Signaling and T Cell Development Differential Requirements for ZAP-70 In

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Differential Requirements for ZAP-70 in TCR Signaling and T Cell Development Theresa A. Kadlecek, Nicolai S. C. van Oers, Leo Lefrancois, Sara Olson, Deborah Finlay, David H. Chu, Kari Connolly, This information is current as Nigel Killeen and Arthur Weiss of September 25, 2021. J Immunol 1998; 161:4688-4694; ; http://www.jimmunol.org/content/161/9/4688 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. Differential Requirements for ZAP-70 in TCR Signaling and T Cell Development1

Theresa A. Kadlecek,* Nicolai S. C. van Oers,2* Leo Lefrancois,§ Sara Olson,§ Deborah Finlay,† David H. Chu,*‡ Kari Connolly,† Nigel Killeen,‡ and Arthur Weiss3*‡

The Syk/ZAP-70 family of protein tyrosine kinases is indispensable for normal lymphoid development. Syk is necessary for the development of B cells and epithelial ␥␦ T cells, whereas ZAP-70 is essential for the normal development of T cells and TCR signaling. In this study, we show that although development of the ␣␤ lineage was arrested in the thymus, CD3-positive T cells, primarily of the ␥␦ lineage, were present in the lymph nodes of mice lacking ZAP-70. Moreover, in the absence of ZAP-70, dendritic epidermal T cells were fewer in number and of abnormal morphology, and intestinal intraepithelial lymphocytes, normally containing a large proportion of ␥␦ T cells, were markedly reduced. These data suggest that ␥␦ T cells show a variable dependence upon ZAP-70 for their development. Biochemical analyses of thymocytes revealed a lack of basal ␨-chain tyrosine Downloaded from phosphorylation. However, several other substrates were inducibly tyrosine phosphorylated following TCR stimulation. Thus, TCR-mediated signaling in ZAP-70-deﬁcient thymocytes is only partially impaired. These studies suggest that Syk compensates only partially for the loss of ZAP-70, and that there is an absolute requirement of ZAP-70 for ␣␤ T cells and epithelial ␥␦ T cells, but not for some ␥␦ T cells in peripheral lymphoid tissues. The Journal of Immunology, 1998, 161: 4688–4694.

ϩ ϩ cell development in the thymus is characterized by a CD4 or CD8 stage (2). These SP thymocytes are the immediate http://www.jimmunol.org/ strictly regulated selection process associated with TCR precursors of the mature peripheral TCR ␣␤-expressing T cells. T rearrangement and a series of phenotypic changes (1, 2). Another lineage of T cells, the TCR ␥␦-expressing cells, can Two developmental checkpoints that depend upon receptor-medi- also develop in the thymus (6). However, although ordered rear- ated signal transduction allow for progression to the next major rangement of TCR ␥ genes and preferential association with dis- developmental stage. The ﬁrst checkpoint occurs when a tinct ␦-chains occur during ontogeny of this lineage (7), distinct Ϫ Ϫ CD4 CD8 (double negative, DN)4 precursor progresses to the ordered developmental checkpoints and phenotypic transitions ϩ ϩ CD4 CD8 (double positive, DP) stage as a result of successful have not been well deﬁned. Moreover, some T cells expressing the TCR ␤-chain gene rearrangement. The expressed TCR ␤-chain ␥␦ ␣␤

