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The Journal of Immunology, 1998, 161: 2888-2894.
Copyright © 1998 by The American Association of Immunologists

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{ddagger}, Hiromitsu Nakauchi*, Oreste Acuto§ and Yousuke Takahama2,*,{dagger}

* Department of Immunology and {dagger} PRESTO Research Project, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan; {ddagger} Department of Immunology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and § Laboratory of Molecular Immunology, Department of Immunology, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Introducing a given gene into developing thymocytes is a powerful technique for analyzing molecular mechanisms regulating T cell differentiation. Transgenic expression of a gene under control of a T cell-specific promoter/enhancer has been widely used for gene manipulation of thymocytes (1, 2). Recently, retroviral gene transfer has been used successfully for a wide variety of cells including hemopoietic cells (3, 4, 5, 6, 7, 8) and developing B lymphocytes (9, 10). Retrovirus-mediated gene transfer has several advantages over the transgenic techniques, including rapid and close analysis of specific cellular events in vitro and its potential application for gene therapy. However, retrovirus-mediated gene transfer often suffers from technical difficulties, such as low efficiency, which hamper applications in various cell types. Consequently, attempts to introduce exogenous genes using retroviruses have had limited success in developing T lymphocytes (11, 12, 13, 14, 15, 16, 17).

The present study reports an effective technique for retroviral gene transfer into developing T cells in fetal thymus organ cultures and for the sensitive detection of gene-transferred cells. We constructed recombinant retroviruses expressing green fluorescence protein (GFP)3 along with a protein of interest, using the internal ribosomal entry site (IRES) sequence. The coexpression of GFP was useful, as gene-transferred cells could be readily detected and sorted using flow cytometry. Immature thymocytes were successfully infected with these retroviruses in suspension culture in the presence of IL-7 and were examined for their developmental capability by transferring to the thymus organ culture. Using this retroviral gene transfer technique, the present study shows that forced expression in developing thymocytes of a kinase-inactive mutant of ZAP-70 inhibits T cell development at the immature CD4-CD8- thymocytes, suggesting that ZAP-70/Syk family tyrosine kinases are involved in the signals that induce the generation of CD4+CD8+ thymocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Retrovirus constructs and virus-producing cells

The S65T mutant of GFP (Clontech, Palo Alto, CA) was cloned into either the BclI site of pGD' (6) or HpaI site of pMSCV (18). Purified plasmids were transfected into GP+E-86-packaging cells (19). G418-resistant cells were clone sorted for GFPhigh clones using a FACSVantage cell sorter (Becton Dickinson, San Jose, CA). Graded 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 selected for subsequent experiments.

GFP-S65T attached downstream of the IRES sequence from encephalomyocarditis virus (20) was cloned into the pGD' vector. Either wild-type human ZAP-70 or its catalytically inactive (kinase-dead) mutant (KD-ZAP; D461N), tagged with the VSV-G sequence (21), was cloned into the XhoI site of pGD'-ires-GFP vector. Resulting retrovirus vectors were transfected into GP+E-86 cells, and virus producing cells were cloned as described above. For pGD'-KDZAP-ires-GFP, virus-producing clones of 106 cfu/ml were selected and were used for subsequent experiments. For pGD'-ZAPwt-ires-GFP, virus-producing cells of more than 105 CFU/ml were not generated. Consequently, to compare the effects of ZAPwt-producing virus and KD-ZAP-producing virus, pGD'-based plasmids were transiently transfected into BOSC23 packaging cells (22) obtained from American Type Culture Collection (Manassas, VA). Two days after the transfection, BOSC23 cells were sorted for GFP+ cells to enrich transfected cells. These GFP+ BOSC23 cells were used for virus-producing cells to infect fetal thymocytes.

All experiments using retroviruses were conducted in accordance with the guidelines of the University of Tsukuba.

