Motif in Mature T Cell Function

A Redundant Role of the CD3γ -Immunoreceptor Tyrosine-Based Activation Motif in Mature T Cell Function

This information is current as Mariëlle C. Haks, Tanina A. Cordaro, Jeroen H. N. van den of September 25, 2021. Brakel, John B. A. G. Haanen, Evert F. R. de Vries, Jannie Borst, Paul Krimpenfort and Ada M. Kruisbeek J Immunol 2001; 166:2576-2588; ; doi: 10.4049/jimmunol.166.4.2576

http://www.jimmunol.org/content/166/4/2576 Downloaded from

References This article cites 70 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/166/4/2576.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. A Redundant Role of the CD3␥-Immunoreceptor Tyrosine-Based Activation Motif in Mature T Cell Function1

Marie¨lle C. Haks,* Tanina A. Cordaro,* Jeroen H. N. van den Brakel,* John B. A. G. Haanen,* Evert F. R. de Vries,† Jannie Borst,† Paul Krimpenfort,‡ and Ada M. Kruisbeek2*

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3␥, CD3␦, and CD3⑀) or three (CD3␨) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3␨ chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-␥␦⑀ module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3␥-, ␦-, and ⑀-ITAMs regulate TCR signaling. We here report on the role of the CD3␥ chain and the CD3␥-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or Downloaded from abrogated in T cells derived from CD3␥ null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3␥. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3␥-ITAM. Loss of integrity of the CD3␥-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3␥-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3␥-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3␦ and/or the CD3⑀ chains endow the TCR with qualitatively unique signaling functions. The http://www.jimmunol.org/ Journal of Immunology, 2001, 166: 2576–2588.

uring their development, T cells encounter several Ag either the CD4 or CD8 coreceptors and eventually exit the thymus receptor-driven checkpoints that are critical for their fur- to populate the peripheral lymphoid organs (21, 22). D ther maturation. The earliest checkpoint thymocytes en- In the periphery, T cells require an Ag receptor-driven signal to counter occurs early in intrathymic development at the transition become activated, proliferate, and exert their effector function. Ϫ Ϫ ϩ ϩ from the CD4 CD8 double negative to the CD4 CD8 double- These events are mediated by the mature clonotypic TCR complex 3 positive (DP) stage. This transition is a control point for produc- (23). This TCR complex is composed of a TCR-␣␤ heterodimer, by guest on September 25, 2021 ␤ tive TCR- rearrangements and is mediated by the pre-TCR (1–5). recognizing Ag in the context of MHC class I and II molecules ␤ The pre-TCR is formed by a TCR- polypeptide, a nonrearranging (24–26), and the CD3 complex, which plays a key role in trans- ␣ pT -chain, as well as noncovalently linked invariant CD3 subunits mitting signals after TCR engagement (27–30). Each TCR-␣␤ het- (6–20). Functioning of the pre-TCR critically depends on the sig- erodimer is linked to at least four different monomorphic CD3 nal transduction capacity of the CD3 complex. components, termed CD3␥, ␦, ⑀, and ␨. According to current The second major Ag receptor-dependent checkpoint in intra- knowledge, per complex, two copies of the CD3⑀ and CD3␨ chains thymic T cell development is at the transition from the ϩ ϩ are present, yet only a single copy of the highly homologous CD3␥ CD4 CD8 DP to the CD4 or CD8 single-positive stage. After and CD3␦ chains (31–33). However, the relative contribution of completion of TCR-␣ rearrangements, the pre-TCR on DP thymocytes is replaced by a mature clonotypic TCR complex and cells the several CD3 components to mature TCR-mediated signaling are subjected to positive and negative selection events. Thymo- has not been completely elucidated. cytes surviving this selection process will shut off expression of CD3 components may have partially overlapping functions, because all CD3 components encompass one or several conserved immunoreceptor tyrosine-based activation motifs (ITAMs) (YxxL/

Ix6–8YxxL/I) within their cytoplasmic domains that fully account *Division of Immunology, †Division of Cellular Biochemistry, and ‡Division of Mo- for their individual signal transduction capacity (30, 34). In sup- lecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands port of the view that multiple ITAMs may provide the capacity to Received for publication July 14, 2000. Accepted for publication November 9, 2000. amplify signals generated by a single mature TCR complex, a di- The costs of publication of this article were defrayed in part by the payment of page ␨ charges. This article must therefore be hereby marked advertisement in accordance rect relationship between the number of CD3 -ITAMs and the ef- with 18 U.S.C. Section 1734 solely to indicate this fact. ficiency of both positive and negative selection was observed (35). 1 This work was supported by Grant 901-07-178 from The Netherlands Organization Furthermore, the intensity of the induction of NF-AT activity by for Scientific Research and Grant RG0335/1998-M from the Human Frontier Science chimeric CD3␨-ITAM-containing polypeptide chains also de- Program Organization (to M.C.H.), and, in part, by Grant 901-02-095 from the Neth- erlands Organization for Scientific Research (to P.K.). pended on the number of ITAMs present, suggesting a quantitative ␨ 2 Address correspondence and reprint requests to Dr. Ada M. Kruisbeek, Division of function of the several ITAMs contained within the CD3 chain Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 C. X. Am- (36). In addition, crippling of the CD3␨-ITAMs did not result in sterdam, The Netherlands. E-mail address: [email protected] obvious abnormalities in the spectrum of activation events and 3 Abbreviations used in this paper: DP, double positive; ITAM, immunoreceptor ty- ϩ rosine-based activation motif; wt, wild type; ES, embryonic stem; PKC, protein ki- effector functions of CD8 peripheral T cells, indicating that at nase C; MLN, mediastinal lymph node. least the CD3␨-ITAMs have no exclusive role in T cell activation

(37). Instead, these findings suggest that the ITAMs in the CD3- ment, a 3.9-kb recombinant fragment still containing the pgk-HPRT cas- ␥␦⑀ module are sufficient for qualitatively normal signaling of the sette, and an 8.6-kb recombinant fragment after removal of the pgk-HPRT TCR (37). Therefore, the different ITAMs may be functionally cassette. One clone was selected of 150 colonies tested and injected into C57BL/6 blastocysts to generate chimeric mice. Male chimeric mice were redundant. subsequently crossed to female FVB mice. Germline transmission was ob- Alternatively, several reports suggest specialized functions for tained and heterozygous mice were intercrossed to produce homozygous individual ITAMs (38, 39), presumably as a result of substantial CD3␥⌬I/⌬I mice. variability in the amino acids flanking the phosphotyrosine resi- Mice dues within the ITAMs, predicting interactions with distinct SH2- domain-containing cytosolic mediators (40, 41). Indeed, ITAMs Mice were maintained under specific pathogen-free conditions in the ani- mal colony of The Netherlands Cancer Institute and analyzed at 8–12 wk derived from distinct CD3 chains bind with varying affinities to ␥ fyn of age. Mice deficient for CD3 and mice expressing the F5-TCR trans- downstream adaptors and enzymes such as Syk, ZAP-70, p59 , genes have been described in detail elsewhere (16, 52). Lyn, Shc, Grb-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase (30, 42–49). Moreover, signaling by rCD3⑀- Virus infection ␨ ITAM- or CD3 -ITAM-containing receptors resulted in differ- Purified recombinant influenza A virus strain A/NT/60/68 was kindly pro- ences in substrate tyrosine phosphorylation patterns (27), induction vided by Dr. R. Consalves (National Institute of Medical Research, Lon- of apoptosis (38), and mobilization of intracellular free calcium don, U.K.). A/NT/60/68 was grown and tested for hemagglutination activ- (39), suggesting that they may couple to distinct signaling path- ity and infectious titers in the Department of Virology, Erasmus University Rotterdam, The Netherlands. ways. The observation that these recombinant receptors containing Mice were anesthetized and subsequently infected intranasally with 50 the cytoplasmic tail of CD3⑀ or CD3␨ were equally capable of ␮l of PBS with or without A/NT/60/68 virus (25 Hau for a primary infec- Downloaded from inducing early and late T cell activation events (27, 37, 50) already tion and 250 Hau for a secondary infection). Draining mediastinal lymph indicates that both options (qualitative vs quantitative differences nodes (MLNs), lungs, and spleens were analyzed at the indicated days between individual ITAMs) are not mutually exclusive and may postinfection. operate simultaneously during TCR-mediated signaling (38). Flow cytometry In all Ag receptor-driven checkpoints, the CD3␥ chain may play Preparation of samples for flow cytometry analysis was performed as de-