TCR may develop extrathymically (8). Like the TCR sub- by guest on September 25, 2021 associates with the pre-T ␣-chain (3, 4). These chains nonco- unit, the TCR ␥␦ subunit associates with the CD3 and ␨ or Fc⑀RI␥ ␨ valently associate with the TCR -chain and CD3 subunits, and the subunits, which mediate the signals leading to cell activation. The resultant expressed pre-TCR complex delivers a signal to allow for TCR ␥␦ T cells are different from TCR ␣␤ T cells in that they may passage beyond the ﬁrst checkpoint. This leads to cellular prolif- have less dependence upon coreceptor (CD4 or CD8) function and ␣ eration and progression to the DP stage, where TCR gene rear- may recognize distinct types of Ags (6). Whereas the functions of rangement is induced (2, 5). The product of the successfully rear- these cells still remain enigmatic, they often appear to localize ␣ ␣ ranged TCR -chain gene replaces the pre-T -chain and preferentially in epithelial tissues. ␣␤ completes the formation of the mature TCR heterodimer. Sig- Biochemical and genetic evidence has demonstrated the require- nals through this TCR are then essential for the passage through ment for two families of PTK, Src and Syk/ZAP-70, in TCR sig- the second checkpoint to the more mature single-positive (SP) naling (reviewed in Refs. 9 and 10). The Syk and ZAP-70 family of PTK share structural features that include tandem SH2 domains and a C-terminal kinase domain. Via their two SH2 domains, *Howard Hughes Medical Institute, Department of Medicine, †Department of Der- ZAP-70 and Syk associate with the constitutively tyrosine-phos- matology, and ‡Department of Microbiology and Immunology, University of Cali- ␨ § phorylated -chain in normal murine thymocytes and with induc- fornia, San Francisco, CA 94143; and Division of Rheumatology, Department of ␨ Medicine, University of Connecticut Health Center, Farmington, CT 06030 ibly phosphorylated - or CD3 chains in T cell lines and clones Received for publication April 17, 1998. Accepted for publication June 30, 1998. (11, 12). The Src-family PTK Lck is required for the constitutive ␨ The costs of publication of this article were defrayed in part by the payment of page and inducible TCR -chain phosphorylation of tyrosine residues charges. This article must therefore be hereby marked advertisement in accordance within the immunoreceptor tyrosine-based activation motif with 18 U.S.C. Section 1734 solely to indicate this fact. (ITAM) (11, 13). The phosphorylation of ZAP-70 as well as its 1 D.H.C. was supported by Medical Scientist Training Program, funded by National catalytic activation is dependent upon TCR stimulation and upon Institute of General Medical Sciences. the function of the Src kinase Lck (11, 14–16). The phosphoryla- 2 Current address: Department of Microbiology and Immunology, University of Texas, Dallas Southwestern, Dallas, TX 75235. tion and activation of Syk may be less dependent upon Src kinase function, suggesting this kinase may be less coreceptor 3 Address correspondence and reprint requests to Dr. Arthur Weiss, Howard Hughes Medical Institute, University of California, 3rd Ave. and Parnassus St., San Francisco, dependent (17, 18). CA 94143-0795. E-mail address: [email protected] Both families of PTK play critical roles in lymphocyte devel- 4 Abbreviations used in this paper: DN, double negative; DETC, dendritic epidermal opment. In Lck-deﬁcient mice, there is a substantial block in TCR T cells; DP, double positive; IEL, intraepithelial lymphocyte; ITAM, immunoreceptor ␣␤ tyrosine-based activation motif; PE, phycoerythrin; PLC, phospholipase C; PTK, pro- T cell development, although DP and SP thymocytes as well as tein tyrosine kinase; SP, single positive. mature T cells do develop in reduced numbers (19). In contrast,

