Distinctive CD3 Heterodimeric Ectodomain Topologies Maximize Antigen-Triggered Activation of βα T Cell Receptors

This information is current as Sun Taek Kim, Maki Touma, Koh Takeuchi, Zhen-Yu J. of September 27, 2021. Sun, Vibhuti P. Dave, Dietmar J. Kappes, Gerhard Wagner and Ellis L. Reinherz J Immunol 2010; 185:2951-2959; Prepublished online 26 July 2010;

doi: 10.4049/jimmunol.1000732 Downloaded from http://www.jimmunol.org/content/185/5/2951

Supplementary http://www.jimmunol.org/content/suppl/2010/07/27/jimmunol.100073 Material 2.DC1 http://www.jimmunol.org/ References This article cites 49 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/185/5/2951.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Distinctive CD3 Heterodimeric Ectodomain Topologies Maximize Antigen-Triggered Activation of ab T Cell Receptors

Sun Taek Kim,*,† Maki Touma,*,† Koh Takeuchi,‡ Zhen-Yu J. Sun,‡ Vibhuti P. Dave,x Dietmar J. Kappes,{ Gerhard Wagner,‡ and Ellis L. Reinherz*,†

The ab TCR has recently been suggested to function as an anisotropic mechanosensor during immune surveillance, converting mechanical energy into a biochemical signal upon specific peptide/MHC ligation of the ab clonotype. The heterodimeric CD3«g and CD3«d subunits, each composed of two Ig-like ectodomains, form unique side-to-side hydrophobic interfaces involving their paired G-strands, rigid connectors to their respective transmembrane segments. Those dimers are laterally disposed relative to

the ab heterodimer within the TCR complex. In this paper, using structure-guided mutational analysis, we investigate the Downloaded from functional consequences of a striking asymmetry in CD3g and CD3d G-strand geometries impacting ectodomain shape. The uniquely kinked conformation of the CD3g G-strand is crucial for maximizing Ag-triggered TCR activation and surface TCR assembly/expression, offering a geometry to accommodate juxtaposition of CD3g and TCR b ectodomains and foster quaternary change that cannot be replaced by the isologous CD3d subunit’s extracellular region. TCRb and CD3 subunit sequence analyses among Gnathostomata species show that the Cb FG loop and CD3g subunit coevolved, consistent with this notion. Further- 2/2 more, restoration of T cell activation and development in CD3g mouse T lineage cells by interspecies replacement can be ratio- http://www.jimmunol.org/ nalized from structural insights on the topology of chimeric mouse/human CD3«d dimers. Most importantly, our findings imply that CD3g and CD3d evolved from a common precursor to optimize peptide/MHC-triggered ab TCR activation. The Journal of Immunology, 2010, 185: 2951–2959.

cell receptors mediate Ag-specific recognition of peptides These results imply a loose association of individual dimeric extra- bound to MHC (pMHC) molecules via their Fab-like het- cellular TCR segments in the resting state. erodimeric clonotypes. The TCR complex consists of ab, How pMHC ligation of the ab heterodimer on the T cell surface

T by guest on September 27, 2021 CD3εg, CD3εd, and CD3zz dimers in a 1:1:1:1 stoichiometry. evokes intracellular signaling via the adjacent CD3 components Whereas the function of the ab clonotype is to bind pMHC, the has been the subject of intense investigation. Previous models associated CD3 subunits mediate signaling through their cytoplas- suggest that TCRs transmit activation signals upon specific pMHC mic (Cyt) tails (1–6). Assembly of the TCR complex is dependent on ligation via clustering, conformational changes, or combining both the transmembrane segment of the individual subunits and their (1, 10–14). Recently we proposed that a common TCR quaternary unique disposition of charged residues in the hydrophobic mem- change rather than conformational alterations of ab clonotypes by brane environment, as detailed elsewhere (7). The ectodomain of pMHCs facilitates signal initiation (15). Given the vast array of ab and the Ig-like CD3εg and CD3εd heterodimers make a limited TCRs and their pMHC ligands, this appears to represent a practi- number of contacts with one another such that, in solution, no inter- cal solution to the T cell immune recognition problem. Clues re- actions are definable by nuclear magnetic resonance (NMR) (8, 9). garding this mechanism were revealed from structural detailing of TCR complex components and by mAb binding studies and anal- ysis of their effects on T cell function. For example, solution struc- *Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber ε ε Cancer Institute, and †Department of Medicine and ‡Department of Biological Chem- tures of CD3 g and CD3 d heterodimers reveal a unique side-to- istry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; side hydrophobic interface with conjoined b-sheets involving xLymphocyte Development Laboratory, Clinical Research Institute of Montreal, { the G-strands of the two Ig-like ectodomains of each pair (8, 9). Montreal, Quebec, Canada; and Blood Cell Development and Cancer Program, Fox Chase Cancer Center, Philadelphia, PA 19111 The squat and rigid CD3 connecting segments contrast sharply Received for publication March 4, 2010. Accepted for publication June 18, 2010. with the long and flexible a and b connecting peptide linking res- pective constant domains to the transmembrane segments. These This work was supported by National Institutes of Health Grants AI19807 (to E.L.R.), CA74620 (to D.J.K.), AI37581, GM47467, and EB002026 (to G.W.), and Canadian opposing features suggested that the ab heterodimer may move Institutes of Health Research Grant 81145 (to V.P.D.). relative to the CD3εg and CD3εd dimers upon pMHC ligation. By Address correspondence and reprint requests to Dr. Ellis L. Reinherz at the current impeding such movement with H57, an Ab that bridges Cb and address: 77 Avenue Louis Pasteur, HIM 419, Boston, MA 02115. E-mail address: CD3εg (15, 16), pMHC-triggered activation is blocked, consistent [email protected] with this view. The online version of this article contains supplemental material. Agonist anti-CD3 mAbs like 2C11 and 500A2 footprint to the Abbreviations used in this paper: 17A2-A647, 17A2-Alexa Fluor 647; 2C11-F, 2C11- ε FITC; Cyt, cytoplasmic; DN, double negative; FTOC, fetal thymic organ culture; membrane distal CD3 lobe that they approach diagonally, adja- hCD3, human CD3; LN, lymph node; mCD3, murine CD3; MFI, mean fluorescence cent to the lever-like Cb FG loop that facilitates pMHC-triggered intensity; mut, mutant; NMR, nuclear magnetic resonance; pMHC, peptide bound to activation (17, 18). This is the most sensitive TCR triggering di- MHC; tg, transgenic; TM, transmembrane; wt, wild-type. rection (15). In contrast, a nonagonist mAb, 17A2, binds to the Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 cleft between CD3ε and CD3g in a perpendicular mode. 17A2 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000732 2952 CD3g AND CD3d G-STRAND GEOMETRIES becomes stimulatory only subsequent to application of an external Retroviral transduction and peptide stimulation ∼ tangential (but not normal) force of 50 pN introduced via optical For retroviral transduction, we used the pLZRS-IRES-eGFP vector encod- tweezers. Specific pMHC, but not irrelevant pMHC, activates a ing the enhanced GFP downstream of an internal ribosome entry site (31). T cell upon application of a similar force. During immune sur- The virus supernatant was prepared as described previously (17, 32, 33). veillance, when specific TCR-pMHC ligation occurs, an extracel- For retroviral transduction with CD3gwt and mutant constructs, total LN 2/2 lular mechanical torque may be applied to the TCR prior to “stop cells from N15TCRtgCD3g mice were stimulated with 4 mg/ml con- canavalin A for 2 d, and T cells were purified by removing I-Ab– movement” of the T cell. In this process, the pMHC on the APC positive cells using anti–I-Ab and magnetic beads. N15TCRtgCD3g2/2 functions as a gaff or hook to pull on the ligated TCR on the op- T cells were placed in a 24-well plate at 106 cells/well (volume of posing T cell. Collectively, these findings suggest that the TCR is 300 ml) in complete RPMI 1640 medium, and 300 ml viral supernatant an anisotropic (i.e., directional) mechanosensor. containing 20 mg/ml Lipofectamin (Life Technologies-BRL, Carlsbad, CA) was added to each well and the plate centrifuged at 2000 rpm for We reasoned that detailed analysis of the distinct topology of 1 h at room temperature. Transduced T cells were cultured for 3 d with CD3 heterodimers would provide further insight into the basis of 50 m/ml human rIL-2 until used for the assay. To assess the TCR signaling signal transduction involving the ectodomain components of the of transduced N15TCRtgCD3g2/2 T cells, cells were washed and stimu- TCR mechanosensor. In addition, the rationale for differences in lated with VSV8 peptide-loaded irradiated splenocytes from C57BL/6 mice CD3 subunit requirements for various T lineage populations and or with plate-coated 2C11 (5 mg/ ml) for 4 h in media containing GolgiPlug reagent (BD Pharmingen). Then, cells were stained for surface CD8 and for developmental stages might emerge (19–28). In this study we have intracellular IFN-g, using Fix/Perm solution (BD Pharmingen). focused on differential G-strand geometry in CD3g and CD3d ecto- domain fragments observed in human and other mammalian spe- Fetal thymic organ culture with retroviral transduction cies. These features make the topology of CD3εg and of CD3εd For retroviral transduction with CD3gwt and mutant constructs, fetal thymi Downloaded from 2 2 distinct from one another and, in turn, mandate a defined quaternary were removed from day 14.5 CD3g / fetuses (observation of vaginal organization for the TCR. The findings suggest that despite the plug is day 0.5), and thymocytes at 100,000 cells/well (volume of 100 ml) in fetal thymic organ culture (FTOC) medium supplemented with 50 loose confederacy of dimeric ectodomains constituting the TCR ng/ml IL-7 and stem cell factor were placed in a 96-well plate. Next, 100 complex, dynamic complementarity of structural elements maxi- ml viral supernatant containing 20 mg/ml Lipofectamin (Life Technolo- mizes sensitivity in the relay of quaternary change essential for gies-BRL) was added to each well and the plate centrifuged at 2000 rpm

