA 93:11842. Finally, our results showing that KD-ZAP inhibits the genera- 17. Crompton, T., K. C. Gilmour, and M. J. Owen. 1996. The MAP kinase pathway ϩ controls differentiation from double-negative to double positive thymocyte. Cell tion of TCR-␥␦ T cells in FTOC suggest a novel possibility that 86:243. ZAP-70/Syk family kinases may also be involved in the generation 18. Hawley, R. G., F. H. L. Lieu, A. Z. C. Fong, and T. S. Hawley. 1994. Versatile Downloaded from of TCR-␥␦-lineage T cells in the thymus. Thus, our results extend retroviral vectors for potential use in gene therapy. Gene Ther. 1:136. Ϫ 19. Markowitz, D., S. Goff, and A. Bank. 1988. A safe packaging line for gene the previous results showing that ZAP-70 mice are selectively transfer: separating viral genes on two different plasmids. J. Virol. 62:1120. ϩ deficient in the intestinal intraepithelial TCR-␥␦ T cells (44), 20. Kim, D. G., H. M. Kang, S. K. Jang, and H. S. Shin. 1992. Construction of a whereas SykϪ mice exhibit selective deficiency in dermal and in- bifunctional mRNA in the mouse by using the internal ribosomal entry site of the ␥␦ϩ encephalomyocarditis virus. Mol. Cell. Biol. 12:3636. testinal TCR- T cells (45, 46). 21. Mege, D., V. Di Bartolo, V. Germain, L. Tuosto, F. Michel, and O. Acuto. 1996.

The present results indicating successful retrovirus gene transfer Mutation of tyrosines 492/493 in the kinase domain of ZAP-70 affects multiple http://www.jimmunol.org/ into immature thymocytes and efﬁcient detection in FTOC of gene- T-cell receptor signaing pathways. J. Biol. Chem. 271:32644. 22. Pear, W. S., G. P. Nolan, M. L. Scott, and D. Baltimore. 1993. Production of transferred cells using GFP provides a powerful method to further high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. analyze the signaling events regulating T cell development in the USA 90:8392. thymus. These gene transfer techniques also have great potential 23. Namen, A. E., S. Lupton, K. Hjerrild, J. Wignall, D. Y. Mochizuki, A. Schmierer, B. Mosley, C. J. March, D. Urdal, and S. Gillis. 1988. Stimulation of B-cell for treating various immunodeﬁciencies to restore T cell progenitors by cloned murine interleukin-7. Nature 333:571. development. 24. Suda, T., and A. Zlotnik. 1991. IL-7 maintains the T cell precursor potential of CD3ϪCD4ϪCD8Ϫ thymocytes. J. Immunol. 146:3068. 25. Jenkinson, E. J., L. L. Franchi, R. Kingston, and J. J. T. Owen. 1982. Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro:

Acknowledgments by guest on September 23, 2021 application in the production of chimeric thymus rudiments. Eur. J. Immunol. We thank Dr. D. Baltimore and R. Hawley for pGDЈ and pMSCV vectors; 12:583. Dr. A. Bank for GPϩE-86 cells; Dr. Y. Katsura for help on high oxygen 26. Tsuda, S., S. Rieke, Y. Hashimoto, H. Nakauchi, and Y. Takahama. 1996. IL-7 submersion culture; Dr. I. Negishi for ZAP-70Ϫ/Ϫ mice; S. Tanaka and Y. supports D-J but not V-DJ rearrangement of TCR-␤ gene in fetal liver progenitor cells. J. Immunol. 156:3233. Morita for technical help; Drs. T. Saito and A. Weiss for critical comments 27. Takahama, Y., S. O. Sharrow, and A. Singer. 1991. Expression of an unusual T during the study; and Drs. D. Alexander, M. Iwashima, and E. W. Shores for cell receptor (TCR)-V ␤ repertoire by Ly-6Cϩ subpopulations of CD4ϩ and/or reading the manuscript. CD8ϩ thymocytes: evidence for a developmental relationship between Ly-6Cϩ thymocytes and CD4ϪCD8ϪTCR-␣␤ϩ thymocytes. J. Immunol. 