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 4689 mice deficient in the Src kinase Fyn have no apparent develop- locus yielded a 6-kb fragment. Mutant cells were injected into wild-type mental phenotypic abnormalities, although some functional defects C57BL/6 blastocysts. Chimeric mice were backcrossed to the C57BL/6 are present in mature thymocytes and peripheral T cells (17, 18, strain of mice (The Jackson Laboratory, Bar Harbor, ME), and wild-type and heterozygous littermates were used for analysis. Mice were pheno- 20, 21). In mice lacking both Lck and Fyn, there is a complete typed by PCR analysis for the wild-type ZAP-70 gene using primers a block in thymic development at the first developmental check- (5Ј-gcacatatgcactgtccctggtcta-3Ј)andc(5Ј-gggtcgctgtagggactctcgtaca-3Ј), point, the transition from the DN stage to the DP stage, suggesting and for the mutant ZAP-70 gene using primers a and b (5Ј-tggctacccgt a defect in pre-TCR signaling function (22, 23). These results also gatattgctgaaga-3Ј). Mice were bred and maintained in Transgenic Animal ␤ Care Facility at University of California, San Francisco. MHC class I ( 2- suggest that Fyn may play a compensatory role if Lck function is microglobulin gene)/MHC class II (Ab ␤ gene) doubly-deficient mice were limiting. In contrast to the profound effects on the TCR ␣␤ lineage, obtained from a commercial source (Taconic, Germantown, NY). T cells expressing TCR ␥␦ still develop in the Lck/Fyn doubly- Stimulation, immunoprecipitation, and Western blotting deficient mice (22, 23). ZAP-70 and Syk PTK also play critical roles in lymphocyte development. In mice deficient in ZAP-70 or Single cell suspensions of lymph node, spleen, and thymus were prepared. carrying a mutation that inactivates ZAP-70 kinase function, there Thymocytes were stimulated with purified anti-CD3 mAb (2C11) for 3 min at 37°C. Pervanadate stimulation of thymocytes was performed at 37°C for is an arrest at the DP to SP stage in the thymus, representing a 3 min, as described (13). Cells were lysed in 1% Nonidet P-40 lysis buffer block at the second developmental checkpoint (24, 25). No defect containing protease and phosphatase inhibitors. Lysates were centrifuged at in TCR ␣␤ thymocyte development is evident in Syk-deficient 10,000 ϫ g, and supernatants were immunoprecipitated with anti-Syk (5F5 mice, although B cell development is arrested, and certain TCR ␥␦ mAb, to be described in detail elsewhere (D. Chu, N. van Oers, and A. T cells in epithelial tissues are absent (26–28). However, a com- Weiss, manuscript in preparation)), anti-Cbl (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PLC-␥ mAb (Upstate Biotechnology, Lake Placid, 1 Downloaded from plete arrest in the progression from the DN stage to DP stage NY), anti-Slp-76 (sheep antisera, a gift from G. Koretzky, University of occurs in mice deficient in both Syk and ZAP-70 (29). This indi- Iowa, Iowa City, Iowa), anti-TCR ␨ (6B10.2 mAb), and anti-Vav (Upstate cates that Syk most likely can only play a compensatory role at the Biotechnology) antisera using either protein A- or protein G-Sepharose DN to DP developmental checkpoint in mice. When overex- beads (Pharmacia Biotech, Piscataway, NJ). Immunoprecipitates were subjected to SDS-PAGE, and proteins were transferred to polyvinyulidene pressed, under the influence of the Lck proximal promoter, Syk difluoride (PVDF) membrane. Membranes were blotted with anti- can rescue the development of SP cells (30). Since the expression phosphotyrosine mAb (4G10 mAb; Upstate Biotechnology), followed by of Syk decreases after the DP stage (31), these results suggest that horseradish peroxidase-conjugated secondary antisera, and developed us- http://www.jimmunol.org/ the expression of Syk limits thymic development of the ␣␤ lineage ing the ECL (Amersham, Arlington Heights, IL) detection system. The of T cells in ZAP-70-deficient mice. Syk appears to play a critical blots were then stripped and reblotted with immunoprecipitating Ab. Ly- ␥␦ sates were diluted with SDS sample buffer and resolved by SDS-PAGE, ZAP-70-independent role in the development of the TCR cells transferred to PVDF membrane, and immunoblotted with anti-ZAP-70 present in epithelial tissues. (1G7 mAb) or anti-phosphotyrosine Ab (4G10). An apparent paradox arises from studies of patients harboring FACS analysis mutations in the ZAP-70 gene. There is an absence of CD8ϩ T cells in their peripheral blood, and the CD4ϩ T cells present are Single cell suspensions of lymph nodes, spleen, and thymus were incubated unable to transduce signals via their TCRs (32–34). Although the with FITC-, PE-, and tri-color-conjugated anti-CD3, anti-CD4, anti-CD8,

