Chains in Immature Thymocytes Ζ Competence of CD3-Independent TCR Surface Expression and Functional

Surface Expression and Functional Competence of CD3-Independent TCR ζ -Chains in Immature Thymocytes

This information is current as Fabio Grassi, Eliane Barbier, Simona Porcellini, Harald von of October 2, 2021. Boehmer and Pierre-André Cazenave J Immunol 1999; 162:2589-2596; ; http://www.jimmunol.org/content/162/5/2589 Downloaded from References This article cites 77 articles, 39 of which you can access for free at: http://www.jimmunol.org/content/162/5/2589.full#ref-list-1

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Surface Expression and Functional Competence of CD3-Independent TCR ␨-Chains in Immature Thymocytes1

Fabio Grassi,2*† Eliane Barbier,† Simona Porcellini,* Harald von Boehmer,‡ and Pierre-Andre´Cazenave†

In recombinase-deficient (RAG-2؊/؊) mice, double-negative thymocytes can be stimulated to proliferate and differentiate by anti-CD3 Abs. CD3 molecules are expressed on the surface of these cells in association with calnexin. In this study, we show that ␨-chains can be recovered as phosphorylated proteins in association with phosphorylated ZAP-70 from anti-CD3-stimulated RAG-2؊/؊ thymocytes, even though they are not demonstrably associated with the CD3/calnexin complex. The lack of a physical association of ␨ dimers with the CD3 complex in RAG-2؊/؊ thymocytes and also in a pre-TCR-expressing cell line, as well as the efficient association of ␨ dimers with ZAP-70 in the RAG-2؊/؊ thymocytes, suggest that these ␨-chain dimers could contribute to pre-TCR signaling. This idea is supported by the finding that in RAG-2؊/؊ ␨-deficient thymocytes, ZAP-70 and p120cbl were only Downloaded from weakly phosphorylated. The Journal of Immunology, 1999, 162: 2589–2596.

evelopment of ␣␤ thymocytes is characterized by the lacking both Lck and Fyn (8, 9) and mice lacking both ZAP-70 and transition of cells through an ordered sequence of dis- Syk (10) exhibit a phenotype that is strikingly similar to the one of D tinct phenotypes, which can be defined by the expression RAGϪ/Ϫ mice (11), implying a critical role for these molecules in of the coreceptor molecules CD4 and CD8. Early in development, the proximal signal transduction by the pre-TCR. The role played http://www.jimmunol.org/ the transition of the most immature double-negative (DN)3 to the by CD3 and ␨-chains in pre-TCR assembly and signaling has been double-positive (DP) stage is largely dependent upon the produc- only partially elucidated. Studies with mutant mice have revealed tive rearrangement of TCR␤ genes and expression of TCR an absolute requirement for CD3⑀-chains (12), no essential re- ␤-chains in association with monomorphic pT␣ chains (1). Cell- quirement for CD3␦-chains (13, 14), and a major role for ␨-chains autonomous signaling by the pre-TCR results in ␤ selection, i.e., (15–18). ␨Ϫ/Ϫ mice have a scarcely populated thymus and display ␤ selection of TCR -expressing cells for survival; other conse- an anomalous progression from the DN to the DP stage, in that the quences include expansion, down-regulation of the IL-2R (CD25), CD4Ϫ8Ϫ25Ϫ stage that precedes the DP stage is absent (19). expression of both coreceptor molecules, and induction of a second Therefore, a specific role for ␨-chain in controlling proliferation ␣ wave of RAG expression, allowing TCR rearrangements to and differentiation of DN cells has been hypothesized. by guest on October 2, 2021 occur (2, 3). In RAG-2-deficient mice, the absence of TCR␤ rearrangements ␣␤ Ligation of the TCR in mature T cells induces activation of leads to a developmental block at the DN CD25ϩ stage (11). Src family kinases, such as Lck and Fyn, as well as phosphoryla- CD25ϩ cells express low levels of CD3␥, ␦, and ⑀ subunits at the tion of the immunoreceptor tyrosine-based activation motifs surface (20, 21). Efficient cellular proliferation, down-regulation of (ITAM) of CD3 and ␨ subunits (4). The phosphorylation of the two CD25, and production of small cortical cells that are characteristic tyrosines present in one ITAM results in recruitment of the ty- of the physiologic transition driven by the pre-TCR can be induced rosine kinase ZAP-70 into the TCR/CD3/␨ complex through the in fetal thymus organ cultures by addition of anti-CD3⑀ mAb (22) binding of tandem SH2 domains (5, 6). This association permits and, in vivo, by injection of mice with anti-CD3⑀ Abs (20, 21). the enzymatic activity of ZAP-70, resulting in phosphorylation of Hence, CD3 appears functionally competent to promote these de- downstream targets (7). A similar sequence of events appears to velopmental steps in the absence of a fully assembled pre-TCR and take place also in signal transduction by the pre-TCR, since mice in fact in the absence of the pre-TCR ␣-chain (23). We found that ␨-chains, despite the lack of detectable physical *Dipartimento di Biologia e Genetica per le Scienze Mediche, Universita`di Milano at association with the surface CD3 complex, are functionally cou- Department of Biological and Technological Research, San Raffaele Scientific Institute pled to the signaling cascade initiated by anti-CD3 treatment of † (HSR), Milan, Italy; Unite´d’Immunochimie Analytique, De´partement d’Immunologie, Ϫ/Ϫ Institut Pasteur, Unite´de Recherche Associeé, Centre National de la Recherche Sci- RAG-2 thymocytes since they become associated with phos- entifique D1961, and Universite´Pierre et Marie Curie, Paris, France; and ‡Institut phorylated ZAP-70. This complex can be immunoprecipitated Necker, Institut National de la Santeét Recherche Medicale, U373, Paris, France from the membrane fraction of anti-CD3-treated RAG-2Ϫ/Ϫ thy- Received for publication August 31, 1998. Accepted for publication November mocytes, suggesting its recruitment to the cell membrane. More- 16, 1998. over, the ␨-chain dimers that are not linked to CD3 can be detected The costs of publication of this article were defrayed in part by the payment of page vav charges. This article must therefore be hereby marked advertisement in accordance in pre-TCR-expressing cells. The comparison of ZAP-70, p95 , with 18 U.S.C. Section 1734 solely to indicate this fact. and p120cbl phosphorylation upon anti-CD3 treatment shows the Ϫ/Ϫ 1 This work was supported by grants from the Ligue Nationale Contre le Cancer and same pattern in RAG-2 thymocytes and pre-TCR-expressing Association pour la Recherche sur le Cancer. cells, and differs from activated RAG-2Ϫ/Ϫ␨Ϫ/Ϫ thymocytes in 2 Address correspondence and reprint requests to Dr. Fabio Grassi, DIBIT-HSR via which ZAP-70 and p120cbl phosphorylation is barely detectable. Olgettina 58 I-20132 Milan, Italy. E-mail address: [email protected] Therefore, an important role of the ␨-chain/ZAP-70 complex in 3 Abbreviations used in this paper: DN, double-negative; DP, double-positive; ECL, enhanced chemiluminescence; IEF, isoelectric focusing; ITAM, immunoreceptor ty- mediating the transition from the DN to the DP thymocyte stage is rosine-based activation motif. postulated.