147:2883. 28. Takahama, Y., and H. Nakauchi. 1996. Phorbol ester and calcium ionophore can References replace TCR signals that induce positive selection of CD4 T cells. J. Immunol. 157:1508. 1. Greaves, D. R., F. D. Wilson, G. Lang, and D. Kioussis. 1989. Human CD2 29. Watanabe, Y., and Y. Katsura. 1993. Development of T cell receptor ␣␤-bearing 3Ј-flanking sequences confer high-level, T cell-specific, position-independent T cells in the submersion organ culture of murine fetal thymus at high oxygen gene expression in transgenic mice. Cell 56:979. concentration. Eur. J. Immunol. 23:200. 2. Chaffin, K. E., C. R. Beals, T. M. Wilkie, K. A. Forbush, M. I. Simon, and R. M. Perlmutter. 1990. Dissection of thymocyte signaling pathways by in vivo 30. Duke, G. M., M. A. Hoffman, and A. C. Palmenberg. 1992. Sequence and struc- expression of pertussis toxin ADP-ribosyltransferase. EMBO J. 9:3821. tural elements that contribute to efficient encephalomyocarditis virus RNA translation. J. Virol. 66:1602. 3. Williams, D. A., I. R. Lemischka, I. R. Nathan, and R. C. Mulligan. 1984. In- troduction of new genetic material into pluripotent haematopoietic stem cells of 31. Davies, M., and R. J. Kaufman. 1992. The sequence context of the initiation the mouse. Nature 310:476. codon in the encephalomyocarditis virus leader modulates efficiency of internal 4. Dick, J. E., M. C. Magli, D. Huszar, R. A. Phillips, and A. Bernstein. 1985. translation initiation. J. Virol. 66:1924. Introduction of a selectable gene into primitive stem cells capable of long-term 32. Heemskerk, M. H. M., B. Blom, G. Nolan, A. P. A. Stegmann, A. Q. Bakker, reconstitution of the hemopoietic system of W/Wv mice. Cell 42:71. K. Weijer, P. C. M. Res, and H. Spits. 1997. Inhibition of T cell and promotion 5. Keller, G., C. Paige, E. Gilboa, and E. F. Wagner. 1985. Expression of a foreign of natural killer cell development by the dominant negative helix loop helix factor gene in myeloid and lymphoid cells derived from multipotent haematopoietic Id3. J. Exp. Med. 186:1597. lck precursors. Nature 318:149. 33. Duplay, P., M. Thome, F. Herve, and O. Acuto. 1994. p56 interacts via its src 6. Daley, G. Q., R. A. Van Etten, and D. Baltimore. 1990. Induction of chronic homology 2 domain with the ZAP-70 kinase. J. Exp. Med. 179:1163. myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chro- 34. Milia, E., M. M. Di Somma, F. Baldoni, R. Chiari, L. Lanfrancone, P. G. Pelicci, mosome. Science 247:824. J. L. Telford, and C. T. Baldari. 1996. The aminoterminal phosphotyrosine bind- 7. Hawley, R. G., A. Z. C. Fong, Z. C. Burns, and T. S. Hawley. 1992. Transplant- ing domain of Shc associates with ZAP-70 and mediates TCR dependent gene able myeloproliferative disease induced in mice by an interleukin 6 retrovirus. activation. Oncogene 13:767. J. Exp. Med. 176:1149. 35. Iwashima, M., B. A. Irving, N. S. van Oers, A. C. Chan, and A. Weiss. 1994. 8. Szilvassy, S. J., and S. Cory. 1994. Efficient retroviral gene transfer to purified Sequential interactions of the TCR with two distinct cytoplasmic tyrosine ki- long-term repopulating hematopoietic stem cells. Blood 84:74. nases. Science 263:1136. 9. Williams, D. E., A. E. Namen, D. Y. Mochizuki, and R. W. Overell. 