anti-␥␦ TCR, anti-TCR-␣␤, and anti-B220 Abs (PharMingen, San Diego, by guest on September 25, 2021 TCRs on the peripheral T cells fail to signal, TCRs in at least some CA). Cells were analyzed on a FACScan (Becton Dickinson, San Jose, CA) DP thymocytes do signal in the one patient studied (35). It is pos- using Cellquest software. DP thymocytes were sorted on a dual argon laser sible that Syk may compensate for ZAP-70 in the signaling func- FACStar (Becton Dickinson, Milpitas, CA). tions of this patient’s thymocytes, but not in the periphery. As Syk Dendritic epidermal T cell staining is not preferentially expressed in CD4 or CD8 lineage T cells, differences in Syk expression in thymic subsets could account for Mouse ears were amputated at the base immediately after sacrifice. Hair removal lotion (Nair; Carter-Wallace, New York, NY) was used to depilate differences between the development of T cells in ZAP-70-defi- the skin, which was then separated from the underlying cartilage using a cient mice versus humans. A comparative analysis of the Syk ex- dissection microscope. The dissected skin was pressed dermal side down pression and TCR signal transduction in ZAP-70-deficient mice onto an adhesive tape window (Instrumedics, Hackensack, NJ), and the and humans, which may help explain the distinct developmental epidermis was lightly coated with cyanoacrylic glue (Superglue; Super phenotypes, remains to be completed. Glue, Hollis, NY) to provide a structural support for the fragile epidermal layer. The skin was then placed dermal side up on an adhesive-coated A detailed analysis of signaling in thymocytes from ZAP-70- microscope slide (Instrumedics) and incubated with 20 mM EDTA in PBS deficient mice has not yet been performed. Therefore, ZAP-70- for3hat37°C. Finally, the dermis was carefully pulled off the epidermis deficient mice were used to address the residual signaling capacity with the aid of fine forceps and a dissecting microscope. Dendritic epider- of murine thymocytes and to perform a more comprehensive anal- mal T cells (DETC) were stained with a 1/100 dilution of FITC-conjugated pan TCR ␦ Ab (PharMingen). ysis of the development of both ␣␤ and ␥␦ T cells. Isolation of IEL Materials and Methods IEL were isolated essentially as described (36). Briefly, the small intestines Cell lines and animals of individual mice were cut into 5-mm pieces and washed twice with me- ␭ dium. The washed intestinal pieces were stirred at 37°C for 20 min in A GT11 library was screened with a cDNA probe to obtain a genomic medium with addition of 1 mM dithioerythritol. This step was repeated, clone of ZAP-70. ZAP-70-deficient mice were generated by homologous and the resultant supernatants were rapidly filtered through nylon wool and recombination of a targeting vector into a 129 ES cell line, clone JM1 the filtrate centrifuged through a 44/67% Percoll step gradient. The cells at (obtained from Roger Pedersen, University of California, San Francisco). Ј ϳ the interface of the Percoll gradient were collected and prepared for flow The targeting vector contained the TK gene at the 5 end followed by 2 cytometry. kb of ZAP-70 genomic sequence, the neomycin resistance gene replacing the ScaI-AatII sequence, followed by another ϳ2 kb of homologous Results genomic sequence. ES cells were selected in growth media containing 2 ␮M gancyclovir (Cytovene; Syntex, Palo Alto, CA) and 200 ␮g/ml G418 ZAP-70 protein is absent in the mutant mice (Geneticin; Life Technologies, Grand Island, NY). Clones were screened ZAP-70 mice were generated by standard gene-targeting technol- by Southern blot analysis of PstI-digested genomic DNA using a ϳ300-bp probe comprised of ZAP-70 genomic sequence immediately 5Ј of the tar- ogy. The targeting vector deleted a segment of the ZAP-70 gene geting construct. Hybridization of this probe to wild-type DNA in Southern (encoding amino acids 221 to 520) that included the C-terminal blots yielded a 10-kb fragment, in which DNA from a properly targeted SH2 domain and replaced it with the neomycin resistance gene 4690 DEVELOPMENT AND SIGNALING IN ZAP-70-DEFICIENT MICE

FIGURE 1. ZAP-70 is absent in genetically targeted mice. A, A sche- Downloaded from matic diagram of the wild-type genomic structure and of the targeting Ϫ/Ϫ vector. The black boxes represent exons within the ZAP-70 gene. a, b, and FIGURE 3. CD3-positive T cells in ZAP-70 mice are primarily of ␥␦ c represent primers used for PCR screening of genomic DNA. B, PCR lineage. Single cell suspensions of lymph node, spleen, and thymus ϩ Ϫ ␣␤ ␥␦ screen of genomic DNA from ZAP-70 / -crossed mice. PCR performed were stained with Abs to TCR (FITC) and TCR (PE). Cells were with primers a and b amplify ZAP-70 genomic DNA in which the targeted gated on lymphocytes using forward and side scatter analysis. The per- neomycin resistance gene replaces ϳ3 kb of genomic sequence. Primers a centages of cells in each quadrant are noted. and c amplify the intact wild-type genomic sequence. C, Anti-ZAP-70 http://www.jimmunol.org/ Western blot analysis of thymocyte lysates from ϩ/ϩ, Ϫ/ϩ, and Ϫ/Ϫ littermates. viously reported (24, 25), an arrest at the DP stage of development was observed in the mutant mice. There were few, if any, CD3high- (Fig. 1A). Mutant ES cells and the mice derived from them were positive cells found in the thymuses of the ZAP-70Ϫ/Ϫ mice. How- identiﬁed by Southern blot and PCR analysis. PCR analysis of ever, the levels of CD3 on wild-type and ZAP-70Ϫ/Ϫ DP thymo- genomic DNA was employed to phenotype mice. One set of PCR cytes were comparable when analyzed by FACS. The spleens of primers detected the wild-type genomic sequence, and the other set the mutant mice were primarily composed of B cells, since ap-