Materials and Methods Deglycosylation Mice, cell lines, and Abs 125I-labeled anti-CD3⑀ immunoprecipitates were boiled twice: 2 min in 1% Young adult (4–6-wk-old) C57BL/6, RAG-2Ϫ/Ϫ, RAG-1Ϫ/Ϫ, and CB17 SDS and 2 min in deglycosylation buffer (20 mM sodium phosphate, pH 7.2, 10 mM NaN3, 50 mM EDTA, 0.5% Nonidet P-40). One sample was scid/scid (SCID) mice were obtained from the animal colony of the Insitut Ϫ Pasteur (Paris, France). ␨Ϫ/ϩ mice were kindly provided by Dr. B. Malissen stored at 20°C, another one was incubated at 37°C for 16 h, and a third (Centre d’Immunologie INSERM-CNRS, Marseille, France). SCB.29 (24) one was incubated at 37°C for 16 h in the presence of 2 mU neuraminidase, and M14T (25) thymocyte cell lines were used. The mouse mAbs em- 2.5 mU O-glycosidase, and 0.4 U N-glycosidase F (Boehringer Mannheim, ployed were anti-CD3⑀ 145-2C11 (26), anti-␨ G3 (27), and anti- Mannheim, Germany). The deglycosylation products were analyzed by phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY). The fol- SDS-PAGE. lowing rabbit antisera were used: anti-calnexin C-terminal peptide (StressGen, Victoria, B.C.), anti-TCR␨ (kindly provided by Dr. L. Samel- son, National Institute of Health, Bethesda, MD), anti-ZAP-70 and anti- Cell fractionation vav cbl p95 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-p120 Ϫ Ϫ After two washes in ice-cold PBS, C57BL/6 and RAG-2 / thymocytes (kindly provided by Dr. A. Veillette, University of Toronto, Canada). were resuspended at 50 ϫ 106/300 ␮l in hypotonic buffer (20 mM Tris-

HCl, pH 7.5, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, and protease inhibitors) and incubated 10 min on ice. Cells were then disrupted by Surface labeling, stimulation, and immunoprecipitation of homogenization on ice with a Dounce homogenizer (30 strokes at low thymocytes speed and 10 strokes at maximum speed). Salt concentration was adjusted to 150 mM NaCl, and intact cells, nuclei, and cytoskeleton were pelleted For cell surface iodination, 50 ϫ 106 thymocytes obtained from C57BL/6 Ϫ Ϫ by centrifugation at 5000 rpm for 5 min in microcentrifuge (Eppendorf, and RAG-2 / mice were treated with 0.1 mg/ml water-soluble Bolton- Hamburg, Germany) at 4°C. After two washes in hypotonic buffer, the

Hunter reagent (Pierce, Rockford, IL) in PBS at 4°C for 30 min, and the Downloaded from pellet (P1) was resuspended in Laemmli sample buffer. The low-speed reaction was quenched by addition of 5% Ig-free FCS and 0.1 mg/ml lysine ϫ in HBSS (28). Cell surface proteins were labeled with 125I by the lactoper- supernatant was centrifuged at 100,000 g for 30 min; the resulting pellet oxidase method (29) and extracted at 4°C in 1% digitonin or 0.5% Triton (P100) was considered the membrane fraction, and the supernantant (S100) X-100 lysis buffer (0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EGTA, 1 mM was considered the soluble proteins fraction. The P100 fraction was dis- ⑀ solved in 0.5% Triton X-100 lysis buffer and immunoprecipitated either MgCl2, and protease inhibitors); anti-CD3 mAb was added to the lysates ⑀ at the ﬁnal concentration of 10 ␮g/ml and immune complexes were pre- with anti-CD3 or anti-ZAP-70 Abs, whereas the S100 fraction was di- cipitated by protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden). rectly immunoprecipitated. All samples were resolved by SDS-PAGE in a