Retrovirus infection to developing thymocytes in suspension cultures

Single-cell suspensions from day 14 fetal thymocytes (0.5–2 x 104/well) were cultured for 2 to 3 days with virus-producing packaging cells (2–4 x 103/well) in the presence of mouse rIL-7 (2–5 ng/well; Genzyme, Cambridge, MA) in 96-well flat-bottom culture plates. IL-7 has been shown to maintain the developmental capability of immature CD4-CD8- thymocytes in suspension culture (23, 24). In some experiments, the cultures also included recombinant mouse SCF (2–5 ng/well; R&D Systems, Minnea-polis, MN). Cells were recovered by gentle pipetting, and viable lymphoid cells, identified by small forward scatter intensity and no propidium iodide staining, were sorted for GFP+ cells using a FACSVantage cell sorter equipped with Clone-Cyt hardware and software (Becton Dickinson). In some experiments, cells were also stained for CD45 to identify lymphoid cells, and GFP+CD45+ cells were sorted out from possibly contaminating virus-producing cells (CD45-). Our preliminary experiments indicated that sorting with and without CD45 selection at this process gives essentially identical results in the efficiency and profiles of the subsequent T cell differentiation. Equal numbers of sorted GFP+ cells (CD45.1-CD45.2+) were transferred into 2-deoxyguanosine (dGuo)-treated B6-Ly5.1 (CD45.1+CD45.2-) fetal thymus lobes in a hanging drop in an inverted Terasaki well and were organ cultured at the interface between a collagen sponge-supported filter and 5% CO2-humidified air. Details for fetal thymal organ culture (FTOC) have been described previously (25, 26). Cells recovered from FTOC were multicolor stained and analyzed on a FACSVantage as described (27, 28).

Retrovirus infection to developing thymocytes in intact organ cultures

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, either freshly prepared or precultured for 1 to 2 days, were mixed with 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 onto 1- to 2-day-precultured virus-producing cells in the bottom of 96-well flat-bottom culture plates. Cultures were conducted for 2 to 3 days in an atmosphere containing 70% O2, 25% N2, and 5% CO2 (29). Thymus lobes were then washed and further cultured under regular FTOC condition.

Immunoblot analysis

GP+E-86-derived packaging cells producing either pGD'-GFP virus, pGD'-ZAPwt-ires-GFP virus, or pGD'-KDZAP-ires-GFP virus were lysed in a buffer containing 1% Nonidet P-40. Cell lysates were electrophoresed in an 8% SDS-polyacrylamide gel, transferred to a nylon membrane, and detected for ZAP-70 using anti-VSV-G polyclonal Ab (21). Signals were visualized using horseradish peroxidase-conjugated anti-rabbit IgG Ab and an enhanced chemiluminescence detection system (ECL; Amersham, Tokyo, Japan).

RT-PCRs

Total cellular RNA from either virus-producing cells or GFP+ fetal thymocytes (5000 cells) sorted out of infection cultures were reverse-transcribed using Superscript II RT (Life Technologies, Gaithersburg, MD) and random oligonucleotide hexamers and were PCR amplified (55 cycles) for either human ZAP-70 (5'-TCTTCTACGGCAGCATCTCG-3' and 5'-AGTAGAACTCGCAGAGCTCTG-3') or mouse ZAP-70 (5'-TCTTCTATGGCAGCATCTCG-3' and 5'-AGTAGAACTGGCAGAGCTCGG-3'). PCR products were electrophoresed on a 7% polyacrylamide gel and were visualized with ethidium bromide staining.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Retrovirus gene transfer into developing thymocytes

