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Importance of the CD3 Ectodomain Terminal -Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly¹

Maki Touma^*,, Zhen-Yu J. Sun, Linda K. Clayton^*,, Wilfred E. Marissen^2,, Ada M. Kruisbeek^3,, Gerhard Wagner and Ellis L. Reinherz^4,*,

^* Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber Cancer Institute and Departments of Medicine and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

	Abstract

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CD3 and CD3 are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal -strands that are linked to individual subunit membrane proximal stalk segments. CD3, CD3, and CD3 stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal -strand, we created a CD3 double mutant CD3_C82S/C85S and a CD3 -strand triple mutant CD3_{Q76S/Y78A/Y79A} for use in retroviral transduction of lymphoid progenitors for comparison with CD3wt. Although both mutant CD3 molecules reduced association with CD3 in CD3 heterodimers, CD3_{Q76S/Y78A/Y79A} abrogated surface TCR expression whereas CD3_C82S/C85S did not. Furthermore, CD3_C82S/C85S rescued thymic development in CD3^–/– fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3_C82S/C85S transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3^–/– thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3wt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3, as evidenced by the loss of reactivity with CD3 N terminus-specific antisera. Single histidine substitution of either CD3 stalk cysteine failed to restore CD3 association and conformation in transient COS-7 cell transfection studies. Thus, CD3_C82 and CD3_C85 residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3_C80 and CD3_C83. The implications of these results for CD3 and TCR structure and signaling function are discussed.

	Introduction

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The TCR is a multimeric complex consisting of eight polypeptides: the Ag-binding clonotypic heterodimer and the invariant CD3 subunit dimers CD3, CD3, and CD3 (1, 2, 3, 4, 5, 6, 7, 8, 9). Specific interaction between an antigenic peptide bound to a MHC molecule and the TCR heterodimer triggers downstream signaling via the ITAM motifs in the cytoplasmic tails of the CD3 subunits (10, 11, 12). In turn, interaction with intracellular adaptors and signaling molecules induces distinct patterns of tyrosine phosphorylation in mature T cells (11, 13, 14, 15, 16). TCR signaling is also critical for thymocyte development, being essential for selection of double-positive (DP)⁵ thymocytes for maturation into single-positive (SP) thymocytes and subsequent peripheral egress (reviewed in Ref. 17). In early thymic development, a surrogate -chain, termed pT, is expressed in double-negative (DN) thymocytes along with the TCR -chain to form the pre-TCR (18, 19). The pre-TCR functions to terminate additional -chain rearrangements and fosters the transition from DN3 to DN4 developmental stages (20, 21, 22). As with the TCR, signal transduction by the pre-TCR is conducted by the noncovalently associated CD3 subunits (5).

Determination of exactly how clonotypic heterodimer recognition of a given pMHC evokes signaling via the associated CD3 subunits is a daunting challenge yet to be resolved. Undoubtedly, this task will require structural elucidation of the TCR complex and its various components. Guided by such structural detail, directed mutational studies in conjunction with functional analyses will reveal the workings of the receptor complex.

In this regard, the solution structure of a heterodimeric murine CD3 complex first revealed a unique side-to-side hydrophobic interface with conjoined sheets between the two Ig-like ectodomains (C2-set folds) (23). Rigidity of the parallel C-terminal G -strands suggested the possibility that a piston-type displacement of CD3 upon TCR ligation might be involved in initiation of T cell signaling. The subsequent solution structure of the heterodimeric murine CD3 complex is also consistent with this view (24). The CD3 subunit conformation of CD3 is virtually identical to that of CD3 in CD3, whereas the CD3 ectodomain adopts a C1-set Ig fold with a narrower GFC front face sheet more parallel to the ABED backface than those sheets in CD3 and . Nonetheless, the dimeric interface between CD3 and CD3 is highly conserved among species and similar in character to that of CD3. Of note, glycosylation sites in CD3 are arranged such that the glycans point away from the membrane and are consistent with a model of TCR assembly, allowing the CD3-chain to be in contact with the TCR -chain. In a similar manner, CD3 and its glycan are on the side of the TCR heterodimer. Recent crystal studies of a human CD3 ectodomain fragment complexed with the Fab of OKT3 and that of human CD3 complexed with a UCHT1 single-chain Ab fragment identify a similar architecture (25, 26).