signal transduction. for 1 h at room temperature, then incubated at 37˚C overnight. The next http://www.jimmunol.org/ day, cells were collected and 30 ml/well placed in a Terasaki plate. One fetal thymic lobe was placed in each well. The plate was inverted and incubated for 2 d. Thymic lobes from C57BL/6 were treated with 1.35 Materials and Methods mM 29-deoxyguanosine (Sigma-Aldrich, St. Louis, MO) in transwell inserts Abs and flow cytometric analysis (Costar, Cambridge, MA) for 5 d before use to remove hematopoietic cells, but not epithelial tissue capable of allowing the differentiation of T cell The following fluorochrome-labeled mAbs were used for surface precursors. After 2 d of hanging drop culture, lobes were transferred to analysis by flow cytometry: FITC–anti-CD3 (2C11), Alexa Fluor 647–anti- ATTP 0.8 mM filters (Millipore, Billerica, MA) on Gelfoam (Pharmacia CD3 (17A2), Pacific Blue-CD4 (H129.19), Pacific Orange anti-CD8a & Upjohn Company, Kalamazoo, MI). After 7 d, thymocytes were counted (53-6.7), PE–anti-Vb5 (MR9.4), and FITC-conjugated anti-TCR Cb and analyzed by FACS. To determine transduction efficiency, 50,000 of the