To evaluate the efficiency of gene transfer into developing thymocytes, we have cloned S65T-GFP into retroviral vectors, either pGD' (6) or pMSCV (18) (Fig. 1GoA). The recombinant vectors were transfected into packaging cell line GP+E-86, and stable virus-producer cells at titers more than >106 cfu/ml were cloned. The mixture of intact mouse fetal thymus lobes with virus-producing cells for 2 to 3 days resulted in GFP expression in only 2% of the thymocytes on average (Table IGo). This low efficiency was consistent for day 12 to day 17 fetal thymus lobes cocultured with virus-producing cells, either in hanging-drop or in high oxygen submersion cultures. Also, this low efficiency of virus infection was not improved by 1) the addition of IL-7 and/or SCF in the cocultures, 2) longer periods of infection cocultures up to 10 days, and 3) graded numbers of virus-producing cells in cocultures (data not shown), despite the fact that a previous study had reported a very efficient (>80%) infection to intact day 13 fetal thymus lobes (17). Nonetheless, we found that a much higher efficiency of gene transfer, ~40% on average, was consistently obtained by 2- to 3-day cocultures of virus-producing cells with fetal thymocytes in single-cell suspension cultures in the presence of IL-7 (Table IGo). Neither virus-containing supernatants nor virus-producing cells separated by filter membrane resulted in successful gene transfer to developing thymocytes. Two retroviral vectors, pGD' and pMSCV, gave a similar efficiency of gene transfer (Table IGo). Expression of GFP upon retroviral transfer is useful, as cells can be readily purified by FACS cell sorting, and their subsequent development in the thymic environment can then be easily analyzed by FTOC (Fig. 1GoB). The sorting of GFP+ thymocytes and transfer into retrovirus-free FTOC also rules out any unwanted side effects by possible gene transfer into other cell types such as thymic epithelial cells, a possibility that could not be ruled out by previous studies (17). Indeed, in most experiments, CD45+GFP+-infected thymocytes were sorted out to distinguish thymocytes from CD45- virus-producing cells and thymic stromal cells. Figure 1GoC shows that IL-7, a growth factor for immature lymphocytes (23, 24), was essential in supporting the infection culture in suspension, as GFP+ cells infected only in the presence of IL-7, but not SCF, retained the capability to enter the CD4/CD8 developmental pathway. The capacity of IL-7-treated thymocytes to undergo T cell development was further ascertained in FTOC by the acquisition of TCR and CD5 and by the down-regulation of CD44 and heat-stable antigen (data not shown). Thus, we have established an efficient method of retroviral gene transfer and gene detection in developing thymocytes.



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  		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 GP+E-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 GP+E-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 pGD'-GFP virus-producing clone (No. 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 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 analyzed by four-color flow cytometry; CD4/CD8 staining profiles of cells within electronically gated gene-transferred (GFP+CD45.1-) 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 CD4-CD8+ cells in fetal thymus cultures represent immature precursor cells for CD4+CD8+ thymocytes, not CD8+ mature T cells expressing TCR at high levels (47–49). Shown are representative results from three individual measurements.

 

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  		Table I. Virus titers and infection efficiencies of cell clones producing GFP-retroviruses1

 
Retroviral introduction of ZAP-70 and GFP into developing thymocytes

Using the method described above, we began introducing genes encoding intracellular signaling molecules into immature thymocytes. To examine the role of ZAP-70 in thymocyte development, we constructed a retrovirus that is capable of producing ZAP-70 and GFP (Fig. 2GoA). The IRES sequence allows cap-independent translation (30, 31), so that GFP-encoding RNA transcripts can be translated even though the IRES-GFP sequence is located downstream of the translation termination sequence of ZAP-70. The recombinant virus indeed produced both ZAP-70 (Fig. 2GoB) and GFP (Fig. 2GoC) in the cells, thereby enabling ZAP-70-expressing cells to be identified and sorted out by virtue of their GFP expression. The lower expression of GFP by ZAP-70-expressing virus than the virus expressing GFP alone (Fig. 2GoC) appeared to be consistent for the IRES-GFP virus construction used in the present study, since we observed a similar decrease of GFP expression in the bicistronic viruses producing MKK1, MKK6, and calcineurin instead of ZAP-70 (T.S. and Y.T., unpublished observation). Nonetheless, GFP+ cells sorted out from the infection culture abundantly expressed virus-transferred human ZAP-70 in addition to endogenous mouse ZAP-70 (Fig. 2GoD), although the limited numbers of fetal thymocytes (5 x 103 to 1 x 104 GFP+ cells in a typical experiment) did not allow us to detect ZAP-70 proteins in gene-transferred thymocytes by immunoblot analysis. The difficulty in obtaining large numbers of cells should be noted as a limitation of the FTOC-based gene transfer approach. A similar gene transfer approach for human CD34+ T/NK-progenitor cells has been recently reported (32).



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  		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 pGD' retrovirus vector containing GFP-S65T attached with IRES sequence. B, Expression of ZAP-70 by virus-producing cells. Lysates from GP+E-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 GP+E-86 control packaging cells indicates the background fluorescence levels. D, Expression of human ZAP-70 by virus-infected thymocytes. RNA transcripts from 5000 GFP+ fetal thymocytes (FT) infected with indicated viruses were reverse transcribed using random oligonucleotide hexamers and amplified for indicated PCR primers. 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.