In this study, we have begun to investigate the functional importance of the CD3 terminal -strand and stalk region with respect to thymic development. To this end, we have created mutants in those segments for use in retroviral transduction of lymphoid progenitors derived from CD3^–/– mice in fetal thymic organ culture (FTOC). The results suggest that these rigidified segments are critical for both pre-TCR- and TCR-linked CD3 assembly and function.

	Materials and Methods

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Mice

C57BL/6 and c^–/–RAG-2^–/– mice were obtained from Taconic Farms. CD3-deficient mice have been described in detail elsewhere (27). Mice were maintained and bred under specific pathogen-free conditions in the animal facility of the Dana-Farber Cancer Institute under a protocol reviewed and approved by the Animal Care and Use Committee. Initial FTOC experiments were performed using mice maintained at the Netherlands Cancer Institute.

cDNA cloning and mutant construction

cDNAs encoding the mouse CD3 and CD3 subunits as well as mutant constructs were generated by PCR using templates obtained from C57BL/6 mice. The PCR products were subcloned into the pCR2.1 plasmid (TA cloning system; Invitrogen Life Technologies) for sequencing and subsequently into the pLZRS-IRES-eGFP vector for retroviral transduction of fetal thymocytes. For COS-7 cell transfections, CD3wt or CD3 mutant constructs, including the FLAG epitope (DYKDDDDK), and a CD3wt construct, including the hemagglutinin (HA) epitope (YPYDVPDYA), were generated by PCR and ligated into the pCDNA1.1 expression vector (Invitrogen Life Technologies).

Preparation of retroviral supernatant

For retroviral transduction, we used the pLZRS-IRES-eGFP vector encoding the enhanced GFP downstream of an internal ribosome entry site (28). The constructs were transfected into a helper-free 293T-based ecotropic packaging cell line, Phoenix-E (29), using a calcium phosphate transfection kit according to the manufacturer’s protocols (Invitrogen Life Technologies). Two days after transfection, selection medium containing 2 µg/ml puromycin was added and cells were grown until 80% confluent. Medium was replaced with puromycin-free medium and cells were incubated for 12 h to wash the puromycin out. Medium was then changed to FTOC medium (Iscove’s medium with 20% FCS, nonessential amino acids, 50 µM 2-ME, 4 mM L-glutamine, penicillin, and streptomycin) and cells were incubated for 12 h at 37°C. Medium including retrovirus was collected, centrifuged, and frozen in cell-free aliquots at –80°C until use (30).

FTOC and retroviral transduction

For retroviral transduction with CD3wt and mutant constructs, fetal thymi were removed from day 14.5 CD3^–/– fetuses (observation of vaginal plug is day 0.5), and thymocytes were placed in a 96-well plate at 100,000 cells/well (volume of 100 µl) in FTOC medium supplemented with 50 ng/ml IL-7 and stem cell factor. One hundred microliters of viral supernatant containing 20 µg/ml Lipofectamine (Invitrogen Life Technologies) was added to each well and the plate was centrifuged at 1800 rpm for 45 min at room temperature, then incubated at 37°C overnight. The next day, cells were collected and 30 µl/well was placed in a Terasaki plate. One freshly isolated day 14.5 c^–/–RAG-2^–/– fetal thymic lobe was placed in each well. The plate was inverted and incubated for 2 days. In some experiments, deoxyguanosine-treated fetal thymic lobes from C57BL/6 mice were used instead of c^–/–RAG-2^–/– fetal thymic lobes. Thymic lobes from C57BL/6 mice were treated with 1.35 mM 2'-deoxyguanosine (Sigma-Aldrich) in Transwell dishes (Costar) for 5 days before use to remove hemopoietic cells, but not epithelium capable of allowing the differentiation of T cell precursors. We confirmed that both methods support T cell development in FTOC and gave similar results. After 2 days of hanging drop culture, lobes were transferred to ATTP 0.8-µm filters (Millipore) on gelfoam (Pfizer). After 7 days, thymocytes were counted and analyzed by FACS. To determine transduction efficiency, 50,000 of the transduced cells were used for the FACS analysis of GFP expression 3 days after the transduction.