of primers detected the mutant ZAP-70 sequence containing the proximately 90% of splenocytes stained positively for B220, and by guest on September 25, 2021 neomycin resistance gene. This PCR strategy allowed for the iden- very few CD3-positive cells were present. The lymph nodes in the tification of wild-type, heterozygous, and homozygous deficient mutant mice were smaller than wild type and were predominantly mice (Fig. 1, A and B). Unlike the wild-type or heterozygotes, no composed of B cells. Surprisingly, unlike the thymus and spleen, Ϫ Ϫ ZAP-70 protein was detected in thymocytes from ZAP-70 / lymph nodes of the ZAP-70-deficient mice contained a significant mice by Western blot analysis (Fig. 1C). The heterozygous mice percentage of CD3-positive cells (5–10%), but few of these cells expressed an intermediate level of ZAP-70 protein in the thymus. expressed CD4 or CD8. The CD3-positive T cells found in the spleen and lymph nodes were primarily ␥␦ T cells (Fig. 3). In Thymocyte and peripheral splenic and lymph node T cell contrast to wild-type littermates, in which less than 1% of CD3ϩ development in ZAP-70-deficient mice T cells were of the ␥␦ lineage, the majority of CD3ϩ cells in lymph Thymocytes from ZAP-70ϩ/ϩ and ZAP-70Ϫ/Ϫ littermate mice nodes of the ZAP-70-deficient mice were of the TCR ␥␦ lineage. were analyzed for expression of CD4 and CD8 (Fig. 2). As pre- This did not appear to reflect a peripheral expansion of TCR ␥␦ T

FIGURE 2. T cell development is arrested in ZAP-70Ϫ/Ϫ mice. Single cell suspensions of lymph node, spleen, and thymus from 6- to 8-wk- old littermates were stained with FITC- and PE-conjugated anti-CD4, anti-CD8, anti-B220, and anti-CD3. The data were collected from lymphocytes gated by forward and side scatter analysis using a Becton Dick- inson FACScan with the CellQuest program. A total of 104 events was collected to generate each dot plot, and the percentage of cells in each quadrant is noted. The quadrant boundaries for dot plots of thymocytes stained for CD3 and B220 were set to quantitate CD3high cells. The Journal of Immunology 4691

FIGURE 5. ZAP-70 is required for CD8ϩ ␥␦ intestinal intraepithelial lymphocyte development. IEL, isolated as described from ZAP-70-deﬁ- cient and heterozygous littermates, were stained with anti-TCR ␤ or anti- TCR ␦ mAbs and analyzed by ﬂow cytometry. Downloaded from

first developmental checkpoint (DN to DP stages) suggests that some ZAP-70-independent TCR signal transduction can occur. To investigate this possibility, thymocytes from ZAP-70Ϫ/Ϫ mice and littermate controls were stimulated in vitro with anti-CD3 Ab. Whole cell lysates from thymocytes of mutant mice blotted with anti-phosphotyrosine mAb showed anti-CD3-dependent induction http://www.jimmunol.org/ of some phosphoproteins, albeit fewer than heterozygote or wild- FIGURE 4. DETC are reduced in number and of abnormal morphology type control littermates (Fig. 6). Note that the greater induction of Ϫ Ϫ ϩ ϩ Ϫ Ϫ in ZAP / mice. Epidermal sheets from ZAP-70 / (A) and ZAP-70 / tyrosine phosphorylation in the heterozygote was not reproducibly ␦ (B) mice were stained with FITC-conjugated anti-pan TCR Ab and an- seen. ϫ alyzed by immunofluorescence (shown at 400 magnification). To determine which proteins were tyrosine phosphorylated in the absence of ZAP-70, immunoprecipitation and Western blot cells, since the absolute number of these cells was comparable with analyses were performed. Phosphorylation of ITAMs within the ␨ that seen in the wild-type mice. -chain is required for ZAP-70 binding. In resting thymocytes, the by guest on September 25, 2021 ␨-chain is constitutively phosphorylated, and receptor stimulation DETC are abnormal in number and appearance in ZAP-70Ϫ/Ϫ leads to phosphorylation of the bound ZAP-70. However, in ex- mice amining long exposures of Western blots of whole cell lysates Ϫ/Ϫ DETC are characterized by the dendritic morphology of their ex- obtained from ZAP-70 mice, there was no evidence for con- ␨ tensions and their surface expression of a V␥3/V␦2 monomorphic stitutive or inducible -chain tyrosine phosphorylation (data not TCR (6). Epidermal sheets were stained with a FITC-conjugated pan TCR ␦ Ab to assess the role of ZAP-70 in the development of this cell population. In ZAP-70-deficient mice, there were somewhat fewer DETC as compared with wild-type littermates (Fig. 4). Moreover, the positively staining cells from the mutant mice had an abnormal morphology that lacked the characteristic dendritic extensions seen in wild-type cells.