Cell surface biotinylation was performed as described (30). In brief, 50 ϫ 5–15% gradient gel and immunoblotted with the indicated Abs. http://www.jimmunol.org/ 106 viable cells were washed in PBS and incubated in 1 ml of PBS, 5 mM NHS-LC-biotin (Pierce, Rockford, IL) for1hat4°C. After washing in PBS, 1 mM glycine, the cells were lysed in 1% digitonin lysis buffer. Results Immunodepletion of CD3⑀-or␨-chain was achieved by three sequential Characterization of the CD3/calnexin complex expressed at the immunoprecipitations of 1% digitonin cell lysates employing G3 and 145- Ϫ/Ϫ 2C11 mAbs, respectively, at 20 ␮g/ml, followed by protein A-Sepharose. cell surface of RAG-2 thymocytes 8 For tyrosine phosphorylation experiments, thymocytes at 10 cells/ml Recently, incomplete endoplasmic reticulum retention has been were either left untreated or incubated at 4°C for 30 min with anti-CD3⑀ mAb. After three washes, cells were incubated at 37°C for 2 min, then demonstrated to take place in immature thymocytes and CD3 has anti-hamster Ig serum was added and cells were incubated at 37°C for 2 been shown to be expressed at the cell surface, devoid of chains of by guest on October 2, 2021 min. For in vivo stimulation, mice were i.v. injected with 50 ␮g of anti- the TCR for Ag and in association with calnexin, a molecular ⑀ CD3 mAb and thymocytes were recovered at day 5 after mAb adminis- chaperone previously considered to reside exclusively in the en- tration. Washed cells (Ͼ85% CD4ϩ8ϩ) were lysed in 0.5 or 1% Triton X-100 lysis buffer. The indicated rabbit antisera (2 ␮l) were added to the doplasmic reticulum (32, 33). To characterize the subunit compo- Ϫ/Ϫ lysates, and immune complexes were precipitated by protein A-Sepharose, sition of CD3 on the surface of thymocytes from RAG-2 mice, run under reducing conditions in SDS-PAGE, and immunoblotted as de- thymocytes from RAG-2Ϫ/Ϫ and C57BL/6 mice were surface la- scribed below. beled by the 125I-lactoperoxidase method and lysed in digitonin, and cell lysates were immunoprecipitated with anti-CD3⑀ mAbs. Electrophoresis and immunoblotting As evident from two-dimensional nonreducing versus reducing SDS/PAGE gel analysis (Fig. 1A), two spots were missing in For two-dimensional nonreducing versus reducing SDS/PAGE, immuno- RAG-2Ϫ/Ϫ mice: one (40–45 kDa) represents the reduced TCR ␣- precipitates were run in SDS sample buffer under nonreducing conditions ␤ in a discontinuous Laemmli SDS-polyacrylamide (5–15% gradient) gel. and -chains in C57BL/6 thymocytes, while the second (16 kDa) The first dimension strips were then equilibrated in reduced SDS sample represents reduced ␨-chains. The latter were undetectable in RAG- buffer for 30 min at room temperature and then run into a second 5–15% 2Ϫ/Ϫ mice even after long exposures. Common to both precipitates gradient SDS-polyacrylamide gel. The gel was then dried and subjected to are four spots that migrate either along (90, 26, and 21 kDa) or autoradiography at Ϫ70°C. For two-dimensional IEF/PAGE, immune complexes were eluted in IEF sample buffer for3hatroom temperature slightly above (25 kDa) the diagonal. While the 26, 21, and 25 kDa and then resolved by IEF in a horizontal apparatus (Pharmacia Biotech), proteins were identified as ␦, ␥, and ⑀ CD3 chains, the 90-kDa followed by SDS-PAGE in a 5–15% gradient gel (31). The gels were molecule is likely to be calnexin. blotted in transfer buffer (100 mM glycine, 0.1% SDS, 10 mM Tris, 25% Indeed, the analysis of an anti-CD3⑀ immunoprecipitate from ethanol, pH 8.3) onto nitrocellulose membrane (Hybond-ECL; Amersham, RAG-2Ϫ/Ϫ 125 I surface-labeled thymocytes by two-dimensional Little Chalfont, U.K.), and the membranes were subjected to autoradiography at Ϫ70°C. To probe the transferred proteins with anti-calnexin rabbit IEF/SDS-PAGE revealed a 90-kDa protein with an acidic isoelec- antiserum, membranes were blocked for1hatroom temperature in PBS tric point together with the CD3␦, ⑀, and ␥ subunits at positions containing 5% nonfat dry milk and 0.1% Tween-20 (PBS-milk), washed expected from their size and charge (Fig. 1B). Immunoblotting three times in PBS, 0.1% Tween-20 (PBS-Tween), followed by overnight with a rabbit anti-calnexin antiserum detected a protein in the same incubation with rabbit antiserum at 4°C. After three washes (30 min each) in PBS-Tween, membranes were incubated for 1 h with horseradish per- position representing the 90-kDa surface-labeled acidic molecule oxidase-conjugated anti-rabbit Igs in PBS-milk and washed three times in (Fig. 1D). Treatment of the anti-CD3⑀ immunoprecipitates with PBS-Tween. After incubation in ECL detection fluid, the blots were ex- peptide-N-glycosidase F and O-glycosidase did not provoke any posed to Hyperfilm ECL (Amersham). For immunoblot of total cell lysates, 7 shift in the electrophoretic mobility of the 90-kDa protein band, 10 thymocytes were lysed in 100 ␮l lysis buffer containing 1% Triton ␥ ␦ X-100. The lysates were run on two-dimensional nonreducing versus re- whereas the CD3 and subunits were sensitive to the same en- ducing SDS-PAGE and immunoblotted with anti-␨ rabbit serum, as de- zymatic digestion (Fig. 1C), as described (26), being resolved as a scribed above. single band with an apparent molecular mass of 20 kDa. The Journal of Immunology 2591