 
Introduction into immature thymocytes of kinase-inactive ZAP-70 mutant inhibits T cell development beyond the CD4-CD8- stage

Immature CD4-CD8- thymocytes from day 14 fetal mice were infected with retroviruses producing GFP and either wild-type ZAP-70 or a catalytically inactive mutant of ZAP-70 (KD-ZAP), which can antagonize ZAP-70 signals in mature T cells (21). As shown in Figure 3Go, the development of CD4-CD8- thymocytes was severely impaired by the introduction of KD-ZAP. Since the developmental arrest appeared to be mapped to the early stage of T cell development before the generation of CD4+CD8+ thymocytes, gene-introduced cells were cultured in FTOC for <10 days so that we could focus our analysis on the effect of KD-ZAP on early T cell development.



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  		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 CD45.1- thymocytes in FTOC were measured on day 6 (Expt. 1) or day 9 (Expt. 2) in FTOC, as described in the legend to Figure 1Go. In parallel staining analyses, >98% of CD45.1- cells expressed CD45.2+, indicating that CD45.1- cells were indeed derived from B6 thymocytes. CD4-CD8+ cells found in KD-ZAP-introduced FTOC (Expt. 2) were mostly TCR-/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 1Go. Expression profiles of GFP and CD45.1 in all cells recovered from FTOC are indicated in the left-hand panels. CD4/CD8 profiles of CD45.1- 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-ß (APC-H57, PharMingen) and TCR-{delta} (PE-GL3, PharMingen); and three-color analyzed for GFP CD45.1 and CD44 (PE-IM7, PharMingen). Shown are fluorescence profiles of indicated molecules by CD45.1--gated B6-derived thymocytes. Data shown in B and C are representative of three individual experiments.

 
The introduction of either wild-type ZAP-70 or GFP alone did not affect the generation of CD4+CD8+ thymocytes (Fig. 3GoA), suggesting that the inhibition by KD-ZAP is caused by the lack of ZAP-70 kinase activity and is not solely due to overexpression of ZAP-70, which is capable of interacting with Lck (33)-, Shc (34)-, and ITAM-containing receptors (35). The block of thymocyte development by KD-ZAP was more severe in GFPhigh cells, i.e., almost complete arrest at CD4-CD8- stage, than in GFPlow cells, i.e., arrest mostly at CD4-CD8+ intermediate precursor stage (Fig. 3GoB and the legend for Fig. 3GoA), and GFPhigh cells indeed expressed higher levels of KD-ZAP transcripts than GFPlow cells (data not shown), suggesting that the kinase-inactive ZAP-70 mutant inhibits T cell development in a dominant negative manner and that the variation in the inhibition by KD-ZAP (Fig. 3GoA, Expt. 1 vs 2) is due to the variation in the levels of KD-ZAP expression. Nonetheless, T cell development was impaired even in GFPlow cells derived from KD-ZAP-introduced GFP+-sorted cells (Fig. 3GoB), suggesting that most cells have expressed KD-ZAP during FTOC. KD-ZAP introduction inhibited T cell development at the immature CD44high stage within the CD4-CD8- compartment, resulting in the developmental arrest of TCR-ß+ TCR-{alpha}ß lineage cells as well as TCR-{delta}+ TCR-{gamma}{delta} lineage cells (Fig. 3GoC). Thus, the introduction of KD-ZAP in immature thymocytes severely inhibits T cell development beyond the CD4-CD8- stage, both for TCR-{alpha}ß and TCR-{gamma}{delta} lineages.