ZAP-70 is required for ␥␦ intestinal intraepithelial lymphocytes (IEL) development The origin of IEL is a subject of considerable controversy (8), but it is likely that a signiﬁcant proportion of TCR ␥␦ IEL do not develop within the thymus, while most TCR ␣␤ IEL require a thymus for maturation (37). Unlike peripheral TCR ␥␦ T cells, which are largely CD4Ϫ/CD8Ϫ, nearly all IEL TCR ␥␦ T cells express a CD8 ␣␣ homodimer (38). In contrast to results obtained with lymph nodes that contained substantial numbers of TCR ␥␦ T cells (Fig. 3), few IEL expressed ␥␦ or ␣␤ TCRs (Fig. 5). The Ϫ/Ϫ small percentage of IEL TCR ␥␦ T cells detected expressed lower FIGURE 6. Reduced induction of tyrosine phosphorylation in ZAP ϫ 6 levels of TCR than their normal counterparts. thymocytes following CD3 stimulation. Thymocytes (5 10 ) from ZAP- 70Ϫ/Ϫ (lanes 1 and 2), ZAP-70Ϫ/ϩ (lanes 3 and 4), and ZAP-70ϩ/ϩ (lanes TCR signal transduction in ZAP-70-deﬁcient thymocytes 5 and 6) were stimulated with anti-CD3 Ab (2C11). Whole cell lysates ϩ from unstimulated (lanes 1, 3, and 5) and CD3-stimulated (lanes 2, 4, and The presence of CD3 T cells in lymph nodes suggests that T cell 6) thymocytes were subjected to SDS-PAGE, transferred to membranes, development may not be blocked completely in the absence of and blotted with anti-phosphotyrosine mAb (4G10). The relative migration ZAP-70. Moreover, the ability of thymocytes to pass through the of m.w. markers is indicated on the left. 4692 DEVELOPMENT AND SIGNALING IN ZAP-70-DEFICIENT MICE