FIGURE 1. Characterization of surface CD3/calnexin complex in RAG-2Ϫ/Ϫ thymocyte. C57BL/6 and RAG-2Ϫ/Ϫ thymocytes (50 ϫ 106 cells) were surface labeled with 125I after treatment with Bolton-Hunter reagent and lysed in digitonin 1%. Anti-CD3⑀ (145- 2C11) mAb was used for immunoprecipitation, and the precipitates were analyzed by two-dimensional nonreducing versus reducing SDS/PAGE. Arrowhead indi- cates the monomeric 90-kDa protein band common to both of the precipitates. The gel was autoradiographed for 5 days; the spot corresponding to ␨-chain was undetectable in RAG-2Ϫ/Ϫ cells even after 6 wk of ex- Downloaded from posure of the gel (A). Anti-CD3⑀ immunoprecipitates were resolved by IEF/SDS-PAGE two-dimensional electrophoresis and then transferred to nitrocellulose. The membrane was autoradiographed (B) and then probed with a rabbit anti-calnexin antiserum. The reaction was revealed by peroxidase-conjugated anti- rabbit Ig Abs and chemoluminescence (D). Digestion http://www.jimmunol.org/ of anti-CD3⑀ immunoprecipitates with peptide-N-glycosidase F and O-glycosidase (C). by guest on October 2, 2021

Functional coupling of CD3/calnexin complex to ␨-chains performed with thymocytes from RAG-2Ϫ/Ϫ mice that were in- ⑀ Although ␨-chains were detectable in total cell lysates from thy- jected with anti-CD3 mAb, with no significant variations upon mocytes of rearrangement-deficient mice (Fig. 2, C–F), we failed restimulation in vitro (lanes 3 and 4). In control experiments, thy- to detect ␨-chains that were associated with CD3⑀ molecules on the mocytes from C57BL/6 mice were analyzed: phosphorylation of cell membrane of RAG-2Ϫ/Ϫ thymocytes in many attempts utiliz- ZAP-70 as well as two ZAP-70-associated phosphoproteins were ing distinct cell surface-labeling procedures, i.e., 125I or biotin la- found. The two proteins with molecular mass of 24 and 28 kDa beling, and using different conditions of lysis. Furthermore, im- correspond most likely to CD3 chains. Furthermore, the expected munoprecipitation of digitonin lysates from unlabeled thymocytes increase in the constitutive phosphorylation of ZAP-70-associated ␨ with anti-CD3⑀ mAb, followed by immunoblotting of immune -chains was observed (lanes 5 and 6) (39). complexes with rabbit anti-␨ antiserum, did not reveal any asso- Since we failed to unambiguously detect phosphorylated CD3 Ϫ/Ϫ ciation of CD3⑀ molecules with ␨-chains even after injection of chains associated with ZAP-70 in activated RAG-2 thymo- mice with anti-CD3⑀ mAb (data not shown). The same protocol cytes, we verified phosphorylation of CD3 chains following Ab has proven to be extremely sensitive in detecting CD3-associated injection, by anti-CD3⑀ mAb immunoprecipitation from the Ϫ/Ϫ ␨-chain, when performed with C57BL/6 thymocytes. These results membrane fraction of RAG-2 thymocytes lysed in 0.5% Tri- are consistent with previous reports demonstrating that ␨-chains do ton X-100. We immunoblotted the immunoprecipitate resolved not associate with calnexin during TCR-CD3 folding (34–37). in two-dimensional nonreducing versus reducing SDS/PAGE We then analyzed a possible functional recruitment of ␨-chains gel, with anti-phosphotyrosine mAb. The resulting autoradio- generated by cross-linking CD3⑀ on the surface of RAG-2Ϫ/Ϫ thy- graph showed the presence of an intensely phosphorylated spot mocytes, in a way analogous to that employed in pre-TCR-expressing corresponding to CD3⑀ and two spots corresponding to CD3␥- cells (38): initially, immunoprecipitation with anti-ZAP-70 Abs of and ␦-chains (Fig. 3B), which are absent in membranes from Ϫ Ϫ lysates of RAG-2Ϫ/Ϫ thymocytes stimulated in vitro with anti-CD3⑀ unstimulated RAG-2 / thymocytes. We conclude therefore mAbs did not reveal an enhancement of the weak constitutive phos- that CD3␥-, ␦-, and ⑀-chains are phosphorylated upon in vivo phorylation of ZAP-70 (Fig. 3A, lanes 1 and 2). However, phos- stimulation of RAG-2Ϫ/Ϫ thymocytes, while phosphorylated phorylation of ZAP-70 as well as the p21 and p23 isoforms of ␨-chains appear to be the dominant recruitment site of ZAP-70 phosphorylated ␨-chains were detected once the experiment was under these experimental conditions. 2592 PHOSPHORYLATION OF CD3-INDEPENDENT ␨-CHAINS IN IMMATURE THYMOCYTES