1990. Clonal 36. Negishi, I., N. Motoyama, K. Nakayama, K. Nakayama, S. Senju, growth of murine pre-B colony-forming cells and their targeted infection by a S. Hatakeyama, Q. Zhang, A. C. Chan, and D. Y. Loh. 1995. Essential role for retroviral vector: dependence on interleukin-7. Blood 75:1132. ZAP-70 in both positive and negative selection of thymocytes. Nature 376:435. 10. Corcoran, A. E., F. M. Smart, R. J. Cowling, T. Crompton, M. J. Owen, and 37. Wiest, D. L., J. M. Ashe, T. K. Howcroft, H. M. Lee, D. M. Kemper, I. Negishi, A. R. Venkitaraman. 1996. The interleukin-7 receptor ␣ chain transmits distinct D. S. Singer, A. Singer, and R. Abe. 1997. A spontaneous arising mutation in the signals for proliferation and differentiation during B lymphopoiesis. EMBO J. DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests thymo- 15:1924. cyte development. Immunity 6:663. 2894 RETROVIRAL TRANSFER OF ZAP-70 INTO DEVELOPING THYMOCYTES IN CULTURE

38. Arpaia, E., M. Shahar, H. Dadi, A. Cohen, and C. M. Roifman. 1994. Defective 44. Gong, Q., L. White, R. Johnson, M. White, I. Negishi, M. Thomas, and T cell receptor signaling and CD8ϩ thymic selection in humans lacking ZAP-70 A. C. Chan. 1997. Restoration of thymocyte development and function in zap- kinase. Cell 76:947. 70Ϫ/Ϫ mice by the Syk protein tyrosine kinase. Immunity 7:369. 39. Elder, M. E., D. Lin, J. Clever, A. C. Chan, T. J. Hope, A. Weiss, and 45. Turner, M., P. J. Mee, P. S. Costello, O. Williams, A. A. Price, L. P. Duddy, T. G. Parslow. 1994. Human severe combined immunodeficiency due to a defect M. T. Furlong, R. L. Geahlen, and V. L. J. Tybulewicz. 1995. Perinatal lethality in ZAP-70, a T cell tyrosine kinase. Science 264:1596. and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 40. Chan, A. C., T. A. Kadlecek, M. E. Elder, A. H. Filipovich, W. L. Kuo, 378:298. M. Iwashima, T. G. Parslow, and A. Weiss. 1994. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 264: 46. Mallick-Wood, C. A., W. Pao, A. M. Cheng, J. M. Lewis, S. Klukarni, 1599. J. B. Bolen, B. Rowley, R. E. Rigelaar, T. Pawson, and A. C. Hayday. 1996. 41. van Oers, N. S., H. von Boehmer, and A. Weiss. 1995. The pre-T cell receptor Disruption of epithelial ␥␦ T cell repertoires by mutation of the Syk tyrosine (TCR) complex is functionally coupled to the TCR-␨ subunit. J. Exp. Med. 182: kinase. Proc. Natl. Acad. Sci. USA 93:9704. 1585. 47. Paterson, D. J., and A. F. Williams. 1987. An intermediate cell in thymocyte 42. Chan, A. C., N. S. van Oers, A. Tran, L. Turka, C. L. Law, J. C. Ryan, differentiation that expresses CD8 but not CD4 antigen. J. Exp. Med. 166:1603. E. A. Clark, and A. Weiss. 1994. Differential expression of ZAP-70 and Syk 48. Nikolic-Zugic, J., and M. J. Bevan. 1988. Thymocytes expressing CD8 differ- protein tyrosine kinases, and the role of this family of protein tyrosine kinases in ϩ TCR signaling. J. Immunol. 152:4758. entiate into CD4 cells following intrathymic injection. Proc. Natl. Acad. Sci. 43. Cheng, A. M., I. Negishi, S. J. Anderson, A. C. Chan, J. Bolen, D. Y. Loh, and USA 85:8633. T. Pawson. 1997. The Syk and ZAP-70 SH2-containing tyrosine kinases are 49. Takahama, Y., and A. Singer. 1992. Post-transcriptional regulation of early T cell implicated in pre-T cell receptor signaling. Proc. Natl. Acad. Sci. USA 94:9797. development by T cell receptor signals. Science 258:1456. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021