Kinase-inactive ZAP-70 Inhibits CD4-CD8- thymocyte development even in the absence of endogenous ZAP-70

ZAP-70-knockout mice (36) and mutant mice expressing unstable ZAP-70 (37) have been shown to exhibit defects in both the positive and negative selection of CD4+CD8+ thymocytes. Immunodeficient patients lacking the expression of ZAP-70 also show a defect in the generation of CD8+ T cells beyond the CD4+CD8+ stage (38, 39, 40). The generation of CD4+CD8+ cells in these ZAP-70-deficient thymocytes appears inconsistent with our results indicating an earlier arrest at the CD4-CD8- stage of thymic development following the introduction of catalytically inactive ZAP-70. Transient introduction of KD-ZAP in Jurkat cells specifically inhibited TCR-induced NFAT activation without exhibiting a generalized suppressive effect on transcription (21), suggesting that KD-ZAP did not inactivate immature thymocytes in a nonspecific manner. It is possible, however, that ZAP-70 signals are involved in, but not essential for, the signals inducing the generation of CD4+CD8+ thymocytes, triggered by the pre-TCR complex containing ITAM motifs (41). In addition to ZAP-70, another member of the Syk/ZAP-70 tyrosine kinase family, Syk, is expressed by CD4-CD8- thymocytes (42). In the absence of ZAP-70, Syk may compensate for the signals inducing CD4+CD8+ thymocytes, whereas KD-ZAP may inhibit those signals by competing for Syk binding sites. This possibility is indeed supported by our results showing that KD-ZAP inhibited the generation of CD4+CD8+ thymocytes even in ZAP-70-knock-out mice (Fig. 4Go), indicating that KD-ZAP can interfere with signaling events not necessarily mediated by ZAP-70. The interchangeable but essential involvement by ZAP-70 and Syk in the signals inducing CD4+CD8+ thymocytes has also been supported by the recent finding that T cell development is arrested at the CD4-CD8- stage of thymocytes in ZAP-70-/Syk- double knock-out mice (43).



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  		FIGURE 4. The effects of KD-ZAP expression in thymocyte development of ZAP-70-knockout mice. Day 14 fetal thymocytes from ZAP-70-/- 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 CD45.1- thymocytes were measured on day 10 in FTOC, as described in the legend to Figure 1Go. Expression profiles of GFP and CD45.1 in all cells recovered from FTOC are indicated in the left-hand panels. CD4/CD8 profiles of CD45.1- ZAP-70-/--derived thymocytes further gated by the expression levels of GFP are indicated in the right-hand panels. CD4-CD8+ cells found in the KD-ZAP-introduced culture were mostly TCR-/low, representing immature precursor cells rather than mature T cells (47–49). Shown are representative results of four individual experiments.

 
Finally, our results showing that KD-ZAP inhibits the generation of TCR-{gamma}{delta}+ T cells in FTOC suggest a novel possibility that ZAP-70/Syk family kinases may also be involved in the generation of TCR-{gamma}{delta}-lineage T cells in the thymus. Thus, our results extend the previous results showing that ZAP-70- mice are selectively deficient in the intestinal intraepithelial TCR-{gamma}{delta}+ T cells (44), whereas Syk- mice exhibit selective deficiency in dermal and intestinal TCR-{gamma}{delta}+ T cells (45, 46).

The present results indicating successful retrovirus gene transfer into immature thymocytes and efficient detection in FTOC of gene-transferred cells using GFP provides a powerful method to further analyze the signaling events regulating T cell development in the thymus. These gene transfer techniques also have great potential for treating various immunodeficiencies to restore T cell development.


   Acknowledgments
 
We thank Dr. D. Baltimore and R. Hawley for pGD' and pMSCV vectors; Dr. A. Bank for GP+E-86 cells; Dr. Y. Katsura for help on high oxygen submersion culture; Dr. I. Negishi for ZAP-70-/- mice; S. Tanaka and Y. Morita for technical help; Drs. T. Saito and A. Weiss for critical comments during the study; and Drs. D. Alexander, M. Iwashima, and E. W. Shores for reading the manuscript.


   Footnotes
 
1 This work was supported by the University of Tsukuba Research Projects, PRESTO Research Project "Unit Process and Combined Circuit," and the Ministry of Education, Science, Sports, and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Y. Takahama, Department of Immunology and PRESTO Research Project, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8577, Japan. E-mail address: Back

3 Abbreviations used in this paper: GFP, green fluorescence protein; IRES, internal ribosomal entry site; KD-ZAP, kinase-dead mutant of ZAP-70; FTOC, fetal thymus organ culture; dGuo, 2-deoxyguanosine; ITAM, immunoreceptor tyrosine-based activation motif. Back

Received for publication April 3, 1998. Accepted for publication May 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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