blots of the same filters. As can be seen in Fig. 3C, at the indicated exposure times we could detect a major signal with anti-ZAP-70 serum in the immunoprecipitated S100 fraction, as shown (40), and confinement of calnexin to the anti-CD3⑀ immunoprecipitate of the P100 membrane fraction of RAG-2Ϫ/Ϫ thymocytes. Immunoprecipitation of CD3-independent surface ␨-chains from pre-TCR-expressing cells To address whether CD3-independent ␨-chains existed at the cell membrane also concomitantly with the pre-TCR complex, SCB.29 cells were surface labeled with biotin, lysed in digitonin, and immunoprecipitated with anti-CD3⑀ and anti-␨-chain Abs. The results of these experiments show that ␨-chains were found in association with CD3␥-, ␦-, and ⑀-chains (Fig. 4, lane 1), as well as isolated or very loosely associated with CD3, as evident by the poor representation of CD3 bands in anti-␨-chain mAb immunoprecipitates (lane 3). The poor coimmunoprecipitation of CD3 proteins by anti-␨ mAb was not a peculiarity of the Ab employed, since the same mAb efficiently coprecipitated CD3 chains from Downloaded from C57BL/6 thymocytes and since anti-␨ rabbit Abs recognizing a different N-terminal ␨-chain epitope (41) gave identical results. The detergent was not responsible since the same results were obtained by lysing cells with Brij96 (data not shown). Finally, anti-CD3⑀ immunoprecipitation of lysates depleted of ␨-chain and anti-␨ immunoprecipitation after CD3 depletion showed that it was impossible to deplete either CD3 or ␨-chain by preclearing with http://www.jimmunol.org/ anti-␨ and anti-CD3⑀ mAbs, respectively (Fig. 4, lanes 2 and 4). The efficiency of the preclearing protocol employed was checked in a thymocyte cell line (M14T) expressing mature TCR (25), in which either anti-␨ or anti-CD3 mAb completely depleted precip- FIGURE 2. Detection of ␨-chain in total thymocyte lysates of rearrange- itable CD3 and ␨-chains, respectively (lanes 6 and 8). ment-deficient mice. Triton X-100 lysates of thymocytes (107 cells), resolved by two-dimensional nonreducing versus reducing SDS-PAGE, were Protein phosphorylation in thymocytes from wild-type, immunoblotted with rabbit anti-␨ antiserum. Cells from the following mice ␨ Ϫ/Ϫ Ϫ/Ϫ recombinase-, -deficient mice, and a pre-TCR-expressing cell strains were used: C57BL/6 (A); ␨ (B); RAG-2 , 7 days after injec- by guest on October 2, 2021 Ϫ Ϫ Ϫ Ϫ line tion with anti-CD3⑀ mAb (C); untreated RAG-2 / (D); RAG-1 / (E); and SCID (F). The prevalence of CD3-independent ␨-chains as recruitment site for ZAP-70 suggested that the ␨-chain/ZAP-70 complex could endow RAG-2Ϫ/Ϫ thymocyte with efficient responsiveness to anti- Immunoprecipitation of CD3-independent ␨-chain/ZAP-70 Ϫ/Ϫ CD3 stimulation and could contribute to pre-TCR-driven signal- complex from the membrane fraction of RAG-2 thymocytes ing. Therefore, we compared ZAP-70 phosphorylation of RAG- To evaluate the subcellular localization of the ZAP-70/␨-chain 2Ϫ/Ϫ, ␨Ϫ/Ϫ, and pre-TCR-expressing cells stimulated by anti-CD3 complex in activated RAG-2Ϫ/Ϫ thymocytes, we separated cell Abs; in addition, we analyzed the phosphorylation of p95vav and lysates from anti-CD3-injected mice into three fractions, contain- p120cbl as downstream targets of Lck and ZAP-70 activation (42– ing nuclei and cytoskeleton (P1), membranes (P100), and cyto- 47). In vivo stimulated RAG-2Ϫ/Ϫ thymocyte and in vitro acti- plasmic proteins (S100). As a control, we applied the same pro- vated SCB.29 cells gave similar results, as all displayed markedly tocol to C57BL/6 thymocytes. The P1 fraction was directly increased phosphorylation of all of the three molecules tested fol- processed for SDS/PAGE; the membrane fraction was dissolved in lowing anti-CD3 stimulation (Fig. 5, B and C). Additionally, anti- lysis buffer containing 0.5% Triton X-100, divided in two aliquots, CD3 administration to RAG-2Ϫ/Ϫ mice resulted in increased ex- and subjected to immunoprecipitation either with anti-ZAP-70 rab- pression of p120cbl (Fig. 5B, lane 6). The same experiment bit serum or anti-CD3⑀ mAb; the S100 soluble phase was directly performed with C57BL/6 thymocytes showed efficient phosphor- immunoprecipitated either with anti-ZAP-70 rabbit serum or anti- ylation of these targets under the experimental conditions em- CD3⑀ mAb. Fig. 3C shows that we could selectively detect the p21 ployed (Fig. 5A). and p23 phosphorylated isoforms of ␨-chain in the anti-ZAP-70 Surprisingly, we found that extensive cross-linking by admin- immunoprecipitate of the membrane fraction derived from acti- istration of anti-CD3 mAb to RAG-2Ϫ/Ϫ␨Ϫ/Ϫ mice resulted in vated RAG-2Ϫ/Ϫ thymocytes and not in the corresponding anti- barely detectable phosphorylation of ZAP-70 (Fig. 5D, lane 2); CD3⑀ immunoprecipitate. Conversely, p21 phosphorylated ␨-chains moreover, p120cbl was only weakly phosphorylated (Fig. 5D, lane were present in both the anti-CD3⑀ and anti-ZAP-70 immunopre- 6), and the amount of protein was unchanged with respect to the cipitates of the membrane fraction from unstimulated C57BL/6 unstimulated counterpart. However, phosphorylation of p95vav was thymocytes, as expected. Therefore, phosphorylated ␨-chains were apparently not affected and was readily detected (Fig. 5D, lane 4). expressed at the cell membrane of activated RAG-2Ϫ/Ϫ thymo- Thus, since anti-CD3⑀ stimulation of ␨Ϫ/Ϫ mice results in thymocytes in association with ZAP-70, but not with CD3. cyte development analogous to the one observed in RAG-2Ϫ/Ϫ The efficacy of cell fractionation and immunoprecipitation was mice (48, 49), subtle differences in proximal signal transduction checked by anti-ZAP-70 and anti-calnexin (in the case of anti- exist that are not critical for the final outcome of extensive CD3 CD3⑀ immunoprecipitates of RAG-2Ϫ/Ϫ thymocytes) immuno- cross-linking in the two mouse strains. The Journal of Immunology 2593 Downloaded from http://www.jimmunol.org/