(H57) (BD Pharmingen, San Diego, CA). For flow cytometry, single-cell transduced cells were used for the FACS analysis of GFP expression. by guest on September 27, 2021 suspensions of thymocytes or lymph node (LN) cells were prepared at 5 3 6 10 cells/ml in PBS containing 2% FCS and 0.05% NaN3. Those cells were triple- or five-color stained with the mAbs at saturating concentrations according to standard procedures. A FACScan or FACSAria (BD Bioscien- Results ces, San Jose, CA) was used for flow cytometric measurements. Data Structural comparison between CD3«g and CD3«d analysis was performed using FlowJo software (Tree Star, Ashland, OR) heterodimeric ectodomains after dead cells were excluded from the analysis by forward and side ε scatter gating. Solution structures of the murine CD3 (mCD3) g heterodimer and a chimeric CD3εd heterodimer (mCD3ε and sheep CD3d) reveal Mice that both CD3ε domains adopt a virtually identical conformation with a root-mean-square-deviation ,1.45 A˚ (8, 9). Each hetero- C57BL/6 mice were purchased from Taconic Farms (Germantown, NY). CD3d2/2 mice and CD3g2/2 mice have been described in detail else- dimer is composed of two Ig-like ectodomains with a hydrophobic where (22, 24). N15TCRtgCD3g2/2 mice were generated by crossing interface brought together by hydrogen bond-paired terminal G- N15TCRtgRAG-22/2 (29) and CD3g2/2 mice. Construction of CD3d strands forming conjoined b-sheets. Unlike CD3ε, the CD3g and mutant mice will be described in detail elsewhere (V.P. Dave and D.J. CD3d ectodomains within these heterodimers are more divergent Kappes, unpublished observations). Mice were maintained and bred from one another. In particular, structural comparison between under specific pathogen-free conditions in the animal facility of the Dana-Farber Cancer Institute under a protocol reviewed and approved by CD3g and CD3d ectodomains shows that there is differential G- the Animal Care and Use Committee. Mutant CD3d cDNAs A–D (Fig. 2) strand geometry resulting in a pronounced cleft between the two were generated by PCR, confirmed by sequencing, and inserted into the CD3 ectodomains in CD3εg that is partially occluded in CD3εd human CD2 minigene cassette (30). Those transgenic (tg) lines were cre- (Fig. 1A). The difference in G-strand disposition likely results from ated by the Fox Chase Cancer Center Transgenic Facility (Philadelphia, PA) according to established protocols. two factors. First, fewer hydrogen bonds are formed between the two G-strands in CD3εg relative to those in CD3εd, at least in part Mutagenesis of murine CD3g owing to an amino acid sequence of CD3g that does not support the optimal packing between CD3ε and CD3g at the N terminus (or top) The cDNA encoding murine CD3g was subcloned into the pUC18 for of the G–G interaction surface. Second, relative to CD3g, the CD3d mutation and sequencing. To generate the CD3g6M mutant in which the six amino acids in the G-strand of CD3g (ETSNPL) were replaced with BC loop is five residues shorter, containing only four amino acids those of CD3d (KVVSSV), we used the QuickChange Site-Directed Mu- (Fig. 1B). The side view of the heterodimers in Fig. 1A (right panel) tagenesis System (Stratagene, La Jolla, CA). CD3gmdecto or CD3ghdecto highlights the difference in CD3g versus CD3d FG and BC loops. mutant was constructed by replacing the whole ectodomain denoted in The longer BC loop and the presence of the C9-strand force the entire Fig. 1 by recombinant PCR methods. CD3g wild-type (wt) or CD3g mu- tant constructs all contained a C-terminal FLAG epitope (DYKDDDDK) GFCC’ face of CD3g, along with the FG loop, to bow away from for quantitation of protein expression. All generated constructs were con- the heterodimeric interface, whereas the shorter BC loop in CD3εd firmed by DNA sequencing. must cut across the two b-sheets (ABED and FGCC9 faces), thereby The Journal of Immunology 2953 Downloaded from http://www.jimmunol.org/ FIGURE 1. Structural comparison between mCD3εg and mCD3εd heterodimers. A, Ribbon diagram showing heterodimeric CD3εg and CD3εd structures superimposed on CD3ε. NMR structures of CD3εg ( code: 1JBJ) and CD3εd (Protein Data Bank code: 1XMW) were used for structural comparison. We compared the lowest energy structure from the ensemble of NMR structures. CD3εg and CD3εd are shown in pink and blue, respectively. Structural differences are highlighted in G-strands, FG loops, and BC loops, as denoted. Another view representing a rotation about the vertical pseudo 2-fold axis (right panel) highlights differences in G-strand geometry between CD3g and CD3d. The key residues (70–75) for unique G- strand geometry in both CD3g and CD3d are shown as stick models. For clarity, the peptide linkers are not shown in the figure. B, Amino acid sequence alignments of CD3g and CD3d from mouse, human, and sheep. The residues conserved among all CD3g and CD3d orthologs are shown in red background. The residues conserved among CD3g or CD3d sequences are shown in green or yellow background, respectively. The b-strands depicted above the sequences are taken from mCD3g and sheep CD3d NMR structures. For each mutA–D shown in Fig. 2, the denoted amino acid sequence of the mCD3d by guest on September 27, 2021 fragment replaced with the corresponding mCD3g fragment is delineated. Mutation sites for the G-strand and BC loop in CD3g are denoted by red and purple brackets, respectively. The corresponding G-strand region in CD3d used for replacement is denoted by blue brackets. preventing the FG loop from bending. The crystal structures of 2060), and this binding is inhibited only by ∼50% upon preincuba- the human CD3 (hCD3)εg and hCD3εd heterodimers (Supplemen- tion with unlabeled 17A2 (see also schematic in Fig. 2A, right tal Fig. 1) identified a similar difference in G-strand geometry, as panel). Similar patterns of reactivity and partial blockade of observed in the mouse orthologs (34, 35). Note that the top of the 2C11-F staining by unlabeled 17A2 preincubation are seen with hCD3εd FG loop (Gly52–Lys57) is missing in the existing crystal introduction of mutA, mutB, and mutC CD3d transgenes. However, structure coordinates, however, most likely owing to the inherent mutD, involving substitution of the G-strand and a portion of the FG flexibility in this region of the human ortholog. loop from CD3g into CD3d, results in T cells whose 2C11 binding is virtually completely blocked by 17A2 (Fig. 2A, mutD; shaded curve). The completeness of this 17A2-mediated inhibition of Differential G-strand geometries of CD3g and CD3d are 2C11-F binding is comparable to that observed in the CD3d2/2 recognized by the 17A2 anti-mCD3«g mAb T cells, indicating that 17A2 is able to bind to this mutant CD3εd Previous NMR binding experiments involving chemical shift and heterodimer (Fig. 2A, right panel). Further competitive binding cross-saturation analyses (15) showed that 17A2 contacts both experiments shown in Fig. 2B indicate that 17A2 inhibits 2C11 CD3ε and CD3g domains, providing a structural explanation for binding completely on ab T cells from mutD mice, whereas in preferential interaction of 17A2 with CD3εg on the T cell surface. contrast, 17A2 inhibits 2C11 binding by only ∼50% on ab T cells To investigate regions of CD3g critical for native 17A2 binding, we from wt mice. Because murine gd T cells lack CD3εd, instead 2/2 used tg mice generated on the CD3d background in which one expressing two CD3εg heterodimers per TCR complex (20), equiv- of four segments of mCD3d was replaced with a corresponding alent 2C11-F blocking activity by 17A2 on gd T cells in both wt and segment from mCD3g (mutant [mut] A–D), as shown in Fig. 1B, mutD mice was expected and is observed (Fig. 2B). and introduced into the mouse germline via transgenesis. The flow Collectively, these findings show that the CD3g G-strand seg- cytometric analysis in Fig. 2A reveals that both CD4 and CD8 ment is critical for 17A2 binding and that this segment can be 2/2 T cells from CD3d mice express low levels of residual 2C11- introduced into CD3d with no loss of TCR expression or function FITC (2C11-F) reactivity (mean fluorescence intensity [MFI] = 127 (Fig. 2). Such is not the case when residues in the CD3d G-strand and 119, respectively; red curves), consistent with low-level CD3εg region are introduced into CD3g (vide infra). surface expression in the absence of CD3εd (24). This 2C11-F staining is completely blocked by unlabeled 17A2 (MFI = 10–14; The CD3g G-strand geometry is critical for TCR signaling shaded blue curves). In contrast, the introduction of wtCD3d on the On the basis of the structural comparison and amino acid sequence CD3d2/2 background restores 2C11-F reactivity (MFI = 1362– alignment in Fig. 1, we generated a number of CD3g mutants, in- 2954 CD3g AND CD3d G-STRAND GEOMETRIES