mocytes allowed for tyrosine phosphorylation of the ␨-chain, although at lower levels than that of wild-type cells (data not shown). The difference in basal ␨ phosphorylation could reflect the absence of ZAP-70 protein and our previously reported (39) function of its SH2 domains in protecting the phosphorylated ITAM tyrosines, or may be due to another yet to be identified function provided by the ZAP-70 protein. Syk can play a compensatory role in cells lacking ZAP-70. Therefore, Syk was analyzed for evidence of its activation following CD3 stimulation. Immunoprecipitation of Syk following stimulation with anti-CD3 mAb demonstrated that Syk was inducibly tyrosine phosphorylated in ZAP-70-deficient, heterozygous, and wild-type thymocytes (Fig. 8). Several other proteins known to be important in TCR signaling were also immunoprecipitated and FIGURE 7. TCR ␨ is not constitutively tyrosine phosphorylated in blotted with anti-phosphotyrosine Abs before and after CD3 stim- ZAP-70 mutant thymocytes. Whole cell lysates from total thymocytes or ulation. Slp-76, Vav, Cbl, and PLC-␥1 proteins were immunopre- sorted CD4/CD8 DP thymocytes from ZAP-70Ϫ/Ϫ and WT mice were cipitated to analyze their state of tyrosine phosphorylation. In all subjected to SDS-PAGE, transferred to membrane, and blotted with anti- cases, the proteins were tyrosine phosphorylated, but at lower phosphotyrosine mAb (4G10), anti-␨ mAb (2F3.2), and sheep anti-mouse Downloaded from Slp-76 (gift from G. Koretzky). (modestly lower in some cases) levels than wild-type (Fig. 8). As a control, thymocytes from MHC class I/MHC class II doubly- deficient mice were analyzed since they are blocked at the same shown). Further studies were performed to analyze the constitutive stage of thymic development as are the ZAP-70-deficient mice. ␥ phospho-␨ levels in cell lysates of total thymocytes and of sorted PLC- 1, Slp-76, and Syk were all tyrosine phosphorylated in these Ϫ/Ϫ MHC-deficient thymocytes following stimulation with anti-CD3 DP thymocytes from ZAP-70 and wild-type mice. There was http://www.jimmunol.org/ no detectable phospho-␨ present in either total thymocytes or Abs. Since there was not a complete blockade of tyrosine-phos- sorted DP thymocytes from the ZAP-70 mutant mice, in contrast to phorylated substrates downstream of ZAP-70, an attempt was the constitutive phospho-␨ present in wild-type thymocytes (Fig. made to identify any missing tyrosine-phosphorylated proteins. An 7). Levels of total ␨ as well as Slp-76, a control for protein loading, anti-phosphotyrosine Western blot of anti-phosphotyrosine immu- were equivalent in all lanes. Thymocytes were also treated with the noprecipitates from unstimulated and stimulated thymocyte lysates phosphatase inhibitor pervanadate to determine whether the failed to show any obvious missing bands, except for bands at the ␨-chain was capable of being phosphorylated in the absence of apparent mobility of ZAP-70 and of TCR ␨ (data not shown). ZAP-70 protein. Pervanadate treatment of ZAP-70-deficient thy- These data suggest that the TCRs or pre-TCRs in DP and/or DN by guest on September 25, 2021

FIGURE 8. Tyrosine-phosphorylated proteins are induced by TCR stimulation in ZAP-70-deficient thymocytes. A, Thymocytes from ZAP-70Ϫ/Ϫ, ZAP-70Ϫ/ϩ, ZAP-70ϩ/ϩ, and MHC class Ϫ/ϪMHC class IIϪ/Ϫ mice were incubated with or without anti-CD3 Ab for 3 min at 37°C. Cells were lysed and immunoprecipitated with Abs against PLC-␥1, Syk, and Slp-76. Immunoprecipitates were subjected to SDS-PAGE, transferred to membrane, and analyzed by Western blot with anti-phosphotyrosine Ab. These immunoblots were then stripped and reprobed with Ab specific to the immunoprecipitating Ab. B, Thymocytes from ZAP-70Ϫ/Ϫ, ZAP-70Ϫ/ϩ, and ZAP-70ϩ/ϩ mice were stimulated and lysed as above, and lysates were immunoprecipitated with Abs against Vav and Cbl. Immunoprecipitates were subjected to SDS-PAGE and anti-phosphotyrosine Western blotting. The blots were stripped and reprobed with Ab against the immunoprecipitating Ab. (Note that the anti-Vav immunoprecipitates were run on the same gel, but were transposed to fit the format of the other blots.) The Journal of Immunology 4693