FIGURE 3. Phosphorylation of ␨-chain, association with ZAP-70, and membrane localization of the complex in stimulated RAG-2Ϫ/Ϫ thymocyte. Thymocytes from untreated, anti-CD3⑀-injected RAG-2Ϫ/Ϫ and C57BL/6 mice were either left untreated or stimulated with anti-CD3⑀ mAb, lysed in 0.5% Triton X-100, and immunoprecipitated with anti-ZAP-70 rabbit Abs. Immune complexes were run under reducing conditions in SDS-polyacrylamide 5–15% gradient gel, transferred to nitrocellulose membrane, and immunoblotted with anti-phosphotyrosine mAb. Membranes were stripped and immu- ⑀ Ϫ/Ϫ

noblotted with anti-ZAP-70 rabbit serum (A). Thymocyte membrane fractions from untreated and anti-CD3 -injected RAG-2 mice were lysed in 0.5% by guest on October 2, 2021 Triton X-100 and immunoprecipitated with anti-CD3⑀ mAb. Immune complexes were resolved in two-dimensional nonreducing versus reducing SDS/ PAGE, transferred to membrane, and immunoblotted with anti-phosphotyrosine mAb (B). Subcellular fractions of thymocytes from C57BL/6 and injected RAG-2Ϫ/Ϫ mice were processed as detailed in the text, resolved in SDS-polyacrylamide 5–15% gradient gel, and immunoblotted with anti-phosphotyrosine mAb. After stripping, membranes were immunoblotted with anti-ZAP-70 or anti-calnexin rabbit sera and revealed by peroxidase-conjugated anti-rabbit Ig Abs and chemoluminescence. Exposure time was 20 s for anti-ZAP-70 and 5 min for anti-calnexin (C).

Discussion ficient allelic exclusion at the TCR ␤-chain locus (50). There- fore, an essential function of the ␨-chain is exerted after rear- The analysis of ␨-deficient mice has revealed the requirement of rangement of TCR␤ genes and is connected to pre-TCR ␨-chain to achieve thymocyte expansion once productive rear- assembly. Transcripts encoding ␨-chains have been detected rangement of TCR␤ genes has occurred (15–18); more recently, very early in thymocyte development (51); in this study, we the absence of ␨-chains has been shown to result also in inef- show the expression of a ␨-chain dimer in the absence of TCR␤ rearrangement and its functional competence in recombinase- deficient thymocyte, in which CD3 chains are sequestered by calnexin and incompletely retained in the endoplasmic reticulum (32). In pre-TCR-expressing cells, we were unable to deplete either surface ␨-chains or CD3 proteins by preclearing with anti-CD3 and anti-␨ Abs, respectively, implying that either ␨-chains are very loosely associated with the pre-TCR/CD3 complex or exist as a physically independent entity in addition to the fully assembled complex. The existence of ␨-chain dimers, in the absence of the pre-TCR and their membrane tar- geting independently of CD3, favors the latter interpretation of ⑀ ␨ FIGURE 4. Immunodepletion of surface CD3 - and -chains in SCB.29 the results. Accordingly, in mature T cells, ␨-chains have been and M14T cells. Biotin-labeled SCB.29 and M14T cells were lysed in 1% shown to be transported to the cell surface independently of the digitonin and either directly immunoprecipitated or after preclearing with the indicated mAb. Immune complexes were resolved in SDS-polyacryl- TCR complex, leading to the hypothesis that the TCR-CD3 ␨ amide 5–15% gradient gel and transferred to nitrocellulose membrane, and complex is transported to the membrane by the turnover path- precipitated surface molecules were revealed by streptavidin-horseradish way (52). Since ␨-chains represent a rate-limiting factor in TCR peroxidase/ECL detection kit. assembly and surface transport (53, 54), expression of ␨-chains 2594 PHOSPHORYLATION OF CD3-INDEPENDENT ␨-CHAINS IN IMMATURE THYMOCYTES