␥␦⑀ http://www.jimmunol.org/ a crucial role, in particular because it was shown that the CD3- scribed (16). Cells were analyzed on a BD Biosciences FACSCalibur module endows the TCR with normal signaling function (37). We (Mountain View, CA). Forward- and side-scatter gating and/or propidium previously reported that mice lacking CD3␥, due to targeted gene iodide gating was used to exclude dead cells from the analysis. disruption, display serious defects in ␣␤ T cell development (16). Biotinylated, FITC-, PE-, or APC-conjugated Abs speciﬁc for murine ␣ ␤ The transition from the CD4ϪCD8Ϫ double negative to the CD4 (clone RM4-5), CD8 (clone 53-6.7), CD8 (clone 53-5.8), CD25 ϩ ϩ ␥ (clone 7D4), CD62L (L-selectin; clone MEL-14), CD69 (clone H1.2F3), CD4 CD8 DP stage is severely impaired, indicating that CD3 CD90.1 (Thy-1.1; clone HIS51), CD90.2 (Thy-1.2; clone 30-H12), is required for the earliest Ag receptor-driven control point (16). TCR-␥␦ (clone GL3), and TCR-␤ (clone H57-597) were obtained from BD Furthermore, the nearly complete absence of CD69-positive DP PharMingen (San Diego, CA). R-PE anti-mouse CD4 (clone CT-CD4) was thymocytes in mice lacking CD3␥ (51) suggests that CD3␥ also purchased from Caltag (South San Francisco, CA). Where appropriate,

streptavidin-Tricolor or streptavidin-PE (Caltag) was used as second-step by guest on September 25, 2021 plays a role during the second major Ag receptor-dependent con- reagent. trol point, because expression of CD69 is normally up-regulated by Preparation of PE-conjugated H-2Db-tetramers containing the nucleo-

DP thymocytes that have been positively selected. Here we explore protein peptide (NP366–374(NT); ASNENMDAM) of the influenza A virus the consequences of CD3␥ deficiency and CD3␥-ITAM deficiency strain A/NT/60/68 has been described previously (53). for the functional capacity of peripheral T cells in vitro and in vivo. T cell purification Single-cell suspensions of total lymph node cells were prepared in IMDM Materials and Methods (Life Technologies) supplemented with 10% FCS (PAA Laboratories Generation of CD3␥-⌬ITAM mutant mice GmbH, Linz, Austria), 2 ϫ 10Ϫ5 M 2-ME (Merck, Darmstadt, Germany), ␮ ␥ 100 U/ml penicillin, and 100 g/ml streptomycin. Erythrocytes were lysed A mouse genomic clone encompassing the CD3 gene was isolated from by incubating the cells in a hypotonic buffer (0.14 M NH Cl, 0.017 M Tris a 129SVJ phage library (Stratagene, La Jolla, CA). To construct the tar- 4 ␥ ⌬ ␥ pH 7.2) for 5 min on ice. Subsequently, cells were washed twice and geting vector CD3 - I, a 10.2-kb fragment comprising CD3 exons 3–7 passaged over a nylon wool column. To remove remaining B cells and was subcloned into plasmid pBS-SKII (Stratagene). A 1.3-kb PvuII-NsiI other MHC class II expressing cells, cells were incubated with saturating fragment encompassing the CD3␥-ITAM was replaced by two comple- Ј concentrations of anti-MHC class II (clone M5/114) mAb for 30 min at mentary oligonucleotides encoding 5 -CTGTGAGTGTCCCCCCTTCTAT 4°C. Cells were washed once and MHC class II-positive cells were nega- CCAGCACCCAGAATCAAAACAATGCA-3Ј and 5Ј-TTGTTTTGATT Ј tively selected twice using a mixture of goat anti-mouse IgG-coated (Ad- CTGGGTGCTGGATAGAAGGGGGGACACTCACAG-3 , restoring the vanced Magnetics, Cambridge, MA) and sheep anti-rat IgG-coated mag- PvuII site in exon 5 and introducing a stop codon immediately after this Ј netic beads (Dynal, Oslo, Norway) in a 5:1 ratio. This procedure resulted particular PvuII site. The oligonucleotide sequence directly 3 of the stop in Ͼ97% Thy-1.1/Thy-1.2 positive T cells as determined by flow codon restores the deleted nucleotide sequence of exon 7 including the NsiI cytometry. site. In addition, a 1.1-kb SacI-SacI fragment contained within intron 4 was replaced by a 3.0-kb pgk-HPRT cassette flanked by loxP sites. The T cell activation, TCR down-regulation, and proliferation assay CD3␥-⌬I targeting vector was electroporated into the HM-1 ES cell line derived from 129OLA mice. Clones resistant to HAT (Life Technologies, Purified lymph node T cells (2 ϫ 105) were cocultured with 4 ϫ 105 (in Paisley, U.K.) were individually screened by Southern blot analysis for case flow cytometry was performed as a readout system: expression anal- homologous recombination events, using a probe located outside the tar- ysis of activation markers and TCR down-regulation) or 1 ϫ 105 (in case geting construct, which recognizes a 10.6-kb wild-type (wt) fragment and [3H]thymidine incorporation was measured as a readout system: prolifer- a 9.3-kb recombinant fragment in case DNA is digested with SacI and an ation assay) irradiated syngeneic spleen cells as a source of APCs in a total 8.2-kb wt fragment and an 11.2-kb recombinant fragment when PstI-di- volume of 200 ␮l per round-bottom microtiter well. Cultures were incu-