FACS analysis of FTOC cells and transfectants

The following Abs were used: rabbit anti-mouse CD3 heterosera, PE- or PE-Cy5- or PE-Cy7-conjugated anti-CD4 (H129.19), PE-Cy5- or allophycocyanin-conjugated anti-CD8 (53-6.7), PE- or biotin-conjugated anti-TCR C (H57-597, referred to as H57), PE- or biotin-conjugated anti-CD3 (145-2C11, referred to as 2C11), PE-conjugated anti-CD25 (PC61), biotin-conjugated anti-CD44 (IM7), biotin-conjugated anti-rabbit Igs (polyclonal), and PE- or allophycocyanin-Cy7-conjugated streptavidin (BD Pharmingen).

Single-cell suspensions of FTOC cells were prepared in FACS buffer (1x PBS containing 2% FCS and 0.05% NaN₃). Cells were pretreated with anti-FcRII/III (clone 2.4G2; 1 µg/ml) to reduce nonspecific staining for 10 min at 4°C. Staining for cell surface Ag expression was performed at saturating Ab concentrations for 20 min at 4°C. Cells were washed once in FACS buffer and incubated with second-step reagent if necessary. A FACScan (BD Biosciences) and CellQuest software were used for analysis of triple-stained samples, and FACSAria and FlowJo software (Tree Star) were used for five-color samples. Dead cells were excluded from the analysis by forward and side scatter gating.

Transfected cells were harvested and assayed by intracellular staining using Fix/Perm solution from BD Pharmingen according to the manufacturer’s recommendations. Anti-HA mAb (Santa Cruz Biotechnology) or anti-CD3 (2C11, 500A2, 17A2; BD Pharmingen) were used to detect the expression of CD3, and anti-FLAG M2 (Sigma-Aldrich) or anti-CD3 heterosera (see below) were used for CD3.

COS-7 cell transfection

COS-7 cells were cultured in DMEM supplemented with L-glutamine, penicillin, streptomycin, and 10% FCS. Cells in 60-mm culture dishes were transfected in 0.2 ml of DMEM containing 12 µl of FuGene 6 transfection reagent (Roche) and 2 µg of expression vector containing CD3wt-HA or FLAG-tagged CD3. The total amount of DNA was kept constant using pCDNA1.1 empty vector DNA. Two days after the transfection, cells were used for FACS analysis and for immunochemistry.

Immunoprecipitation and Western blotting

At 48 h posttransfection, the medium was removed and the plates were washed twice in ice-cold PBS and cells were solubilized in a buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40 (v/v), 1 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 1 mM iodoacetamide. Cell lysates were precleared for 2 h by incubation with 20 µl of a 50% (v/v) slurry of normal mouse purified IgG (Sigma-Aldrich) coupled to cyanogen bromide-activated Sepharose 4B or 20 µl of GammaBind G-Sepharose beads (50% slurry; Amersham Biosciences). After removing the insoluble material, the precleared supernatants were incubated for 4–16 h at 4°C with 20 µl of a 50% (v/v) slurry of anti-HA beads (Santa Cruz Biotechnology) or 20 µl of a 50% (v/v) slurry of 2C11 coupled to cyanogen bromide-activated Sepharose 4B beads at 5 mg/ml. The beads were subsequently washed four times with a buffer containing 150 mM NaCl and 50 mM Tris-HCl (pH 7.4) (TBS), and resuspended in Laemmli sample buffer.

SDS-PAGE was performed on 12% polyacrylamide gels under reducing conditions and the proteins were then transferred onto polyvinylidene difluoride membranes at 100 V for 1 h. The membranes were blocked using a TBS buffer containing 5% (w/v ratio) nonfat milk and 0.05% (v/v) Tween 20 blocking media (TBS-(BM)) at 4°C overnight. The blots were next washed with TBS containing 0.05% Tween 20 (TBST) and incubated with primary Ab diluted in TBS-BM at room temperature for 1 h. The membranes were subsequently washed with TBST, and incubated with 1/2000 diluted HRP-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) in TBS-BM at room temperature for 2 h. Finally, the blots were washed with TBST, and protein bands were visualized with ECL (Amersham Biosciences).