FIGURE 2. Introduction of the CD3g G-strand into CD3d creates an mCD3εg chimera reactive with 17A2. A, Binding of 17A2 and 2C11 to ab T cells of wt, CD3d2/2, and CD3d mutant mice. For each CD3d mutA–D, a denoted amino acid segment of mCD3d was replaced by the corresponding mCD3g fragment (Fig. 1B). Peripheral lymphocytes from mutA, mutB, mutC, mutD, wtCD3d, and CD3d2/2 mice were examined by flow cytometry. 2C11-F staining was determined with or without prior unlabeled 17A2 blockade (100 mg/ml). The red line represents 2C11-F staining without blockade, whereas the blue line Downloaded from represents 2C11-F staining after 17A2 blockade. Numbers denote MFI. The curves shaded blue show similarity in 17A2 blockade, reducing 2C11-F to essentially background staining levels. 2C11-F binding on ab T cells withor without prior unlabeled17A2 blockade is depicted schematically. B, Binding competition between 17A2 and 2C11 on the T cell surface from wtCD3d and mutD mice. LNs were isolated and stained with mAbs against T cell surface markers for flow cytometric analysis. Each dot represents 2C11-F and 17A2-Alexa Fluor 647 binding after unlabeled 17A2 blocking (blue), 2C11 blocking (green), or no preincubation (red). ab TCR+ (CD4SP+)orgd TCR+ populations were gated for analysis. Data are representative of three experiments. 17A2-A647, 17A2-Alexa Fluor 647. http://www.jimmunol.org/ cluding CD3g6M, a variant in which the six amino acids in the G- in which the entire CD3g ectodomain is replaced with that of strand of CD3g (ETSNPL) at positions 70–75 were replaced with CD3d (CD3gmdecto) also fails to reconstitute TCR surface expres- those of CD3d (KVVSSV) (Fig. 1A). This swap is predicted to sion. Cellular protein expression for each of the mutant CD3g force the preceding segment and loop of the CD3g6M mutant to transductants was comparable to that of wtCD3g, as assessed adopt a more vertical trajectory than that of the CD3gwt counter- by intracellular staining using the FLAG-tag appended to the part. To test the functional significance of the CD3g ectodomain Cyt tail of each construct and anti-FLAG Ab (Supplemental Fig. modification, we exploited N15TCR tg mice specific for the ve- 3). However, we cannot exclude that in the case of CD3g6M- sicular stomatitis virus nuclear protein octapeptide (VSV8) bound DBC, incorrect protein folding may contribute to absent surface to Kb (29, 36) and bred onto a CD3g2/2 genetic background (22). expression. by guest on September 27, 2021 Retroviral transduction of peripheral T cells from these animals Next, following retroviral transduction, N15 T cells were stim- with pLZRS-IRES-eGFP–containing CD3gwt or CD3g mutant ulated with VSV8-pulsed Kb-expressing APCs. As shown by the cDNAs encoding schematically depicted in Fig. 3A representative experiment in Fig. 3C, no significant IFN-g pro- gives rise to GFP-expressing transductants (GFP+). As shown in duction was obtained from GFP+ empty vector control transduc- Fig. 3B, CD3gwt retrovirally transduced N15tg CD3g2/2 T cells tants, whereas N15 T cells transduced with wtCD3g cDNA showed restores TCR surface expression, as monitored by flow cytometric specific responsiveness to VSV8 peptide in a concentration- analysis with each of four anti-TCR mAbs (MR 9.4, anti-Vb5; dependent manner. N15 T cells expressing the CD3g6Μ cDNA in- 2C11, anti-CD3ε; H57, anti–TCR-Cb; and 17A2, anti-CD3εg). duced less IFN-g production compared with that of CD3gwt, Note that this increase occurs only in the GFP+ population (Fig. emphasizing the importance of the unique CD3g G-strand geometry 3B). Although the CD3g6M mutant restores much of the surface for Ag-triggered activation. As expected, VSV8 stimulation failed TCR expression (∼60% of wtCD3g), as assessed by MR9.4, to induce IFN-g production in N15 T cells transduced with 2C11, and H57 reactivity, 17A2 mAb binding to TCRs incor- CD3g6M-DBC or CD3gmdecto. porating the CD3g6M mutant is lost (Fig. 3B). These results sup- Fig. 3D represents a composite histogram of IFN-g production port the notion that CD3g6M adopts an extended G-strand more from multiple experiments using VSV8 stimulation of retrovirally like mCD3d, thereby preventing 17A2 mAb from binding in the transduced N15tgCD3g2/2 T cells. Both CD3gwt and CD3g6M cleft between CD3ε and CD3g. Because the direct interaction transductants (GFP+) produce IFN-g upon VSV stimulation, but residues mapped by proton-induced relaxation are exclusively in with cytokine production from CD3gwt greater than that from the CD3ε side (15), the trivial explanation that mutants abrogate CD3g6M transductants. Note the lack of detectable activation by 17A2 binding per se is excluded. Subsequently, we shortened the pMHC in GFP2 cells in either set of cultures. In contrast, with anti- BC loop (LTDKT) in CD3g by deleting the LTDKT sequence to CD3ε mAb triggering, IFN-g production is observed in transduced create a second mutant, CD3g6M-DBC. This foreshortened BC (GFP+) and nontransduced (GFP2) populations, implying that 2C11 loop likely reinforces the upward trajectory of the FG loop in the stimulation via CD3εd induces detectable IFN-g production. CD3g6M-DBC variant. Strikingly, the surface TCR expression Nonetheless, transduction of CD3gwt significantly augments that rescue observed with CD3g6M was not detected by any of the 2C11-stimulated response, compared with the vector control. Sim- four Abs tested after CD3g6M-DBC transduction. Collectively, ilar 2C11-stimulated IFN-g production in CD3gwt and CD3g6M these data suggest that this kinked CD3g G-strand geometry is transductants suggests that differential responsiveness to pMHC is important for TCR surface expression, permitting CD3εg to oc- not a consequence of TCR expression level. Together, the findings cupy a position beneath the Cb FG loop of the b subunit and imply that TCRb-CD3εg juxtaposition involving the kinked consistent with our previous TCR quaternary model (15) (Supple- CD3g G-strand affords an optimized geometry for effective Ag- mental Fig. 2). In further support of this hypothesis, a third mutant triggered T cell activation. The Journal of Immunology 2955 Downloaded from http://www.jimmunol.org/

FIGURE 3. The unique G-strand geometry of CD3g is important for TCR complex surface expression and signaling. A, Schematic drawing of mCD3gwt and CD3g ectodomain mutant constructs. All constructs contain transmembrane and Cyt domains of mCD3g, as well as a C-terminal FLAG-tag (d). The mutation positions in CD3g ectodomain are denoted in the accompanying ribbon diagrams (CD3ε, cyan; CD3g, green; and CD3d, yellow). B, Surface TCR expression on N15TCRtgCD3g2/2 T cells after CD3gwt and CD3g ectodomain mutant transduction. N15 T cells were stained with each indicated mAb and analyzed by flow cytometry after retroviral transduction. Data are representative of three independent experiments. C, pMHC/TCR-mediated signaling in N15TCRtgCD3g2/2 T cell transfectants. Transduced N15 T cells were stimulated with VSV8 peptide-loaded APCs for 4 h. The number of cytokine- by guest on September 27, 2021 producing cells was measured using flow cytometry after gating each GFP+CD8+ population in one representative experiment. D, Statistical analysis of VSV8- or 2C11-stimulated N15TCRtgCD3g2/2 transduced populations. Transduced T cells were stimulated by VSV8 peptide (1 mM) or plate-bound 2C11 Ab (5 mg/ml). GFP2CD8+ and GFP+CD8+ populations were gated for IFN-g+ analysis. The number of cytokine-producing cells was normalized by setting the IFN-g amount from the CD3gwt transduced N15TCRtgCD3g2/2 T cells as 1 and no stimulation as 0. Two-sided exact Wilcoxon -sum test was used to compare. Values are average 6 SD (n = 3). pp # 0.05; ppp # 0.01. TM, transmembrane.