gested DNA is used. Two homologous recombinants were identified of 600 bated in a 37°C, 5% CO2 humidified incubator. For immobilized, plate- colonies tested. Subsequently, clones were transiently transfected with Cre bound Ab induced stimulation, anti-CD3⑀ (clone 145-2C11) (54) or control recombinase to remove the pgk-HPRT cassette from the embryonic stem hamster IgG (Jackson ImmunoResearch, West Grove, PA) was diluted in (ES) cell genome. Clones surviving counterselection by 6-Thioguanine PBS, and 50 ␮l of diluted Ab was added per well. Plates were incubated for (Sigma, St. Louis, MO) were individually screened by Southern blot anal- 6 h at 37°C and washed three times with PBS before use. After 24 h, cells ysis for loss of the pgk-HPRT cassette, using EcoRV-digested DNA and a were stained for flow cytometric analysis. Alternatively, 100 ␮l of culture probe located inside the targeting construct recognizing an 11.0-kb wt frag- supernatant was collected at indicated time points for cytokine analysis and 2578 FUNCTION OF THE CD3␥-ITAM IN TCR SIGNALING cultures were pulsed with 0.5 ␮Ci/well [3H]thymidine. Cultures were har- TCR-␤, reaching only 5–10% of the levels observed on control vested 18 h later, and [3H]thymidine incorporation was measured using populations (16). This finding suggests that in the absence of liquid scintillation counting. The analysis was performed in triplicate and CD3␥, assembly and/or efficient transport of the mature TCR com- SDs were Ͻ10% of the mean. plex to the cell surface is severely impaired. Indeed, biochemical IL-2 assay analysis of the mature TCR complex after surface radio-iodination ␥ IL-2 production was analyzed using the IL-2-responsive cell line CTLL-2 confirmed that lymph node T cells lacking CD3 express strongly (American Type Culture Collection, Manassas, VA). After three washes reduced levels of both the TCR-␣␤ heterodimer as well as CD3 with complete medium, 5 ϫ 103 CTLL-2 cells/well were cultured in a components compared with their wt counterparts (Fig. 1A). It flat-bottom microtiter plate with 75 ␮l supernatant in a total volume of 100 should be noted, in this respect, that the exposure time of the ␮l. Human rIL-2 (Cetus, Emeryville, CA) was used as a control. At indi- ␥ cated time points, cultures were pulsed with 0.5 ␮Ci/well [3H]thymidine CD3 -deficient TCRs shown in Fig. 1A is three times as long as for 18 h and thymidine incorporation was measured by liquid scintillation the exposure time of the wt peripheral TCRs. Furthermore, only counting. All assays were performed in triplicate and SDs were Ͻ10% of anti-CD3␦ and anti-CD3⑀ but not anti-CD3␥ Abs immunoprecipi- the mean. tated the TCR complex expressed on lymph node T cells from ␥ Mixed lymphocyte reaction CD3 -deficient mice, while all three antisera used could easily immunoprecipitate the TCR complex expressed by control T cells For primary MLRs, 2 ϫ 105 purified lymph node T cells (for suboptimal ␥ ϫ 5 (Fig. 1A). These data indicate that with the exception of CD3 , conditions: 1 10 purified lymph node T cells) were cocultured with ␥ indicated numbers of irradiated MHC-mismatched SJL/J spleen cells in a lymph node T cells derived from CD3 null-mutant mice display round-bottom microtiter plate in a total volume of 200 ␮l. After 4 days, the normal complement of TCR subunits. In agreement with these 3 cultures were pulsed with 0.5 ␮Ci/well [ H]thymidine for 18 h and thy- results, two-dimensional nonreduced/reduced SDS-PAGE re- Downloaded from midine incorporation was measured by liquid scintillation counting. All vealed once more the presence of CD3⑀ and CD3␦ proteins in assays were performed in triplicate and SDs were Ͻ10% of the mean. addition to the disulfide-linked TCR-␣␤ heterodimer at the cell CTL effector function assay surface of T cells lacking CD3␥ (Fig. 1B). Polyclonal induction of CTL activity was established by coculturing 2 ϫ 106 purified lymph node T cells with 4 ϫ 106 irradiated MHC-mismatched 6 T cell development in CD3␥-⌬ITAM mutant mice SJL/J spleen cells (for suboptimal conditions: 1 ϫ 10 purified lymph node http://www.jimmunol.org/ T cells with 2 ϫ 106 irradiated MHC-mismatched SJL/J spleen cells) in a Because the CD3␥ protein is clearly required for efficient surface 24-well plate in a total volume of 2 ml. After 4 days, effector T cells were expression of the TCR, besides endowing the TCR with signaling harvested and CTL activity was assayed as described (54). Briefly, effector cells were cocultured with 51Cr-labeled (Amersham, Little Chalfont, U.K.) capacity, quantitative or qualitative causes for any signaling defect non-Ag-bearing, Fc receptor-positive K562 cells for 18 h in the presence or must be distinguished. For this purpose, CD3␥-⌬ITAM mutant absence of 5 ␮g/ml of soluble anti-CD3⑀ (clone 145-2C11). The percent- mice lacking only the ITAM of the CD3␥ chain (Fig. 2A) were age of specific lysis was calculated as the ratio of 100 ϫ (cpm experimental generated by gene targeting in ES cells, using CD3␥-⌬I as a tar- release Ϫ cpm spontaneous release)/(cpm 1% Triton X-100 release Ϫ cpm spontaneous release). geting vector containing a pgk-HPRT cassette flanked by loxP sites (Fig. 2B). Two homologous recombinants were identified of Generation of anti-peptide sera 600 colonies tested and subsequently transiently transfected with by guest on September 25, 2021 Peptides corresponding to the C-terminal regions of CD3␥ (EYDQY Cre recombinase to remove the pgk-HPRT cassette from the ES SHLQGNQLRKK) and CD3␦ (TQYSRLGGNWPRNKKS) were synthe- cell genome. One clone was selected of 150 colonies tested and sized and coupled to cationized BSA using the Imject Activated Super- used for the generation of chimeric mice. Germline transmission Carrier System according to the manufacturer’s protocol (Pierce, Rockford, was obtained and heterozygous mice were intercrossed to produce IL). The peptide-cationized BSA conjugates were used to immunize ␥ ⌬ rabbits. mice homozygous for the CD3 - ITAM mutation (Fig. 2C). Biochemical analysis of the mature TCR complex expressed on Radiolabeling and immunoprecipitation lymph node T cells derived from CD3␥-⌬ITAM mice revealed the Lactoperoxidase-catalyzed surface labeling of 108 purified lymph node T presence of all known TCR components: the TCR-␣␤ heterodimer cells with Na125I (Amersham) was performed and cells were subsequently as well as CD3␥, CD3␦, CD3⑀, and CD3␨ polypeptides (Fig. 1A lysed in lysis buffer (1% Brij96, 150 mM NaCl, 10 mM triethanolamine and B). The apparent contradiction that the CD3␥ chain lacking the (pH 7.8), 5 mM EDTA, 1 mM PMSF, 20 ␮g/ml trypsin inhibitor, 20 ␮g/ml leupeptin, and 20 ␮g/ml TLCK) for 30 min on ice. Nuclear debris was ITAM can be detected by two-dimensional nonreduced/reduced removed by centrifugation at 14.000 rpm for 15 min at 4°C. Cell lysates SDS-PAGE (Fig. 1B), while the anti-CD3␥ serum does not pre- were extensively precleared by incubation with normal hamster serum or cipitate the TCR complex expressed by CD3␥-⌬ITAM T cells normal rabbit serum and protein A-Sepharose-CL4B beads (Pharmacia, (Fig. 1A), can be explained by the fact that the anti-CD3␥ serum Uppsala, Sweden). Subsequently, extracts were immunoprecipitated with ⑀ ␦ ␥ recognizes epitopes present in that particular cytoplasmic region of Abs specific for CD3 , CD3 ,orCD3 (54) in the presence of protein ␥ ␥ ⌬ A-Sepharose-CL4B beads for2hat4°C. After five to six washes in lysis the CD3 chain that has been removed in CD3 - ITAM mice. buffer, immunoprecipitates were resuspended in 30 ␮l of sample buffer At face value, ␣␤ T cell development proceeds undisturbed in (10% glycerol, 3% SDS, 62.5 mM Tris pH 6.8, 0.005% Bromophenol blue, the absence of the CD3␥-ITAM. Indeed, as illustrated in Fig. 3, A and, if applicable, 5% 2-ME), heated for 5 min at 95°C, cooled to room and B, by CD4 vs CD8 staining (top), mature ␣␤ T cells can easily temperature, and loaded on one-dimensional 10–15% gradient SDS-PAGE be detected in normal numbers in the thymus and lymph nodes of gels or first on a 12.5% SDS polyacrylamide tube gel under nonreducing ⌬I/⌬I conditions, followed by a 12.5% SDS-PAGE slab gel under reducing con- CD3␥ mice. Consistent with our previous report, mice lacking ditions. After fixation and drying of the gels, signals were visualized and the complete CD3␥ chain exhibit strongly reduced thymic and quantified by phospho-imaging or autoradiography at Ϫ70°C. lymph node cellularity and reduced CD3⑀ and TCR-␤ surface expression on mature T cells (Fig. 3, A and B) (16). The expression Results level of the mature ␣␤ TCR complex on T cells from CD3␥- Subunit composition of the mature TCR complex expressed by ⌬ITAM mice, in contrast, is similar to that on control T cells (Fig. ␥ CD3 -deficient peripheral T cells 3, A and B, bottom). We also analyzed to which extent loss of the In contrast to peripheral T cells derived from wt mice, the few CD3␥-ITAM affects development of the TCR-␥␦ lineage and find peripheral T cells that are exported to the periphery in mice lacking apparently normal maturation of this T cell lineage as well, quite CD3␥ display severely reduced expression levels of CD3⑀ and in contrast to the situation in CD3␥-deficient mice (Fig. 3C) (16). The Journal of Immunology 2579 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Biochemical analysis of the clonotypic TCR complex expressed on peripheral T cells lacking CD3␥. Lymph node T cells of 8- to 12-wk-old CD3␥ϩ/ϩ, CD3␥⌬I/⌬I, and CD3␥Ϫ/Ϫ mice were surface iodinated, lysed, and immunoprecipitated with the indicated Abs. Immunoprecipitates were analyzed (A) under nonreducing conditions by SDS-PAGE on a 10–15% gradient gel or (B) by 2-D nonreduced/reduced SDS-PAGE. The positions of TCR-␣␤, CD3␥, CD3␦, CD3⑀, and CD3␨ chains are indicated. Poor visualization of the CD3␨-␨ dimers is due to inefﬁcient labeling by surface radio- iodination of this particular chain.