FIGURE 5. Proximal phosphorylation potentials in thymocyte from wild-type, recombinase-, ␨-deficient mice and a pre-TCR-expressing cell line. Ex vivo thymocytes from C57BL/6 (A), RAG-2Ϫ/Ϫ (B), RAG- 2Ϫ/Ϫ␨Ϫ/Ϫ (D) mice, and SCB.29 cells (C) were lysed in 1% Triton X-100, treated as detailed, and immunoprecipitated with the indicated Abs. SDS-PAGE-resolved immune complexes were probed with anti- phosphotyrosine mAb; stripped Downloaded from membranes were sequentially immunoblotted with anti-ZAP-70, anti- p95vav, and anti-p120cbl rabbit Abs. The results shown for each group have been obtained from the same experiment, and samples were analyzed on the same gel. http://www.jimmunol.org/ by guest on October 2, 2021 before the pre-TCR might allow immediate and efficient assem- is preferentially recruited by phosphorylated ␨-chains present at bly/transport of pre-TCR/CD3 complex. This hypothesis would the cell surface physically unlinked to CD3 molecules. In the phys- be further strengthened by the detection of surface TCR iologic cellular environment, loosely associated and CD3-indepen- Ϫ Ϫ ␨-chains in CD3⑀ / thymocytes. However, the impossibility to dent ␨-chain could constitute a preferential recruitment site for stimulate in vivo these cells makes this demonstration particu- ZAP-70 and endow the cell with the optimal stoichiometry of larly difficult to obtain. available signaling complexes at this stage of thymocyte Ϫ/Ϫ In vivo stimulation through anti-CD3⑀ mAb of RAG-2 thy- differentiation. mocytes leads to phosphorylation of ZAP-70, which is found in Efficient transition to the DP stage can be obtained in ␨Ϫ/Ϫ mice association with phosphorylated ␨-chains. The detection of CD3⑀- ⑀ ␨ Ϫ Ϫ by anti-CD3 mAb administration (48, 49), suggesting that -chain ␨ / Ϫ Ϫ independent -chain/ZAP-70 complexes in RAG-2 mice as recruitment in stimulated RAG-2 / thymocytes is nonessential well as in pre-TCR-expressing cells (38) could imply a physiologic and replaceable without functional impairment of the signaling relevance for the functional recruitment of ␨-chain, not stably as- cascade. However, it has been shown that a number of different sociated to pre-TCR complex. The dual role played by ␨-chains as stimuli, such as irradiation (60–63), Ras activation (64), p53 in- rate-limiting factor in TCR assembly (53, 54) and as signaling activation (65, 66), as well as abrogation of the Fas function (67) module (55, 56) makes it difficult to dissociate these two functions promotes the development of DP cells in rearrangement-deficient during normal T cell development, but it is clear that in TCR␤- mice, thereby providing evidence for the existence of diverse sig- deficient mice, ␨-chains can be recruited at the cell membrane as ␨/ZAP-70 complex. nals that enable the same developmental program. The proximal ␨Ϫ/Ϫ Restoration of TCR surface expression in ␨-deficient mice by signal-transduction events occurring in activated thymocytes truncated ITAM-less ␨-chains has been shown to overcome defi- are subtly different from the ones taking place in stimulated RAG- Ϫ/Ϫ cient signaling at this stage of development (57). However, in an 2 thymocytes and pre-TCR-bearing cells, but apparently apt in analogous experimental model, a transgenic mutant ITAM-less these circumstances, to determine the same developmental course. vav ␨-chain could not rescue differentiation of DN cells into DP cells The efficient phosphorylation of p95 in the absence of marked of ␨Ϫ/Ϫ mice, leading to the hypothesis that ␨-chains contribute ZAP-70 recruitment might be enabling and sufficient per se for signals that cannot be replaced by CD3 chains (58). It has been such a differentiation to occur. Accordingly, Vav has been shown hypothesized that CD3 subunits might become more accessible to to be critically involved in thymocyte proliferation (68–71). Lck in the absence of full-length ␨-chains (59); indeed, we have It has also been shown that coreceptor expression in immature shown that in RAG-2Ϫ/Ϫ mice, CD3⑀ is not associated with thymocytes results in poor ZAP-70 phosphorylation after TCR ␨-chains and is phosphorylated following mAb injection. ZAP-70 stimulation due to diversion of available Lck; this effect has been The Journal of Immunology 2595 postulated to be dependent on the association of Lck to cytoplas- 13. Dave, V. P., Z. Cao, C. Browne, B. Alarcon, G. Fernandez-Miguel, J. Lafaille, ␦ mic tails of coreceptors (72). The absence of coreceptor expression A. de la Hera, S. Tonegawa, and D. J. Kappes. 