Coevolution of the elongated Cb FG loop and duplication of structure for pMHC-triggered ab TCR activation. TCRab hetero- CD3g plus CD3d from a single precursor dimer assembly studies with various CD3 complexes, using a phy- Whereas the striking elongation of the Cb FG loop among mam- logenetic approach with TCR complex subunits from mammalian malian species is well conserved, sequence comparison with non- and nonmammalian vertebrates, support this conclusion (41). mammalian vertebrate species (chicken, fish, and frog) reveals that the lengthy Cb FG loop is not observed in the latter (Fig. 4A). This Restoration of T cell signaling and development by rigid Cb FG loop in mouse has been shown to facilitate both selec- heterologous replacement of the mCD3g ectodomain tion of thymocytes and activation of T cells (17, 18). The absence of Despite the above results and conclusions, it has been reported that distinct CD3g and CD3d subunits in the above-mentioned nonmam- hCD3d can partially restore thymic development in the absence of malian species and expression of a single precursor CD3g/d gene mCD3g in CD3g2/2 mice (42, 43), suggesting that the hCD3d have been shown based on genomic and biochemical analyses, as ectodomain may be accommodated in juxtaposition to the murine well as theoretical predictions dating the required CD3 duplication b subunit. We therefore assessed whether a chimeric CD3g pro- event (37–39). Although recently it has been demonstrated that the tein containing the hCD3d ectodomain fused with transmembrane jawless vertebrates (agnathans) have alternative adaptive immune and Cyt tail segments of mCD3g (CD3ghdecto) could restore TCR systems with variable lymphocyte receptors, all jawed vertebrates surface expression and T cell activation in N15tgCD3g2/2 T cells. (gnathostomates) possess fully developed adaptive immune systems In contrast to the inability of CD3gmdecto chimera to restore TCR with TCR and Ig genes (40). Both the elongated Cb FG loop and the expression and function, CD3ghdecto was competent to do both. distinct CD3g and CD3d genes are unique in the mammalian species As shown in Fig. 5A, CD3ghdecto transduction induced surface among Gnathostamata (Fig. 4B). These analyses support the notion TCR expression comparable to that of CD3gwt, although, not that TCRCb and CD3g have been evolutionarily coupled for TCR surprisingly, with loss of 17A2 mAb reactivity. In vitro stimulation assembly and signaling in the mammalian species. Furthermore, experiments likewise showed that CD3ghdecto possesses a signal- these findings imply that the distinct topology of CD3 heterodimers ing capacity equivalent to that of CD3gwt to induce IFN-g coevolved with TCRC domains to optimize the quaternary TCR production upon pMHC- (Fig. 5B) or 2C11- mediated stimulation 2956 CD3g AND CD3d G-STRAND GEOMETRIES Downloaded from

FIGURE 4. Coevolution of the elongated TCRCb FG loop and CD3g plus CD3d genes from a single precursor in Gnathostamata, a group of vertebrates with adaptive immunity involving a recombinatorial system (VDJ). A, Sequence comparison of the TCRCb FG loop regions among various species. The position of the F- and G-strands is defined based on the N15 TCR structure (16). The bracket region defines the elongated FG loop in mammalian species with well-conserved key residues (L219, W225, and P232) forming the hydrophobic core. The two cysteines contributing to the intrachain and an interchain disulfide bond are indicated by d and s, respectively. The conserved lysine residue in the transmembrane region of Cb is also highlighted in yellow. B,

Schematic representation of evolutionary relationships between TCRb and CD3 gene products. Possession of adaptive immunity with recombinatorial- http://www.jimmunol.org/ based immune receptors is known for agnathans and gnathostomates. Gnathostomates possess a developed adaptive supporting various VDJ recombinations for Ig and TCR rearrangement, whereas Agnathans do not but contain variable lymphocyte receptors. Distinctions among features of mammals and those of birds, amphibians, reptiles, and bony fish are described and shown schematically.

2/2 (Fig. 5C). Thus, CD3ghdecto serves as a suitable structural surro- employed. Thymocyte progenitors from CD3g fetal mice were gate for both VSV8- and 2C11-triggered activation. transduced with CD3gwt or CD3ghdecto cDNA containing retro- To next assess whether this CD3ghdecto chimera can provide viruses of comparable viral titer. Subsequently, thymocytes gen- differentiation signals during development, an FTOC system was erated within the reconstituted FTOC after 7 d of culture were by guest on September 27, 2021