The absolute numbers of TCR-␥␦-positive T cells that could be ␥␦ TCR complex are expressed in wt and CD3␥-ITAM mutant T detected in the thymus as well as in the periphery of CD3␥-ITAM cells (Fig. 3C; data not shown). Importantly, these data indicate mutant and wt mice are comparable, and equivalent levels of the that in CD3␥-deﬁcient mice, the observed defects in ␣␤ and ␥␦ T 2580 FUNCTION OF THE CD3␥-ITAM IN TCR SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Disruption of the CD3␥-ITAM by homologous recombination. A, Schematic representation of the intracellular tail of the wt CD3␥ chain (top) and the CD3␥-⌬ITAM chain (bottom). B, Partial organization of the CD3␥ locus (top), structure of the targeting vector CD3␥-⌬I(middle), and structure of the disrupted CD3␥ gene following homologous recombination and transient transfection of the Cre recombinase (bottom). The probes used for the hybridizations and the predicted fragment sizes generated by the endogenous and targeted alleles after EcoRV digestion are depicted. Exons are denoted as ﬁlled boxes and numbered 3–7. The location of the CD3␥-ITAM is indicated by Y LYL above exons 5 and 6, respectively. Restriction enzymes: B, BamHI; EV, EcoRV; N, NsiI; P, PstI; Pv, PvuII; S, SacI. C, Southern blot analysis of EcoRV-digested tail DNA derived from wt (CD3␥ϩ/ϩ), heterozygous mutant (CD3␥ϩ/⌬I), and homozygous mutant (CD3␥⌬I/⌬I) mice.

cell development are not the result of impaired CD3␥-ITAM-spe- null-mutant mice were stimulated with plate-bound anti-CD3⑀ or cific signaling, but of defective assembly of the pre-TCR in case of control hamster IgG and stained for CD25, CD69, or CD62L (Fig. maturation of ␣␤ T cells, and impaired assembly of a pre-TCR- 4). Within 24 h, ϳ75–95% of the wt peripheral T cells express ␥␦-type structure or the mature TCR-␥␦ complex in case of ␥␦ T CD25 or CD69 and down-regulate CD62L in response to anti- cell differentiation. CD3⑀ treatment. In the absence of an intact CD3␥-ITAM, the percentage of T cells exhibiting an activated phenotype drops to ␥Ϫ/Ϫ Expression of surface activation markers is impaired in CD3 ϳ50–65%, suggesting a quantitative contribution of the CD3␥- ␥ ⌬ as well as CD3 - ITAM peripheral T cells albeit to different ITAM with respect to the induction or reduction in expression of extents these particular activation markers. Moreover, only a relatively To investigate the functional capacity of primary T cells deficient small fraction (ϳ30–35%) of the CD3␥-deficient T cells display a for CD3␥ or expressing CD3␥ subunits devoid of a functional similar activated phenotype. This reduced number is likely to be ITAM, lymph node T cells from wt, CD3␥-⌬ITAM, and CD3␥ related to the recent report that T cell activation requires a certain The Journal of Immunology 2581 Downloaded from

FIGURE 3. Development of both the ␣␤

and ␥␦ T cell lineage is unaffected in CD3␥- http://www.jimmunol.org/ ⌬ITAM mice. (A) Thymocytes and (B) lymph node cells of 8- to 12-wk-old CD3␥ϩ/ϩ, CD3␥⌬I/⌬I, and CD3␥Ϫ/Ϫ mice were analyzed by three-parameter ﬂow cytometry for the expression of CD4-PE vs CD8-Biotin plus SA- Tricolor (top) and for the expression of TCR␤- FITC (gray) or an irrelevant mAb (white) in the distinct cell populations that are character- ized, based on the differential expression of CD4 vs CD8 (bottom). The percentage of cells by guest on September 25, 2021 within each quadrant is indicated. The absolute number of cells detected in the different geno- types is depicted above the corresponding dot display. C, Total thymocytes were analyzed for the expression of TCR␥␦-FITC vs CD3⑀-PE. The percentage of cells within the depicted gates is indicated in each dot display. 2582 FUNCTION OF THE CD3␥-ITAM IN TCR SIGNALING Downloaded from

FIGURE 3. (Continued) number of TCRs to be triggered, irrespective of the nature of the obtained using control peripheral T cells (Fig. 5A), clearly indi- triggering ligand (55). The capacity to reach this activation thresh- cating that the ITAM of the CD3␥ subunit is dispensable for in- old is severely compromised by a reduction in the number of TCRs duction of these late T cell activation events. In contrast, peripheral http://www.jimmunol.org/ expressed at the cell surface. Therefore, the reduced TCR surface T cells lacking CD3␥ display a delayed onset of proliferation and expression on lymph node T cells lacking CD3␥ (16) (Figs. 1A and IL-2 production in response to CD3⑀-mediated triggering (Fig. 3B) will impair the ability to reach the optimal activation threshold 5A). Two days after stimulation, CD3␥-deficient T cells show a and may provide an explanation for the small proportion of acti- strongly impaired capacity to proliferate and to produce IL-2 com- vated CD3␥Ϫ/Ϫ T cells detected after exposure to anti-CD3⑀ mAb. pared with control T cells. However, 3 days poststimulation, Importantly, the activated CD3␥Ϫ/Ϫ T cell population expresses CD3␥-deficient T cells clearly have started to proliferate and to normal levels of CD25 and CD69 (Fig. 4). This suggests that, at secrete levels of IL-2 comparable to those observed in wt and ⌬I/⌬I least with respect to the expression levels of these cell surface CD3␥ mice (Fig. 5A). These data are in accordance with the by guest on September 25, 2021 markers, T cells that finally have reached the activation threshold view that only a certain proportion of the CD3␥-deficient T cells can become fully activated in the absence of CD3␥. gets activated in response to CD3⑀-mediated cross-linking and would suggest that also with respect to proliferation and IL-2 pro- ␥ Proliferation and cytokine secretion is delayed only in CD3 - duction, CD3␥-deficient T cells are not dramatically hampered in deficient peripheral T cells their functional ability, once they get activated by anti-CD3⑀ mAb We next evaluated the capacity of peripheral T cells that developed triggering. Another possibility is that 3 days after stimulation with in CD3␥ϩ/ϩ, CD3␥⌬I/⌬I,orCD3␥Ϫ/Ϫ mice to proliferate and se- anti-CD3⑀ mAb, all CD3␥-deficient T cells have become acti- crete IL-2 in response to anti-CD3⑀-mediated cross-linking (Fig. vated. However, also this latter explanation would suggest that 5). Lymph node T cells harboring signaling-defective CD3␥ chains once activated, CD3␥-deficient T cells are not defective in the displayed dose-response curves almost superimposable to those execution of these late T cell activation events. In contrast to anti-

FIGURE 4. Regulation of expression of activation markers is impaired in CD3␥-deficient as well as CD3␥-⌬ITAM-deficient peripheral T cells, albeit to different extents. Lymph node T cells of 8- to 12-wk-old CD3␥ϩ/ϩ, CD3␥⌬I/⌬I, and CD3␥Ϫ/Ϫ mice were analyzed by flow cytometry for the expression of CD25, CD69, and CD62L (open areas) or an irrelevant mAb (gray areas) in response to stimulation with 10 ␮g/ml plate-bound control hamster IgG (thin line) or 145–2C11 (anti-CD3⑀) (fat line) for 24 h. In several histograms, the staining with irrelevant mAb and the specific staining after stimulation with control hamster IgG overlap. The Journal of Immunology 2583

the TCR, whereas T cells derived from mice lacking CD3␥ were completely defective in this respect (Fig. 6A, top), despite the fact that ϳ90% of the CD3␥-deficient lymph node T cells were activated according to the expression of the early activation marker CD69 (Fig. 6A, bottom). In sharp contrast, the CD3␥-ITAM seemed dispensable for efficient internalization of the TCR in response to PMA treatment, because the dose-response curves of TCRs deprived of a functional CD3␥-ITAM and their wt counterparts were overlapping (Fig. 6A). Ab-mediated cross-linking of CD3⑀ on control lymph node T cells also resulted in a profound down-modulation of the TCR in a dose-dependent manner to ϳ25% of the initial expression levels at the highest anti-CD3⑀ mAb concentration used (Fig. 6B, top). Internalization of the TCR occurred irrespective of the presence of a functional CD3␥-ITAM, excluding a role for the CD3␥-ITAM in this process (Fig. 6B). Surprisingly, although 30–35% of the CD3␥-deficient peripheral T cells expressed CD69 in response to CD3⑀-mediated cross-linking, TCR down-modulation was se-

verely compromised (Fig. 6B). Taken together, in contrast to ear- Downloaded from lier reports in cell lines (59, 60), these data in primary T cells point toward a role of the CD3␥ chain in both PMA-induced as well as Ab-mediated TCR internalization. Because we ﬁnd no role for the CD3␥-ITAM in this process, a possible role for the CD3␥ di- leucine motif in internalization remains to be addressed. Further

clariﬁcation of this issue awaits the generation of CD3␥-deﬁcient http://www.jimmunol.org/ mice only lacking the di-leucine motif of the CD3␥ protein (M. C. Haks, C. Geisler, and A. M. Kruisbeek, manuscript in preparation).