1997. CD3 deficiency arrests development of the ␣␤ but not the ␥␦ T cell lineage. EMBO J. 16:1360. at the DN stage would permit a free dispersion of the membrane- 14. Berger, M. A., V. Dave, M. R. Rhodes, G. C. Bosma, M. J. Bosma, D. J. Kappes, associated Lck pool, rendering it more available for ITAM phos- and D. L. Wiest. 1997. Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3␦ is physically associated but not func- phorylation and subsequent ZAP-70 activation. In addition, a basal tionally required. J. Exp. Med. 186:1461. Lck activity might also result in the observed weak phosphoryla- 15. Love, P. E., E. W. Shores, M. D. Johnson, M. L. Tremblay, E. J. Lee, tion of ZAP-70 before RAG-2Ϫ/Ϫ thymocyte stimulation and be A. Grinberg, S. P. Huang, A. Singer, and H. Westphal. 1993. T cell development ␨ in mice that lack the ␨ chain of the T cell antigen receptor complex. Science dependent on weak phosphorylation. This hypothesis is sup- 261:918. ported by the absence of constitutive ZAP-70 phosphorylation in 16. Liu, C. P., R. Ueda, J. She, J. Sancho, B. Wang, G. Weddell, J. Loring, ␨Ϫ/Ϫ mice. The pool of phosphorylated ZAP-70 molecules could C. Kurahara, E. C. Dudley, A. Hayday, C. Terhorst, and M. Huang. 1993. Ab- normal T cell development in CD3-␨Ϫ/Ϫ mutant mice and identification of a lower the threshold for subsequent signaling by the assembled pre- novel T cell population in the intestine. EMBO J. 12:4863. TCR, rendering the cell appropriately sensitive. These potential 17. Malissen, M., A. Gillet, B. Rocha, J. Trucy, E. Vivier, C. Boyer, F. Koentgen, functions of CD3-independent ␨-chains could depend on their asso- N. Brun, G. Mazza, E. Spanopoulou, D. Guy-Grand, and B. Malissen. 1993. T cell development in mice lacking the CD3-␨/␩ gene. EMBO J. 12:4347. ciation to developmentally regulated membrane proteins. Among 18. Ohno, H., T. Aoe, S. Taki, D. Kitamura, Y. Ishida, K. Rajewsky, and T. Saito. potential candidates, Thy-1 could play a role since it has been 1993. Developmental and functional impairment of T cells in mice lacking CD3␨ ␨ chains. EMBO J. 12:4357. shown to use -chains for efficient signaling (73). CD16 is ex- 19. Crompton, T., M. Moore, H. R. MacDonald, and B. Malissen. 1994. Double- pressed very early in thymocyte development and has been found negative thymocyte subsets in CD3␨ chain-deficient mice: absence of ϩ Ϫ Ϫ associated to ␨ homodimers (74). Interestingly, CD16 and CD2 are HSA CD44 CD25 cells. Eur. J. Immunol. 24:1903. 20. Jacobs, H., D. Vandeputte, L. Tolkamp, E. de Vries, J. Borst, and A. Berns. 1994. coordinately expressed during T cell development with loss of CD3 components at the surface of pro-T cells can mediate pre-T cell development Downloaded from CD16 and acquisition of CD2 expression characterizing a late DN in vivo. Eur. J. Immunol. 24:934. stage immediately before the conversion into DP thymocytes (75); 21. Shinkai, Y., and F. W. Alt. 1994. CD3⑀-mediated signals rescue the development of CD4ϩCD8ϩ thymocytes in RAG-2Ϫ/Ϫ mice in the absence of TCR ␤ chain since CD16 could substitute for the TCR in coupling CD2 to sig- expression. Int. Immunol. 6:995. naling pathways by contributing ␨-chains (76), it has been pro- 22. Levelt, C. N., P. Mombaerts, A. Iglesias, S. Tonegawa, and K. Eichmann. 1993. ␤ posed that CD16 may serve a role similar to the TCR early in Restoration of early thymocyte differentiation in T-cell receptor -chain-deficient mutant mice by transmembrane signaling through CD3⑀. Proc. Natl. Acad. Sci. thymocyte ontogeny by coupling CD2 and Thy-1 to downstream USA 90:11401. signaling (77). The efficient transition to DP stage observed in 23. Fehling, H. J., B. M. Iritani, A. Krotkova, K. A. Forbush, C. Laplace, http://www.jimmunol.org/ Ϫ/Ϫ R. M. Perlmutter, and H. von Boehmer. 1997. Restoration of thymopoiesis in stimulated RAG-2 thymocytes could benefit from the artefac- pT␣Ϫ/Ϫ mice by anti-CD3⑀ antibody treatment or with transgenes encoding ac- tual recruitment of these alternative forms of surface ␨-chain, tivated Lck or tailless pT␣. Immunity 6:703. thereby mimicking the assembly and signaling of pre-TCR. 24. Groettrup, M., K. Ungewiss, O. Azogui, R. Palacios, M. J. Owen, A. C. 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