Preparation of anti-CD3 heterosera

Anti-mCD3 rabbit heterosera were raised by injecting rabbits with 100 µg keyhole limpet hemocyanin conjugated with mouse CD3 peptide, to which a C-terminal cysteine was added for ease of coupling (QTNKAKNLVC). Boosted heterosera were tested for specificity by ELISA and Western blotting. The specificity of heterosera for CD3 on the cell surface was tested by flow cytometry using normal T cells at a 1/400 dilution. Pretreatment of the heterosera with 100 ng/ml of the specific peptide used to prepare the antisera completely blocked the binding to T cells from normal mice.

	Results

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Mutations in the CD3 G -strand and stalk region residues

Fig. 1a shows the sequence alignment of CD3 Ig-like domains from mouse, rat, sheep, cow, monkey, and humans with -strand assignments based on the structure of the murine CD3 heterodimer. In addition, the stalk region immediately distal to the G-strand and proximal to the transmembrane (TM) segment is shown. As previously reported (23), a strong correlation was noted between the conserved residues in CD3 subunits and their locale at the dimerization interface and/or involvement in the Ig-like fold. In contrast, many surface-exposed residues showed considerable variability. The G-strand harbors one cluster of conserved residues. As shown in the sequence alignment and depicted graphically in Fig. 1b (center), CD3 Q76, Y78, and Y79 lie toward the carboxyl end of the G-strand, sitting at the interface between CD3 and CD3. A cavity in CD3 accommodates CD3 Y78 and CD3 Y79, which engage in an aromatic ring-aliphatic chain hydrophobic interaction with CD3; CD3 Q76 on the G-strand hydrogen bonds to the carbonyl of CD3 Y74 (23). The side chains from CD3 and CD3 G-strands interlock like zipper teeth to create a stable interface and shield hydrophobic residues from solvent. This interdigitation is shown most clearly in the open book configuration view of the interface in Fig. 1b (left and right). A similar architecture is observed in the CD3 heterodimer interface (24).

View larger version (62K): [in this window] [in a new window]   FIGURE 1. Sequence alignment and structural features of CD3 heterodimers. a, Sequence alignments of CD3 ectodomains with conserved cysteine residues shown in red and other conserved residues highlighted in blue. The mutated residues are denoted by green replacement residues give in single-letter amino acid code above. The secondary structure elements of mouse CD3 and CD3 are shown with yellow ribbons. The N-terminal segment of the G-strand in CD3 is poorly defined in the solution structure and is therefore boxed but not shaded yellow. Alignments with mouse CD3 and CD3 are also provided for reference. b, The CD3 heterodimer molecular complex (center panel) with the five mutated CD3 residues shown in space-filled spheres. The parallel paired G -strands (dark green) are followed by the membrane proximal CxxC motifs, in turn, leading to the TM helices as represented by two cylinders (drawn to scale) that extend into the lipid bilayer region colored in yellow. Open book views of the interface are provided with CD3 rotated –90 degrees clockwise (left panel) and CD3 rotated +90 degrees counterclockwise (right panel). Note that in the right panel, the three mutated residues are colored in red and in the left panel, the G-strand of CD3 containing the same residues is shown as a stick model. The surface of CD3 is colored in pink and that of CD3 is colored green. The N-terminal CD3 epitope region identified by heteroantisera is colored in blue.

Critical role of CD3 interface and stalk residues for thymic development in FTOC

To address the role of CD3 Ig-like domain interface residues and stalk region residues in T cell development, we performed FTOC after transduction into CD3-deficient thymocytes of retroviruses carrying CD3wt or CD3 mutants described above. This retroviral system uses fetal thymocytes from CD3^–/– fetuses and viruses encoding CD3wt or variants plus GFP to mark the transduced cells (28, 31). Thymocytes from day 14.5 CD3^–/– fetuses that are in the DN stage were used for transduction and then repopulation into fetal thymic lobes from c^–/– RAG-2^–/– mice in which there are no hemopoietic cells but normal stromal cell populations capable of supporting thymocyte development. The titers of each of these viral supernatants were comparable as tested by analysis of the percentage of GFP⁺ thymocytes in suspension culture 3 days after the transduction (data not shown).