Cytotoxic T cell responses are affected in T cells lacking CD3␥ when stimulated under suboptimal conditions To examine the cytolytic capability of effector T cells deficient for CD3␥ or expressing CD3␥ subunits lacking ITAMs, polyclonal induction of CTL activity was established by a primary MLR. T cells deficient for CD3␥ or T cells lacking the CD3␥-ITAM dis- by guest on September 25, 2021 played a similar proliferative response induced in MLR to those induced in T cells encompassing the normal complement of CD3 FIGURE 5. Proliferation and IL-2 secretion are delayed only in CD3␥- deficient peripheral T cells. Proliferation and IL-2 production were ana- ITAMs (Fig. 7A, top), suggesting that at least this aspect of the lyzed in lymph node T cells of 8- to 12-wk-old CD3␥ϩ/ϩ (Ⅺ), CD3␥⌬I/⌬I induction phase of CTL activity did not depend on the presence of (छ), and CD3␥Ϫ/Ϫ (F) mice in response to (A) stimulation with 145-2C11 (anti-CD3⑀) for 48 h (top)or72h(bottom), or (B) treatment with PMA for 24h(top)or48h(bottom).

CD3⑀ exposure, stimulation with PMA, bypassing external triggering of the TCR complex, results in comparable proliferation and IL-2 production between CD3␥-deﬁcient and CD3␥-⌬ITAM or wt peripheral T cells (Fig. 5B). These results indicate that the observed defects in inducing activation are primarily at the level of initiation of the signal-transduction pathway by the TCR complex expressed at the cell surface.

TCR down-regulation is completely abrogated in CD3␥-deficient T cells In addition to the ITAM, a di-leucine motif has been identified in the cytoplasmic domain of the CD3␥ chain (56–58). Studies in cell lines have suggested that this motif may be involved at least in protein kinase C (PKC)-mediated TCR down-regulation but is irrelevant for TCR down-modulation resulting from exposure to anti- FIGURE 6. Induction of TCR down-regulation is defective only in CD3␥-deficient T cells. Lymph node T cells of 8- to 12-wk-old CD3␥ϩ/ϩ CD3⑀ (59, 60). After T cell activation, TCR internalization ulti- ⌬ ⌬ Ϫ Ϫ (Ⅺ), CD3␥ I/ I (छ), and CD3␥ / (F) mice were analyzed by flow cy- mately results in extinction of the signaling process, allowing the tometry for the expression of TCR␤-FITC (top) or CD69-biotin plus cells to calibrate the response to the level of the stimulus. Consis- SA-PE (bottom) in response to (A) treatment with PMA for5hor(B) tent with the view that the CD3␥ di-leucine motif is important for stimulation with anti-CD3⑀ for 24 h. In both A and B, 100% value corre- PKC-induced internalization, stimulation of lymph node T cells of sponds to the mean fluorescence of the TCR-␤ staining on unstimulated control mice with PMA resulted in a clear-cut down-modulation of peripheral T cells. 2584 FUNCTION OF THE CD3␥-ITAM IN TCR SIGNALING

FIGURE 7. The effector function of cytotoxic T cells is affected in T cells lacking CD3␥. A, Prolifer- ation was assayed in a primary mixed lymphocyte reaction by coculturing lymph node T cells derived from 8- to 12-wk-old CD3␥ϩ/ϩ (Ⅺ), CD3␥⌬I/⌬I (छ), and CD3␥Ϫ/Ϫ (F) mice with indicated numbers of MHC-mismatched SJL/J spleen cells under optimal conditions (top) or suboptimal conditions (bottom) for 4 days. B, Effector T cells of 8- to 12-wk-old CD3␥ϩ/ϩ (Ⅺ), CD3␥⌬I/⌬I (छ), and CD3␥Ϫ/Ϫ (F) mice generated in an MLR under optimal conditions (top) or suboptimal conditions (bottom) for 4 days were analyzed for their cytolytic capacity in a 51Cr-release assay in the presence of 145-2C11 (anti-CD3⑀). Downloaded from

␥ an intact CD3 chain. CTL activity of the effector T cells was these mice, albeit at lower frequencies (1.5–2.5% and 0.5–1.5%, http://www.jimmunol.org/ 51 assayed by coculturing these cells with Cr-labeled non-Ag-bear- respectively) (data not shown) (53). In sharp contrast, NP366–374- ing, Fc receptor-positive K562 cells in the presence or absence of specific CD8 T cells were entirely undetectable in the lungs, soluble anti-CD3⑀. In this assay, Abs directed against the TCR spleens, and MLNs of mice deficient for CD3␥ (Fig. 8A, top; data complex trigger the lytic function by binding to the TCR complex not shown). This is not simply due to a delay in the response, on the effector T cells and provide cell-cell contact with the target because even at 21 days postinfection, NP366–374-specific CD8 T K562 cells through Fc receptor binding (54). As expected, in the cells are still undetectable in the lungs of influenza A infected absence of anti-CD3⑀, cytolytic activity was below detection levels CD3␥-deficient mice (Fig. 8A, bottom). Moreover, even a primary ⌬I/⌬I in control as well as CD3␥ and CD3␥-deficient effector T cells infection with influenza A virus followed 8 wk later by induction

⑀ by guest on September 25, 2021 (data not shown). In the presence of anti-CD3 , cytolytic activity of a memory T cell response by a second infection with influenza could easily be detected in all effector T cells tested, even in the A virus did not result in any detectable Ag-specific CD8 T cells in ␥ ϳ complete absence of a CD3 chain, reaching 50–60% specific the lungs of CD3␥-deficient mice, while, in the lungs of control lysis at the highest E:T ratios used (Fig. 7B, top). However, poly- mice, Ag-specific CD8 T cells now comprised 25–30% of the total clonal induction of CTL activity under suboptimal conditions (by CD8 T cell population (Fig. 8B). Thus, although the in vitro pro- decreasing both the absolute number of stimulator and responder liferative response induced by anti-CD3⑀ or MLR in CD3␥-defi- cells) resulted in a reduction in the proliferative response of cient T cells is slightly delayed (only in the case of anti-CD3⑀) but CD3␥Ϫ/Ϫ T cells compared with wt and CD3␥-⌬ITAM T cells in otherwise comparable in magnitude (Fig. 5A; Fig. 7A), the in vivo MLR and completely abolished cytolytic activity of only the expansion of an Ag-specific CD8 T cell population is completely CD3␥-deficient effector T cells (Fig. 7, A and B, bottom). There- abrogated by lack of CD3␥ (Fig. 8, A and B). fore, more stringent culture conditions reveal that loss of the CD3␥ Interestingly, the lack of expansion of Ag-specific CD3␥-defi- chain does result in a severe loss of the capacity to generate ef- cient CD8 T cells is not the consequence of a CD3␥-ITAM-spe- fector T cells. Nevertheless, this is not a consequence of a unique ␥ contribution of CD3␥-ITAM. cific signaling function. In mice lacking the CD3 -ITAM, the percentage of NP366–374-specific CD8 T cells in the lung after CD3␥ deficiency and CD3␥-ITAM deficiency have opposite influenza infection is 10–14% of the total CD8-positive T cell effects on the appearance of NP366–374-specific CD8 T cells in population (Fig. 8D), i.e., even higher than in control mice. This response to influenza infection increase was specific for the lung, because absence of the CD3␥- Collectively, the results shown this far indicate that CD3␥-defi- ITAM did not seem to affect the percentage of NP366–374-specific cient T cells function relatively normal in some respects (induction CD8 T cells detected in spleens and MLN (data not shown). The b of cytokine production and proliferation), although they are defec- inability to detect H-2D -NP366–374(NT) tetramer-positive CD8 T ␥ tive in others. Particularly notable is their inability to perform cy- cells in CD3 -deficient mice is unlikely to result from reduced tolytic effector function under suboptimal conditions. Because this expression levels of the TCR complex on peripheral T cells of response is dependent on CD8 T cells, we explored the ability of these mice (16) (Fig. 1A; Fig. 3B): because the staining intensity of ␤ b Ag-specific CD8 T cells to respond in vivo to a viral infection by the anti-TCR mAb and the H-2D -NP366–374(NT) tetramers is b expansion. Mice were infected intranasally with influenza A virus comparable (Fig. 8, A and C), staining with H-2D -NP366–374(NT) strain A/NT/60/68 and at the peak of the infection (8 days postin- tetramers should be able to visualize NP366–374-specific CD8 T fection), 4–7% of the CD8-positive T cells obtained from inflamed cells easily above background levels in CD3␥-deficient mice. The b lung tissue derived from control mice stained positive for H-2D - possibility that recognition of NP366–374-specific CD8 T cells by b NP366–374(NT) tetramers (Fig. 8A, top).NP366–374-specific CD8 T H-2D -NP366–374(NT) tetramers is abrogated as a consequence of a cells could also be detected in the spleens and draining MLNs of conformational change of the mature TCR complex in the absence The Journal of Immunology 2585 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