FIGURE 5. Restoration of T cell signaling by replacing the ectodomain of mCD3g with that of hCD3d, but not mCD3d. A, Surface TCR expression on N15TCRtgCD3g2/2 T cells resulting from chimeric CD3g transduction. N15 T cells were stained with the indicated anti-TCR mAb for flow cytometric analysis after retroviral transduction with results of one of three representative experiments shown. B, pMHC/TCR-mediated signaling in N15 T cells. Transduced N15TCRtgCD3g2/2 T cells were stimulated with VSV8 peptide-loaded APCs for 4 h. The number of cytokine-producing cells was measured using flow cytometry after gating GFP+CD8+ populations. C,Anti-CD3ε mAb stimulation. Transduced N15 T cells were stimulated by plate-bound 2C11 Ab (5 mg/ml) for 3 h. GFP+ + + 2/2 CD8 populations weregatedfor IFN-g analysis. D, Thymocytedevelopment in CD3g FTOC byretroviraltransduction of CD3ghdecto or CD3gwt cDNAs.The transduced (GFP+) and nontransduced (GFP2) cells were separately analyzed. The percentages of cells in the CD4SP,CD8SP,and DP quadrants are shown inthe top panels. The percentagesof cells intheDN3 (CD442CD25+) and DN4 (CD442CD252) quadrants are shown inthe middle panels after DN population(CD42CD82) gating. Histograms on the bottom panels show H57 staining for DN3 and DN4 subpopulations combined (CD42CD82CD442). The Journal of Immunology 2957 prepared and analyzed for GFP expression and surface phenotype. As shown by CD4 and CD8 surface expression patterns in Fig. 5D (top), thymic development in the CD3ghdecto transduced thymo- cytes was equivalent or better than that of CD3gwt transduced thymocytes. In contrast to nontransduced cells (GFP2) within the same FTOC cultures, the number of double positive thymocytes was increased 2- to 5-fold. Fig. 5D (middle) shows that double negative (DN) cell development from DN1 (CD44+CD252) plus DN2 (CD44+CD25+) stages to DN3 (CD442CD25+) and DN4 (CD442CD252) stages was also accelerated by the transduction of CD3ghdecto. Thus, for example, in CD3ghdecto transductants, DN3 = 9.8% and DN4 = 42.2%, compared with 1.1 and 3.9%, respectively, for the GFP2 control. Furthermore, surface TCRb expression on DN3/4 stage cells was only slightly higher than on nontransduced cells (Fig. 5D, bottom), suggesting that pre-TCR expression is supported by CD3ghdecto, as it is with wtCD3g without nonphysiological overexpression of the pre-TCR. These results imply that the CD3ghdecto protein can replace that of the wtCD3g component in the pre-TCR during T cell development. Downloaded from Our findings with the chimeric CD3ghdecto protein are in ag- reement with results generated through transgenesis in CD3g2/2 mice using the hCD3d (human only) construct (42, 43). Why, then, might these mouse-human interspecies CD3εd heterodimers function to foster signaling (Fig. 6)? In this regard, it is notewor- 89 FIGURE 6. The abTCR quaternary complex and T cell signaling. A, ab thy that the aromatic ring of Phe in human CD3ε makes hydro- http://www.jimmunol.org/ ε phobic interactions with both the g-methyl of Thr62 and the TCR quaternary structure model. This model includes ab (red/blue), CD3 g ε hydrophobic b-methylene position of the Gln64 side chain of (cyan/green), and CD3 d (cyan/yellow) heterodimers. The high-mannose N- glycans [-(N-acetylglucosamine) -(mannose) ] shown in brown are taken human CD3d. This interaction creates a tightly packed interface 2 7 ε from models solved by Wyss et al. (49). The protruding Cb FG loop is between hCD3 and hCD3d molecules at the top of the G–G- denoted by an asterisk and overrides the top of the CD3εg ectodomain, as strand interface (Supplemental Fig. 4). This preferential interac- shown in the figure and Supplemental Fig. 2. B, Schematic representations of ε 89 tion would not be formed in the mCD3 ghdecto dimer, as the Phe TCR assembly and signaling involving the TCRb–CD3εg module. The in hCD3ε is a Thr in the corresponding position mCD3ε. As a re- models show that an intact TCRb–CD3εg module geometry is required sult, the top of the G-strand in CD3ghdecto and hCD3d proteins, for both TCR surface expression and signaling. Steric clash prevents the when dimerized with mCD3ε, can bend and slot into the area Cb FG loop juxtaposition with mCD3εd, but not mCD3εg or the by guest on September 27, 2021 normally accommodating mCD3εg in the TCRb–CD3εg junction, interspecies mCD3ε/hCD3d heterodimer. For simplicity, CD3z and the without steric clash. Furthermore, hCD3d contains four alternating Cyt tail regions of the depicted CD3 subunits are not visualized. charged residues at the end of the FG loop (K57/D58/K59/E60). This charge cluster would destabilize a straight-up b-strand con- loop coevolved with appearance of the CD3g gene from a CD3g/d formation with two positively charged lysines on one side and two precursor are strong support for this notion. By contrast, the more negatively charged residues on the back side. Thus, the FG loop of vertical CD3d FG loop trajectory and greater number of N-linked hCD3d is most likely unstructured, consistent with its crystal glycan adducts in CD3εd heterodimers assume a more extended structure (Supplemental Fig. 1) (35), and could be readily bent geometry that cannot fit into the homologous TCRb–CD3εg inter- toward the back ABED face when heterodimerized with mCD3ε action site (15). The CD3εd disposition on the TCRa “side” of the in a fashion analogous to that of mCD3g. Differential glycosyla- complex occupies intervening space between the coreceptor (CD4 tion of mCD3d versus hCD3d with one fewer N-linked adduct in or CD8) and the ab heterodimer (44, 45). This bulky CD3εd com- the human ortholog may also contribute to the functionality of this ponent may also be entropically advantageous to help precon- replacement. figure the coreceptor as a TCR, pMHC, and coreceptor ternary com- plex forms. The crystal structure of a gd TCR heterodimer reveals a Cg–Cd Discussion domain symmetry, in contradistinction to the Ca–Cb domain Analysis of CD3 sequence divergence indicates that CD3g, CD3d, asymmetry observed in ab TCRs (16, 46, 47). The gd TCR het- and CD3ε genes arose from a common ancestor in a two-step pro- erodimer also differs by lacking an elongated C b FG loop equiv- cess of gene duplication (38). Mammals have three CD3 genes (g, alent. gd TCR lineage commitment is associated with more robust d, and ε), whereas nonmammalians (birds, fish, and amphibians) signaling relative to that of the ab TCR, with greater TCR copy have only two: a CD3gd precursor and a CD3ε gene. Protein number and/or ligand density likely affecting gd T lineage sig- sequence comparison indicates that each CD3g and CD3d subunit naling strength (22, 48). In contrast, ab TCR pMHC ligands are evolved with highly homologous heterodimeric interfaces and present at low levels, mandating additional TCR modifications to membrane proximal segments for efficient and specific signaling compensate for weak signals promoting ab T cell fate and func- transfer when paired with CD3ε. The compact orientation of the tion. During immune surveillance, continued cell movement fol- CD3g FG loop and single horizontally attached glycan in the mouse lowing ligation of the TCR ab clonotype by specific pMHC fosters is a feature of the CD3εg heterodimer that affords lateral support of quaternary change; the Cb FG loop interacts with CD3εg on one the C region domains for ab as well as gd TCRs. In addition, side of the TCR and the Ca domain with the bulky CD3εd heter- CD3εg appears to have adapted to optimally interact with the Cb odimer on the other. It is the tangential rather than normal (i.e., FG loop. Our findings that the elongation of the structured Cb FG perpendicular to the membrane) directional force that triggers TCR 2958 CD3g AND CD3d G-STRAND GEOMETRIES activation post-pMHC ligation, as shown by optical tweezer experi- 5. Irving, B. A., and A. Weiss. 1991. The cytoplasmic domain of the T cell receptor ments (15). zeta chain is sufficient to couple to receptor-associated signal transduction path- ways. Cell 64: 891–901. Our current results emphasize how the ab TCR quaternary 6. Reth, M. 1989. Antigen receptor tail clue. Nature 338: 383–384. structure is optimal for surface expression and signaling. Fig. 6A 7. Call, M. E., and K. W. Wucherpfennig. 2007. Common themes in the assembly and architecture of activating immune receptors. Nat. Rev. Immunol. 7: 841–850. gives a side view of the surface-exposed TCR complex based upon 8. Sun, Z. Y., S. T. Kim, I. C. Kim, A. Fahmy, E. L. Reinherz, and G. Wagner. 2004. existing structural information of individual components and mo- Solution structure of the CD3epsilondelta ectodomain and comparison with lecular modeling (8, 15, 49). The substantial N-linked glycosy- CD3epsilongamma as a basis for modeling T cell receptor topology and sig- naling. Proc. Natl. Acad. Sci. USA 101: 16867–16872. lation of TCR subunits is indicated by the brown space-filling 9. Sun, Z. J., K. S. Kim, G. Wagner, and E. L. Reinherz. 2001. Mechanisms con- molecular representations. On the TCRb “side,” the Cb FG loop tributing to T cell receptor signaling and assembly revealed by the solution (17, 18) and the compact orientation of the CD3g FG loop (Fig. 1, structure of an ectodomain fragment of the CD3 epsilon gamma heterodimer. Cell 105: 913–923. Supplemental Fig. 2) are key features contributing to the asymmetry 10. Beddoe, T., Z. Chen, C. S. Clements, L. K. Ely, S. R. Bushell, J. P. Vivian, optimizing TCR signaling. Lateral movement of the TCRab heter- L. Kjer-Nielsen, S. S. Pang, M. A. Dunstone, Y. C. Liu, et al. 2009. Antigen odimer can apply a torque on CD3εg via the Cb FG loop appendage. ligation triggers a conformational change within the constant domain of the alphabeta T cell receptor. Immunity 30: 777–788. Fig. 6B schematically demonstrates that the extended CD3d subunit 11. Ma, Z., K. A. Sharp, P. A. Janmey, and T. H. Finkel. 2008. Surface-anchored ectodomain would sterically clash with the Cb FG loop above, monomeric agonist pMHCs alone trigger TCR with high sensitivity. PLoS Biol. whereas that of CD3g or the chimeric heterodimer does not. On 6: e43. 12. Minguet, S., M. Swamy, B. Alarco´n, I. F. Luescher, and W. W. Schamel. 2007. the TCRa “side,” the bulky glycans and vertical CD3d FG loop Full activation of the T cell receptor requires both clustering and conformational disposition may also likewise relay quaternary change to CD3εd changes at CD3. Immunity 26: 43–54. after tangential force-induced torque. Alternatively, the torque on 13. Alarco´n, B., M. Swamy, H. M. van Santen, and W. W. Schamel. 2006. T-cell Downloaded from ε antigen-receptor stoichiometry: pre-clustering for sensitivity. EMBO Rep. 7: CD3 d could be applied through the highly conserved connecting 490–495. peptide at the base of the TCRCa domain (30). Further studies 14. Choudhuri, K., D. Wiseman, M. H. Brown, K. Gould, and P. A. van der Merwe. aimed at rigidifying or de-rigidifying segments of the TCR complex, 2005. T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand. Nature 436: 578–582. without altering pMHC binding, should show an impact on TCR 15. Kim, S. T., K. Takeuchi, Z. Y. Sun, M. Touma, C. E. Castro, A. Fahmy, signaling, consistent with a mechanosensor mechanism of action. M. J. Lang, G. Wagner, and E. L. Reinherz. 2009. The alphabeta T cell receptor is an anisotropic mechanosensor. J. Biol. Chem. 284: 31028–31037. The ability of the hCD3d ectodomain to pair with mCD3ε and http://www.jimmunol.org/ 16. Wang, J., K. Lim, A. Smolyar, M. Teng, J. Liu, A. G. Tse, J. Liu, R. E. Hussey, foster TCR complex expression signaling, as well as murine thy- Y. Chishti, C. T. Thomson, et al. 1998. Atomic structure of an alphabeta T cell mocyte development, might appear, at first glance, contradictory to receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody. EMBO J. 17: 10–26. our view that CD3g and CD3d ectodomains evolved to occupy 17. Touma, M., H. C. Chang, T. Sasada, M. Handley, L. K. Clayton, and a different side of the TCR complex. However, that is not the case. E. L. Reinherz. 2006. The TCR C beta FG loop regulates alpha beta T cell Isologous subunit ectodomain substitution is not permitted, where- development. J. Immunol. 176: 6812–6823. 18. Sasada, T., M. Touma, H. C. Chang, L. K. Clayton, J. H. Wang, and as the heterologous hCD3d ectodomain can replace that of mCD3g. E. L. Reinherz. 2002. Involvement of the TCR Cbeta FG loop in thymic selection That functional substitution, as noted in Supplemental Fig. 4, is and T cell function. J. Exp. Med. 195: 1419–1431. possible because mCD3ε has a Thr residue in lieu of Phe89 in 19. Hayes, S. M., E. W. Shores, and P. E. Love. 2003. An architectural perspective