ZAP-70 thymocytes can couple to Syk and to several other down- ZAP-70-deficient thymocytes. This is particularly relevant since stream substrates. However, it is possible that the inducibly phos- thymocytes from ZAP-70-deficient humans can signal in response phorylated substrates observed when whole thymocytes were stim- to TCR stimulation (35). However, a detailed analysis of tyrosine ulated reflect those present only in a subset of cells, for instance in phosphoproteins has not previously been performed. In this study, the few less mature DN cells present in the unfractionated thymo- we show that a number of substrates, including Syk, Vav, PLC-␥1, cyte populations. Slp-76, and Cbl, are tyrosine phosphorylated following CD3 stimulation in the thymocytes from ZAP-70-deficient mice. Syk may compensate for the loss of ZAP-70 in the DP and/or DN thymo- Discussion cytes and phosphorylate these downstream proteins. Indeed, we These studies support the view that ZAP-70 is strictly required for could detect Syk-inducible phosphorylation following CD3 stim- the development of ␣␤ T cells, but is variably required for ␥␦ T ulation. However, the proteins that are phosphorylated may not be cells. Although few or no CD3ϩ T cells were present in the thy- phosphorylated to the same extent or on the same tyrosine residues muses or spleens of the mutant mice, a distinct population of that are normally phosphorylated by ZAP-70, thus explaining the CD3ϩ cells was reproducibly present in the lymph nodes. In the blockade in the signaling events necessary for the DP to SP tran- mutant mice, these cells were primarily TCR ␥␦ lineage T cells. sition. However, relevant to these observations are recent studies This observation also held true for the few CD3ϩ cells that were in our laboratory of mouse thymocytes that demonstrate a down- present in the spleens. In contrast, the absence of ZAP-70 pro- regulation of Syk as DN thymocytes transit to DP thymocytes foundly affected the development of IEL and DETC. A similar (Chu et al., manuscript in preparation). Thus, the observed Syk requirement for ZAP-70 in the development of IEL was reported phosphorylation may have occurred in response to CD3 stimula- Downloaded from recently for another ZAP-70-deficient mouse (30). Interestingly, in tion in the DN thymocytes, and this activation led to the induced contrast to the absence of IEL, DETC were reduced in number, but tyrosine phosphorylation of downstream signaling proteins. Such more strikingly exhibited a morphologic change in which dendritic signaling by Syk is likely to be responsible for thymocytes to pass processes were absent. Thus, ZAP-70 is not only required for the the checkpoint from DN to DP cells in these mutant mice (29). development of conventional TCR ␣␤-expressing cells, but also A very proximal protein in the TCR signaling pathway that was for the development, proliferation, and/or maturation of ␥␦ cells not detectably phosphorylated in ZAP-70-deficient thymocytes http://www.jimmunol.org/ that reside in epithelial tissues. was the TCR ␨-chain. In ex vivo murine thymocytes, TCR ␨ is The development of the TCR ␥␦ lineage was variably affected constitutively tyrosine phosphorylated and associated with by the absence of ZAP-70. This suggests that development of dis- ZAP-70 via the ZAP-70 SH2 domains (11–13). Lck is required for tinct subsets of TCR ␥␦-expressing cells may not have the same this constitutive phosphorylation of ␨, and it has been proposed TCR signaling requirements. However, it is important to note, that ZAP-70 association can protect the dephosphorylation of there is no evidence that the lymph node TCR ␥␦ T cells in the phospho-␨ since overexpression of the tandem ZAP-70 SH2 do- mutant mice are functionally normal. mains alone is sufficient to increase ␨-chain phosphorylation (39). The requirement for ZAP-70 in TCR ␣␤ T cell development is Syk may not be able to sustain this association or may not be not surprising and is consistent with observations made in other present at sufficient levels in DP thymocytes and, consequently, ␨ by guest on September 25, 2021 ZAP-70-deficient or ZAP-70 kinase mutant mice (24, 25). How- would be dephosphorylated in the absence of ZAP-70. ever, its relative requirement for development of TCR ␥␦ T cells As more detailed analyses of TCR signaling and developmental in epithelial tissues is somewhat surprising. A similar requirement regulation are performed, the complexity of the signaling pathways for Syk has previously been reported for the development of these responsible for developmental decisions is revealed. Through mul- epithelial T cells (28). The requirement for Syk is consistent with tiple checkpoints in the developmental process, an intact immune the notion that this kinase has less dependence upon Src kinases system matures. Kinase families with multiple members can com- (17, 18), and, by inference, coreceptor function. Since most TCR pensate for the loss of one another in some, but not all, cases, ␥␦ T cells lack the CD4 and CD8 coreceptors, it was not surprising allowing for normal development and signaling. These studies to find that Syk is important for the development of these T cells show that although Syk may be able to compensate for the lack of in epithelial tissues. By contrast, a similar requirement for ZAP-70 ZAP-70 to some degree in some ␣␤ lineage thymocytes, ZAP-70 in the development of these same cells is surprising. These unex- and Syk PTK are both required for the normal development of pected findings suggest that ZAP-70 and Syk may not play com- certain ␥␦ lineage T cells. pletely overlapping functions in the development of these cells. It is unclear whether this reflects distinct functions of these PTK or Acknowledgments distinct temporal expression of these PTK during the development We thank Lena Dilacio for technical assistance in breeding ZAP-70-defi- of TCR ␥␦ epithelial T cells. Perhaps the presence of one of the cient mice. family members is sufficient for immature cells to migrate to the appropriate tissue, such as the skin, but differentiation cannot occur References without both ZAP-70 and Syk present. An alternative, but less likely possibility is that these PTK simply have additive identical 1. Malissen, B., and M. 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