␥ ␥ FIGURE 8. CD3 deficiency and CD3 -ITAM deficiency have opposite effects on the appearance of NP366–374-specific CD8 T cells in response to influenza infection. CD3␥ϩ/ϩ, CD3␥⌬I/⌬I, and CD3␥Ϫ/Ϫ mice were infected with influenza A virus strain A/NT/60/68. At the indicated days postinfection b ␤ electronically gated CD8-APC-positive T cells from the lung were stained with H-2D -NP366–374(NT)-PE conjugated tetramers (A, B, and D)orTCR -FITC ␥ ␥ b (C). E, DP thymocytes derived from CD3 -deficient mice and F5-TCR transgenic CD3 -deficient mice were stained with H-2D -NP366–374(NT)-PE- conjugated tetramers (top)orTCR␤-FITC (bottom). F, Single-cell suspensions from influenza infected lung tissue were stained with CD8-APC plus b H-2D -NP366–374(NT)-PE-conjugated tetramers. of the CD3␥ polypeptide seems unlikely, because these tetramers normal signaling capabilities. The spectrum of T cell activation, do stain DP thymocytes derived from CD3␥-deficient mice crossed proliferation, and differentiation events that was examined in this ␣␤ ␥ with F5-TCR transgenic mice recognizing the NP peptide (Fig. report did not depend on the integrity of the CD3 -ITAM, with the 8E, top). Importantly, the expression level of the CD3␥-deficient exception of the expression of activation markers in which a slight

F5-TCRs on DP thymocytes is comparable to the expression level reduction could be observed. In contrast, expression of activation of nontransgenic CD3␥-deficient TCRs on T cells derived from markers and induction of proliferation, IL-2 secretion, and TCR lung tissue (Fig. 8, C and E, lower). This indicates that the ex- down-regulation were all affected in lymph node T cells lacking pression level of TCRs on CD3␥-deficient T cells is sufficient for the complete CD3␥ chain. The small fraction of T cells up-regu- b ␥ H-2D -NP366–374(NT) tetramer binding. Thus, either the reduced lating CD25 and CD69 and down-regulating CD62L in CD3 - TCR expression levels of CD3␥ null-mutant T cells or absence of deficient T cells, as well as the delayed onset of proliferation and other signaling functions (such as di-leucine motif-mediated sig- IL-2 secretion, is presumably the result of impaired cell surface naling) are responsible for the total lack of NP366–374-specific CD8 expression of the TCR complex (16) (Fig. 1A). In accordance with Ϫ Ϫ T cells in influenza-infected CD3␥ / mice. The CD3␥-ITAM, in this view, it has recently been shown that T cells count the number contrast, has no role in generating this response; it proceeds nor- of triggered TCRs on their cell surface and respond only when a mally (and is even slightly elevated) in mice lacking CD3␥-ITAM. certain threshold is reached (55). Because reduced TCR expression levels will compromise the capacity to reach this activation thresh- Discussion old, this predicts that fewer peripheral T cells in CD3␥Ϫ/Ϫ mice The present study explores the consequences of CD3␥ deficiency will be activated in response to a certain stimulus. Importantly, the and CD3␥ ITAM deficiency for TCR-induced activation and dif- normal expression levels of CD25 and CD69 and the massive pro- ferentiation of peripheral T cells. These experiments are particu- liferation and IL-2 secretion observed at later time points after larly relevant in view of the recent finding (35, 37) that the CD3- stimulation suggest that, once activated, these in vitro parameters ␥␦⑀ module is sufficient for endowing the TCR with qualitatively of T cell activation become independent of an intact CD3␥ chain. 2586 FUNCTION OF THE CD3␥-ITAM IN TCR SIGNALING

find that the ability of CD3␥-deficient mice to mount an in vivo response to an antigenic challenge is completely abrogated. This was not predicted by their in vitro behavior, and several possible (not mutually exclusive) explanations for the absence of a response to the main NP-epitope of influenza virus in CD3␥-deficient mice can be put forward. First, in the absence of CD3␥, positive and/or negative selection may be disturbed, resulting in absence of an NP-specific T cell repertoire. Second, influenza A virus infection may be unable to activate and/or expand NP-specific CD8 T cells in the absence of CD3␥. Third, CD3␥-deficient NP-specific CD8 T cells may be activated by influenza A virus, but due to a delayed expansion (Fig. 5A), they may remain below detection level. How- ever, the observations that NP-specific CD8 T cells can still not be detected in the lungs of CD3␥-deficient mice at later time points or in the memory response render this explanation rather unlikely. Fourth, activated NP-specific CD8 T cells lacking CD3␥ may be unable to home to the lung. This also seems unlikely because CD3␥-deficient T cells that do get activated express normal levels

of activation markers and homing receptors, such as LFA-1 (Fig. Downloaded from 4 and data not shown) and an influx of CD8 T cells can be observed in the lungs of influenza infected CD3␥-deficient mice (Fig. 8F). Finally, CD3␥Ϫ/Ϫ NP-specific CD8 T cells may get activated FIGURE 8. (Continued) in vivo but then die more quickly due to activation-induced cell death than normal CD8 T cells. Because CD3␥-deficient T cells

are defective in down-regulation of the TCR, the extinction of the http://www.jimmunol.org/ Studies using (transformed) T cell lines derived from CD3␥- signaling process may be defective and make the cells more prone deficient patients revealed that whereas proliferation was com- to activation-induced cell death. Several of these issues will be pletely normal in these cells, IL-2 secretion was severely impaired addressed in further analyses of F5-TCR transgenic mice express- (61, 62). However, in these studies, a kinetic analysis of the re- ing a TCR specific for influenza nucleoprotein (52) crossed to the sponse was not performed, complicating the interpretation of the CD3␥Ϫ/ϪRAGϪ/Ϫ background. The augmentation in the percent- results and providing an explanation for the differences between age of NP366–374-specific CD8 T cells in the lungs of influenza our observations and these earlier data. Furthermore, the response A-infected mice lacking a CD3␥-ITAM compared with control of a (transformed) T cell line to a certain stimulus may not be mice might indicate a defect in activation-induced cell death, be- representative of the response of a polyclonal peripheral T cell cause the lungs are the site for accumulation of previously acti- by guest on September 25, 2021 population. For example, the diversity in the expression of activa- vated apoptotic T cells. This option will also be the subject of tion markers in response to CD3⑀-mediated cross-linking (Fig. 4) further investigation. can never be mimicked by a T cell line. Overall, these studies indicate that proper TCR functioning does Several studies have implicated the CD3␥ di-leucine motif spe- require the CD3␥ chain but not the CD3␥-ITAM, at least not in the cifically in PKC-mediated TCR internalization (57) and not in li- presence of the full collection of other ITAMs. Both quantitative gand- or anti-CD3⑀-induced TCR down-modulation (59, 60, 63). and qualitative differences may exist between the various CD3- Ligand-induced TCR down-modulation has been suggested to de- ITAMs with respect to their ability to interact with distinct kinases pend on p56lck and p59fyn (59, 64). These Src family protein ty- and adaptors (69, 70), and the present data predict that CD3␥ con- rosine kinases are involved in phosphorylation of the ITAMs after tributes primarily in a quantitative manner to TCR signaling. It ligation of the TCR complex (29). Interestingly, it has been sug- remains to be investigated whether a TCR lacking the CD3␥- gested using chimeric TAC/CD3␥ or CD3␦ cytoplasmic domain- ITAM couples differentially to cytosolic substrates and signaling containing polypeptide chains that besides the di-leucine motif pathways. If it does, dissecting how distinct biochemical events also ITAMs may be involved in TCR down-modulation (56). couple the TCR to similar functions will also require further study. Moreover, clathrin-coated vesicles mediate endocytosis of transmembrane receptors (65), and ITAM regions contain a tyrosine- Acknowledgments based sorting signal able to interact with a subunit of the AP-2 We thank Hergen Spits for critically reviewing the manuscript; B. Malissen clathrin-associated protein complex (66). Together, these studies for providing the genomic probe used to screen the phage library; predict that in mice lacking CD3␥ as well as in CD3␥-⌬ITAM M. Toebes, M. Hoffmann, and M. Beumkes for their excellent technical mutant mice, there will be no TCR down-modulation due to lack assistance; and M. A. van Halem for help in preparing the manuscript. of the di-leucine motif and/or the CD3␥-ITAM, respectively. In contrast to these predictions, internalization of the TCR complex References after exposure to anti-CD3⑀ mAb occurred irrespective of the pres- 1. Dudley, E. C., H. T. Petrie, L. M. Shah, M. J. Owen, and A. C. Hayday. 1994. ␤ ence of intact CD3␥-ITAMs, excluding a unique role for this par- T cell receptor chain gene rearrangement and selection during thymocyte development in adult mice. Immunity 1:83. ticular ITAM in internalization (Fig. 6B). 2. Levelt, C. N., and K. Eichmann. 1995. Receptors and signals in early thymic The in vitro activation studies with T cells lacking CD3␥ indi- selection. Immunity 3:667. 3. Malissen, B., and M. Malissen. 1996. Functions of TCR and pre-TCR subunits: cate a deficiency mainly in induction of cytolytic activity, while lessons from gene ablation. Curr. Opin. Immunol. 8:383. induction of cytokine secretion and proliferation are delayed but 4. Fehling, H. J., and H. Von Boehmer. 1997. Early ␣␤ T cell development in the otherwise intact. These findings of only mild defects contrast thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263. 5. Haks, M. C., M. A. Oosterwegel, B. Blom, H. Spits, and A. M. Kruisbeek. 1999. sharply with the dramatic immune deficiency syndrome observed Cell-fate decisions in early T cell development: regulation by cytokine receptors in CD3␥-deficient patients (67, 68). Therefore, it was surprising to and the pre-TCR. Semin. Immunol. 11:23. The Journal of Immunology 2587