ε on signaling by the pre-, alphabeta and gammadelta T cell receptors. Immunol. by guest on September 27, 2021 hCD3 , allowing the top of the G-strand of hCD3d, when paired Rev. 191: 28–37. with mCD3ε, to avoid steric clash with the TCRb subunit. We 20. Hayes, S. M., and P. E. Love. 2002. Distinct structure and signaling potential of emphasize that the geometry of mCD3g and mCD3d G-strand N- the gamma delta TCR complex. Immunity 16: 827–838. 21. Wang, B., N. Wang, M. Salio, A. Sharpe, D. Allen, J. She, and C. Terhorst. 1998. terminal residues (residues 70–75 and residues 58–63) are distinct Essential and partially overlapping role of CD3gamma and CD3delta for de- from each other, as are the corresponding segments in the respec- velopment of alphabeta and gammadelta T lymphocytes. J. Exp. Med. 188: tive human orthologs. 1375–1380. 22. Haks, M. C., P. Krimpenfort, J. Borst, and A. M. Kruisbeek. 1998. The As structural and functional analyses of these and other Ig-like CD3gamma chain is essential for development of both the TCRalphabeta and domains of receptors become more sophisticated, additional sub- TCRgammadelta lineages. EMBO J. 17: 1871–1882. tleties and their biological implications will be revealed. The details 23. DeJarnette, J. B., C. L. Sommers, K. Huang, K. J. Woodside, R. Emmons, K. Katz, E. W. Shores, and P. E. Love. 1998. Specific requirement for CD3ep- as described in this study for CD3 heterodimers demonstrate the silon in T cell development. Proc. Natl. Acad. Sci. USA 95: 14909–14914. important functional consequences of structural evolution. Under- 24. Dave, V. P., Z. Cao, C. Browne, B. Alarcon, G. Fernandez-Miguel, J. Lafaille, standing these differences will help with elucidating the function of A. de la Hera, S. Tonegawa, and D. J. Kappes. 1997. CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage. EMBO J. multisubunit receptors, such as the TCR. 16: 1360–1370. 25. Malissen, M., A. Gillet, L. Ardouin, G. Bouvier, J. Trucy, P. Ferrier, E. Vivier, and B. Malissen. 1995. Altered T cell development in mice with a targeted Acknowledgments mutation of the CD3-epsilon gene. EMBO J. 14: 4641–4653. We thank Haesook Kim for statistical analysis, Maris Handley for flow 26. Malissen, M., A. Gillet, B. Rocha, J. Trucy, E. Vivier, C. Boyer, F. Ko¨ntgen, N. Brun, G. Mazza, E. Spanopoulou, et al. 1993. T cell development in mice cytometry, and Jia-huai Wang for careful review of this manuscript. lacking the CD3-zeta/eta gene. EMBO J. 12: 4347–4355. 27. Love, P. E., E. W. Shores, M. D. Johnson, M. L. Tremblay, E. J. Lee, A. Grinberg, S. P. Huang, A. Singer, and H. Westphal. 1993. T cell development Disclosures in mice that lack the zeta chain of the T cell antigen receptor complex. Science The authors have no financial conflicts of interest. 261: 918–921. 28. Liu, C. P., R. Ueda, J. She, J. Sancho, B. Wang, G. Weddell, J. Loring, C. Kurahara, E. C. Dudley, A. Hayday, et al. 1993. Abnormal T cell development in CD3-zeta-/- mutant mice and identification of a novel T cell population in the References intestine. EMBO J. 12: 4863–4875. 29. Ghendler, Y., M. K. Teng, J. H. Liu, T. Witte, J. Liu, K. S. Kim, P. Kern, 1. Smith-Garvin, J. E., G. A. Koretzky, and M. S. Jordan. 2009. T cell activation. H. C. Chang, J. H. Wang, and E. L. Reinherz. 1998. Differential thymic selection Annu. Rev. Immunol. 27: 591–619. outcomes stimulated by focal structural alteration in peptide/major histocom- 2. Rudolph, M. G., R. L. Stanfield, and I. A. Wilson. 2006. How TCRs bind MHCs, patibility complex ligands. Proc. Natl. Acad. Sci. USA 95: 10061–10066. peptides, and coreceptors. Annu. Rev. Immunol. 24: 419–466. 30. Ba¨ckstro¨m, B. T., U. Mu¨ller, B. Hausmann, and E. Palmer. 1998. Positive se- 3. Romeo, C., M. Amiot, and B. Seed. 1992. Sequence requirements for induction lection through a motif in the alphabeta T cell receptor. Science 281: 835–838. of cytolysis by the T cell antigen/ zeta chain. Cell 68: 889–897. 31. Jaleco, A. C., A. P. Stegmann, M. H. Heemskerk, F. Couwenberg, A. Q. Bakker, 4. Letourneur, F., and R. D. Klausner. 1992. Activation of T cells by a tyrosine K. Weijer, and H. Spits. 1999. Genetic modification of human B-cell develop- kinase activation domain in the cytoplasmic tail of CD3 epsilon. Science 255: ment: B-cell development is inhibited by the dominant negative helix loop helix 79–82. factor Id3. Blood 94: 2637–2646. The Journal of Immunology 2959

32. Touma, M., Z. Y. Sun, L. K. Clayton, W. E. Marissen, A. M. Kruisbeek, 41. Gouaillard, C., A. Huchenq-Champagne, J. Arnaud, C. L. Chen Cl, and G. Wagner, and E. L. Reinherz. 2007. Importance of the CD3gamma ectodomain B. Rubin. 2001. Evolution of T cell receptor (TCR) alpha beta heterodimer terminal beta-strand and membrane proximal stalk in thymic development and assembly with the CD3 complex. Eur. J. Immunol. 31: 3798–3805. receptor assembly. J. Immunol. 178: 3668–3679. 42. Pan, Q., J. F. Brodeur, K. Drbal, and V. P. Dave. 2006. Different role for mouse 33. Kinsella, T. M., and G. P. Nolan. 1996. Episomal vectors rapidly and stably and human CD3delta/epsilon heterodimer in preT cell receptor (preTCR) produce high-titer recombinant retrovirus. Hum. Gene Ther. 7: 1405–1413. function: human CD3delta/epsilon heterodimer restores the defective preTCR 34. Kjer-Nielsen, L., M. A. Dunstone, L. Kostenko, L. K. Ely, T. Beddoe, function in CD3gamma- and CD3gammadelta-deficient mice. Mol. Immunol. 43: 1741–1750. N. A. Mifsud, A. W. Purcell, A. G. Brooks, J. McCluskey, and J. Rossjohn. 43. Ferna´ndez-Malave´, E., N. Wang, M. Pulgar, W. W. Schamel, B. Alarco´n, and 2004. Crystal structure of the human T cell receptor CD3 epsilon gamma C. Terhorst. 2006. Overlapping functions of human CD3delta and mouse heterodimer complexed to the therapeutic mAb OKT3. Proc. Natl. Acad. Sci. CD3gamma in alphabeta T-cell development revealed in a humanized USA 101: 7675–7680. CD3gamma-mouse. Blood 108: 3420–3427. 35. Arnett, K. L., S. C. Harrison, and D. C. Wiley. 2004. Crystal structure of a hu- 44. Wang, J. H., R. Meijers, Y. Xiong, J. H. Liu, T. Sakihama, R. Zhang, man CD3-epsilon/delta dimer in complex with a UCHT1 single-chain antibody A. Joachimiak, and E. L. Reinherz. 2001. Crystal structure of the human CD4 N- fragment. Proc. Natl. Acad. Sci. USA 101: 16268–16273. terminal two-domain fragment complexed to a class II MHC molecule. Proc. 36. Shibata, K., M. Imarai, G. M. van Bleek, S. Joyce, and S. G. Nathenson. 1992. Natl. Acad. Sci. USA 98: 10799–10804. Vesicular stomatitis virus antigenic octapeptide N52-59 is anchored into the 45. Moody, A. M., Y. Xiong, H. C. Chang, and E. L. Reinherz. 2001. The CD8al- groove of the H-2Kb molecule by the side chains of three amino acids and the phabeta co-receptor on double-positive thymocytes binds with differing affinities main-chain atoms of the amino terminus. Proc. Natl. Acad. Sci. USA 89: 3135– to the products of distinct class I MHC loci. Eur. J. Immunol. 31: 2791–2799. 3139. 46. Adams, E. J., Y. H. Chien, and K. C. Garcia. 2005. Structure of a gammadelta Science 37. Go¨bel, T. W., E. L. Meier, and L. Du Pasquier. 2000. Biochemical analysis of the T cell receptor in complex with the nonclassical MHC T22. 308: 227– 231. Xenopus laevis TCR/CD3 complex supports the “stepwise evolution” model. 47. Garboczi, D. N., P. Ghosh, U. Utz, Q. R. Fan, W. E. Biddison, and D. C. Wiley. Eur. J. Immunol. 30: 2775–2781. 1996. Structure of the complex between human T-cell receptor, viral peptide and 38. Go¨bel, T. W., and J. P. Dangy. 2000. Evidence for a stepwise evolution of the HLA-A2. Nature 384: 134–141. CD3 family. J. Immunol. 164: 879–883. 48. Hayes, S. M., L. Li, and P. E. Love. 2005. TCR signal strength influences Downloaded from 39. Krissansen, G. W., M. J. Owen, P. J. Fink, and M. J. Crumpton. 1987. Molecular alphabeta/gammadelta lineage fate. Immunity 22: 583–593. cloning of the cDNA encoding the T3 gamma subunit of the mouse T3/T cell 49. Wyss, D. F., J. S. Choi, J. Li, M. H. Knoppers, K. J. Willis, A. R. Arulanandam, antigen receptor complex. J. Immunol. 138: 3513–3518. A. Smolyar, E. L. Reinherz, and G. Wagner. 1995. Conformation and function of 40. Cooper, M. D., and M. N. Alder. 2006. The evolution of adaptive immune the N-linked glycan in the adhesion domain of human CD2. Science 269: 1273– systems. Cell 124: 815–822. 1278. http://www.jimmunol.org/ by guest on September 27, 2021