6. Groettrup, M., K. Ungewiss, O. Azogui, R. Palacios, M. J. Owen, A. C. Hayday, 35. Shores, E. W., T. Tran, A. Grinberg, C. L. Sommers, H. Shen, and P. E. Love. and H. Von Boehmer. 1993. A novel disulfide-linked heterodimer on pre-T cells 1997. Role of the multiple T cell receptor (TCR)-␨ chain signaling motifs in consists of the T cell receptor ␤ chain and a 33 kd glycoprotein. Cell 75:283. selection of the T cell repertoire. J. Exp. Med. 185:893. 7. Saint-Ruf, C., K. Ungewiss, M. Groettrup, L. Bruno, H. J. Fehling, and 36. Irving, B. A., A. C. Chan, and A. Weiss. 1993. Functional characterization of a H. Von Boehmer. 1994. Analysis and expression of a cloned pre-T cell receptor signal transducing motif present in the T cell antigen receptor ␨-chain. J. Exp. gene. Science 266:1208. Med. 177:1093. 8. Love, P. E., E. W. Shores, M. D. Johnson, M. L. Tremblay, E. J. Lee, 37. Ardouin, L., C. Boyer, A. Gillet, J. Trucy, A.-M. Bernard, J. Nunes, J. Delon, A. Grinberg, S. P. Huang, A. Singer, and H. Westphal. 1993. T cell development A. Trautmann, H.-T. He, B. Malissen, and M. Malissen. 1999. Crippling of in mice that lack the ␨ chain of the T cell antigen receptor complex. Science CD3-␨ ITAMs does not impair T cell receptor signaling. Immunity 10:409. 261:918. 9. Jacobs, H., D. Vandeputte, L. Tolkamp, E. De Vries, J. Borst, and A. Berns. 1994. 38. Combadie`re, B., M. Freedman, L. Chen, E. W. Shores, P. Love, and CD3 components at the surface of pro-T cells can mediate pre-T cell development M. J. Lenardo. 1996. Qualitative and quantitative contributions of the T cell ␨ in vivo. Eur. J. Immunol. 24:934. receptor -chain to mature T cell apoptosis. J. Exp. Med. 183:2109. 10. Malissen, M., A. Gillet, L. Ardouin, G. Bouvier, J. Trucy, P. Ferrier, E. Vivier, 39. Jensen, W. A., C. M. Pleiman, P. Beaufils, A.-M. K. Wegener, B. Malissen, and and B. Malissen. 1995. Altered T cell development in mice with a targeted mu- J. C. Cambier. 1997. Qualitatively distinct signaling through T cell antigen re- tation of the CD3-⑀ gene. EMBO J. 14:4641. ceptor subunits. Eur. J. Immunol. 27:707. 11. Berger, M. A., V. Dave´, M. R. Rhodes, G. C. Bosma, M. J. Bosma, D. J. Kappes, 40. Songyang, Z., S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, and D. L. Wiest. 1997. Subunit composition of pre-T cell receptor complexes F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, et al. 1993. SH2 domains expressed by primary thymocytes: CD3␦ is physically associated but not func- recognize specific phosphopeptide sequences. Cell 72:767. tionally required. J. Exp. Med. 186:1461. 41. Songyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X. R. Bustelo, 12. Van Oers, N. S. C., H. Von Boehmer, and A. Weiss. 1995. The pre-T cell receptor M. Barbacid, H. Sabe, H. Hanafusa, T. Yi, et al. 1994. Specific motifs recognized ␨ (TCR) complex is functionally coupled to the TCR- subunit. J. Exp. Med. 182: by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. 1585. Mol. Cell. Biol. 14:2777.

13. Malissen, M., A. Gillet, B. Rocha, J. Trucy, E. Vivier, C. Boyer, F. Ko¨ntgen, 42. Osman, N., H. Turner, S. Lucas, K. Reif, and D. A. Cantrell. 1996. The protein Downloaded from N. Brun, G. Mazza, E. Spanopoulou, D. Guy-Grand, and B. Malissen. 1993. T interactions of the immunoglobulin receptor family tyrosine-based activation mo- cell development in mice lacking the CD3-␨␩ gene. EMBO J. 12:4347. tifs present in the T cell receptor ␨ subunits and the CD3 ␥, ␦, and ⑀ chains. Eur. 14. Ohno, H., T. Aoe, S. Taki, D. Kitamura, Y. Ishida, K. Rajewsky, and T. Saito. J. Immunol. 26:1063. 1993. Developmental and functional impairment of T cells in mice lacking CD3␨ chains. EMBO J. 12:4357. 43. Exley, M., L. Varticovski, M. Peter, J. Sancho, and C. Terhorst. 1994. Associ- ation of phosphatidylinositol 3-kinase with a specific sequence of the T cell 15. Liu, C.-P., R. Ueda, J. She, J. Sancho, B. Wang, G. Weddell, J. Loring, receptor ␨-chain is dependent on T cell activation. J. Biol. Chem. 269:15140. 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 44. Isakov, N., R. L. Wange, W. H. Burgess, J. D. Watts, R. Aebersold, and http://www.jimmunol.org/ novel T cell population in the intestine. EMBO J. 12:4863. L. E. Samelson. 1995. ZAP-70 binding specificity to T cell receptor tyrosine- 16. Haks, M. C., P. Krimpenfort, J. Borst, and A. M. Kruisbeek. 1998. The CD3␥ based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct ty- chain is essential for development of both the TCR␣␤ and TCR␥␦ lineages. rosine-based activation motifs with varying affinity. J. Exp. Med. 181:375. EMBO J. 17:1871. 45. Johnson, S. A., C. M. Pleiman, L. Pao, J. Schneringer, K. Hippen, and 17. Shores, E. W., M. Ono, T. Kawabe, C. L. Sommers, T. Tran, K. Lui, M. C. Udey, J. C. Cambier. 1995. Phosphorylated immunoreceptor signaling motifs (ITAMs) J. Ravetch, and P. E. Love. 1998. T cell development in mice lacking all T cell exhibit unique abilities to bind and activate Lyn and Syk tyrosine kinases. J. Im- receptor ␨ family members (␨, ␩, and Fc⑀RI␥). J. Exp. Med. 187:1093. munol. 155:4596. 18. DeJarnette, J. B., C. L. Sommers, K. Huang, K. J. Woodside, R. Emmons, 46. Ravichandran, K. S., K. K. Lee, Z. Songyang, L. C. Cantley, P. Burn, and K. Katz, E. W. Shores, and P. E. Love. 1998. Specific requirement for CD3⑀ in S. J. Burakoff. 1993. Interaction of Shc with the ␨-chain of the T cell receptor T cell development. Proc. Natl. Acad. Sci. USA 95:14909. upon T cell activation. Science 262:902. 19. Borst, J., H. Jacobs, and G. Brouns. 1996. Composition and function of T-cell 47. Cambier, J. C., and S. A. Johnson. 1995. Differential binding activity of ARH1/ by guest on September 25, 2021 receptor and B-cell receptor complexes on precursor lymphocytes. Curr. Opin. TAM motifs. Immunol. Lett. 44:77. Immunol. 8:181. 48. De Ao´s, I., M. H. Metzger, M. Exley, C. E. Dahl, S. Misra, D. Zheng, 20. Shores, E. W., and P. E. Love. 1997. TCR ␨ chain in T cell development and L. Varticovsky, C. Terhorst, and J. Sancho. 1997. Tyrosine phosphorylation of selection. Curr. Opin. Immunol. 9:380. ⑀ ϩ ϩ the CD3- subunit of the T cell antigen receptor mediates enhanced association 21. Guidos, C. J. 1996. Positive selection of CD4 and CD8 T cells. Curr. Opin. with phosphatidylinositol 3-kinase in Jurkat T cells. J. Biol. Chem. 272:25310. Immunol. 8:225. 49. Ottinger, E. A., M. C. Botfield, and S. E. Shoelson. 1998. Tandem SH2 domains 22. Kisielow, P., and H. Von Boehmer. 1995. Development and selection of T cells: confer high specificity in tyrosine kinase signaling. J. Biol. Chem. 273:729. facts and puzzles. Adv. Immunol. 58:87. 23. Weiss, A., and D. R. Littman. 1994. Signal transduction by lymphocyte antigen 50. Van Oers, N. S. C., P. E. Love, E. W. Shores, and A. Weiss. 1998. Regulation of ␨ receptors. Cell 76:263. TCR signal transduction in murine thymocytes by multiple TCR -chain signaling motifs. J. Immunol. 160:163. 24. Samelson, L. E., J. B. Harford, and R. D. Klausner. 1985. Identification of the components of the murine T cell antigen receptor complex. Cell 43:223. 51. Haks, M. C., P. Krimpenfort, J. H. N. Van den Brakel, and A. M. Kruisbeek. 25. Dembic, Z., W. Haas, S. Weiss, J. McCubrey, H. Kiefer, H. Von Boehmer, and 1999. Pre-TCR signaling and inactivation of p53 induces crucial cell survival M. Steinmetz. 1986. Transfer of specificity by murine ␣ and ␤ T-cell receptor pathways in pre-T cells. Immunity 11:91. genes. Nature 320:232. 52. Mamalaki, C., J. Elliott, T. Norton, N. Yannoutsos, A. R. Townsend, P. Chandler, 26. Saito, T., A. Weiss, J. Miller, M. A. Norcross, and R. N. Germain. 1987. Specific E. Simpson, and D. Kioussis. 1993. Positive and negative selection in transgenic antigen-Ia activation of transfected human T cells expressing murine Ti ␣␤- mice expressing a T-cell receptor specific for influenza nucleoprotein and endog- human T3 receptor complexes. Nature 325:125. enous superantigen. Dev. Immunol. 3:159. 27. Letourneur, F., and R. D. Klausner. 1992. Activation of T cells by a tyrosine 53. Haanen, J. B. A. G., M. Toebes, T. A. Cordaro, M. C. Wolkers, A. M. Kruisbeek, kinase activation domain in the cytoplasmic tail of CD3⑀. Science 255:79. and T. N. M. Schumacher. 1999. Systemic T cell expansion during localized viral 28. Wegener, A.-M. K., F. Letourneur, A. Hoeveler, T. Brocker, F. Luton, and infection. Eur. J. Immunol. 29:1. B. Malissen. 1992. The T cell receptor/CD3 complex is composed of at least two 54. Leo, O., M. Foo, D. H. Sacha, L. E. Samelson, and J. A. Bluestone. 1987. Iden- autonomous transduction modules. Cell 68:83. tification of a monoclonal antibody specific for murine T3 polypeptide. Proc. 29. Tybulewicz, V. L. J. 1998. Analysis of antigen receptor signalling using mouse Natl. Acad. Sci. USA 84:1374. gene targeting. Curr. Opin. Cell. Biol. 10:195. 55. Viola, A., and A. Lanzavecchia. 1996. T cell activation determined by T cell 30. Wange, R. L., and L. E. Samelson. 1996. Complex complexes: signaling at the receptor number and tunable thresholds. Science 273:104. TCR. Immunity 5:197. 56. Letourneur, F., and R. D. Klausner. 1992. A novel di-leucine motif and a ty- 31. De la Hera, A., U. Mu¨ller, C. Olsson, S. Isaaz, and A. Tunnacliffe. 1991. Structure ⑀ rosine-based motif independently mediate lysosomal targeting and endocytosis of of the T cell antigen receptor (TCR): two CD3 subunits in a functional TCR/ CD3 chains. Cell 69:1143. CD3 complex. J. Exp. Med. 173:7. 57. Dietrich, J., X. Hou, A.-M. K. Wegener, and C. Geisler. 1994. CD3␥ contains a 32. Punt, J. A., J. L. Roberts, K. P. Kearse, and A. Singer. 1994. Stoichiometry of the T cell antigen receptor (TCR) complex: each TCR/CD3 complex contains one phosphoserine-dependent di-leucine motif involved in down-regulation of the T TCR ␣, one TCR ␤, and two CD3⑀ chains. J. Exp. Med. 180:587. cell receptor. EMBO J. 13:2156. 33. Rutledge, T., P. Cosson, N. Manolios, J. S. Bonifacino, and R. D. Klausner. 1992. 58. Dietrich, J., X. Hou, A.-M. K. Wegener, L. Østergaard Pedersen, N. Ødum, and Transmembrane helical interactions: ␨-chain dimerization and functional associ- C. Geisler. 1996. Molecular characterization of the di-leucine-based internaliza- ation with the T cell antigen receptor. EMBO J. 11:3245. tion motif of the T cell receptor. J. Biol. Chem. 271:11441. 34. Cambier, J. C. 1995. Antigen and Fc receptor signaling: the awesome power of 59. Lauritsen, J. P. H., M. D. Christensen, J. Dietrich, J. Kastrup, N. Ødum, and the immunoreceptor tyrosine-based activation motif (ITAM). J. Immunol. 155: C. Geisler. 1998. Two distinct pathways exist for down-regulation of the TCR. 3281. J. Immunol. 161:260. 2588 FUNCTION OF THE CD3␥-ITAM IN TCR SIGNALING

60. Salio, M., S. Valitutti, and A. Lanzavecchia. 1997. Agonist-induced T cell re- 66. Ohno, H., J. Stewart, M.-C. Fournier, H. Bosshart, I. Rhee, S. Miyatake, T. Saito, ceptor down-regulation: molecular requirements and dissociation from T cell A. Gallusser, T. Kirchhausen, and J. S. Bonifacino. 1995. Interaction of tyrosine- activation. Eur. J. Immunol. 27:1769. based sorting signals with clathrin-associated proteins. Science 269:1872. 61. Pe´rez-Aciego, P., B. Alarcoń, A. Arnaiz-Villena, C. Terhorst, M. Timoń, 67. Arnaiz-Villena, A., P. Perez-Aciego, C. Ballestin, T. Sotelo, C. Perez-Seoane, O. G. Segurado, and J. R. Regueiro. 1991. Expression and function of a variant J. M. Martin-Villa, and J. R. Regueiro. 1991. Biochemical basis of a novel T T cell receptor complex lacking CD3-␥. J. Exp. Med. 174:319. lymphocyte receptor immunodeficiency by immunohistochemistry: a possible 62. Pacheco-Castro, A., D. Alvarez-Zapata, P. Serrano-Torres, and J. R. Regueiro. CD3␥ abnormality. Lab. Invest. 64:675. 1998. Signaling through a CD3␥-deficient TCR/CD3 complex in immortalized 68. Arnaiz-Villena, A., M. Timon, A. Corell, P. Perez-Aciego, J. M. Martin-Villa, mature CD4ϩ and CD8ϩ T lymphocytes. J. Immunol. 161:3152. and J. R. Regueiro. 1992. Primary immunodeficiency caused by mutations in the 63. Luton, F., M. Buferne, V. Legendre, E. Chauvet, C. Boyer, and A.-M. Schmitt- gene encoding the CD3-␥ subunit of the T-lymphocyte receptor. N. Engl. J. Med. Verhulst. 1997. Role of CD3␥ and CD3␦ cytoplasmic domains in cytolytic T 327:529. lymphocyte functions and TCR/CD3 down-modulation. J. Immunol. 158:4162. 69. Wu, L., R. Scollay, M. Egerton, M. Pearse, G. J. Spangrude, and K. Shortman. 64. D’Oro, U., M. S. Vacchio, A. M. Weissman, and J. D. Ashwell. 1997. Activation 1991. CD4 expressed on earliest T-lineage precursor cells in the adult murine of the Lck tyrosine kinase targets cell surface T cell Ag receptors for lysosomal thymus. Nature 349:71. degradation. Immunity 7:619. 70. Shores, E. W., S. O. Sharrow, I. Uppenkamp, and A. Singer. 1990. TCR-negative 65. Schmid, S. L. 1997. Clathrin-coated vesicle formation and protein sorting: an thymocytes from SCID mice can be induced to enter the CD4/CD8 differentiation integrated process. Annu. Rev. Biochem. 66:511. pathway. Eur. J. Immunol. 20:69. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021