Thymocytes generated within the reconstituted FTOC after 7 days of culture were prepared and analyzed for GFP expression. The GFP⁺ subpopulation contains the transduced cells, while the GFP^– fraction represents nontransduced thymocytes, serving as an internal control in immunophenotyping experiments. As shown in Fig. 2 (middle), transduced and nontransduced thymocytes were analyzed for CD4 and CD8 expression. Thymic development in the LZRS-eGFP vector-only transduced thymocytes (Fig. 2, upper row) is similar to that reported in CD3-deficient mice (27), with thymocyte development beyond the DN stage being severely impaired. Not surprisingly, there is little difference between GFP^– and GFP⁺ subsets induced with the LZRS-eGFP control vector. In contrast, retroviral transduction of CD3wt resulted in an increased percentage of the DP population in GFP⁺ cells compared with vector control (62% DN, 13% DP in LZRS-eGFP empty vector control, and 21% DN, 43% DP in the CD3wt-containing vector). In contrast, no augmentation of DP cell development was observed following transduction with CD3_{Q76S/Y78A/Y79A}- or CD3_5M-containing vector into the CD3-deficient thymocytes (Fig. 2 and data not shown). Transduction of CD3_C82S/C85S yielded an increased percentage of DP cells compared with vector control (25% DP in CD3_C82S/C85S), although this value is clearly lower than that following transduction with CD3wt.

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Thymocyte development in CD3–/– FTOC is partly restored by retroviral transduction of a CD3C82S/C85S stalk region mutant but not by transduction of the G-strand mutant CD3Q76S/Y78A/Y79A. Day 14.5 fetal thymocytes from CD3–/– fetuses were transduced with LZRS-eGFP empty vector or LZRS-CD3 constructs. Transduced thymocytes were placed in a hanging drop culture with a fetal thymic lobe from c–/–RAG-2–/– mice. Two days later, thymic lobes were placed on filter membranes on gelfoam sponges and incubated for 7 days before FACS analysis. Dead cells were gated out from forward scatter/side scatter profiles and both the GFP+ and GFP– cells were separately analyzed for CD4 and CD8. Histograms on the left show GFP expression in total living cells. Dot plots show CD4/CD8 profiles of GFP– or GFP+ thymocytes, respectively. Top row panels are transduced with LZRS-eGFP, second row panels with LZRS-CD3wt, third row panels with LZRS-CD3Q76S/Y78A/Y79A, and fourth row panels with LZRS-CD3C82S/C85S. The percentages of cells in the DN and DP quadrants are shown. Histograms on the right represent H57 staining of corresponding DP and CD4SP thymocytes. Bold lines show cold block with excess unlabeled mAb on GFP+ cells. Thin lines are GFP– cells and filled histograms are GFP+ cells. Arrows highlight a clear increase in H57 reactivity in transduced populations. Results are representative of five independent transduction experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Generation of DP and SP thymocytes by CD3C82S/C85S transduction. The total number of cells in FTOC (a) and number of GFP+ cells in FTOC (b) relative to vector-only transduced cultures from FACS analysis of five independent transduction experiments are shown. Likewise, the percentages of GFP+ DP cells (c) and GFP+ CD8 SP cells (d) were obtained. Cell numbers of GFP+ DP cells (e) and GFP+ CD8SP cells (f) were calculated by multiplication of total cell number and percentage of GFP+ cells and percentage of DP or CD8 SP cells, respectively. To normalize the data from five independent experiments, the value of the vector control is referred to as 1 and the relative values of others are shown in the graphs. Statistical differences between vector control and others are indicated (*, p < 0.05) and differences between CD3wt and CD3C82S/C85S are indicated (#, p < 0.05).

CD3_C82S/C85S supports pre-TCR surface expression but not normal thymocyte development

To investigate differences among CD3wt, CD3_{Q76S/Y78A/Y79A}, and the CD3_C82S/C85S in their ability to support early thymic development, we have also analyzed the DN subpopulations in the same FTOC. Expression of CD44 and CD25 was used to assess development of the DN1-DN4 subsets. As shown in Fig. 4a, transduction of CD3wt increased CD44^–CD25⁺ DN3 and CD44^–CD25^– DN4 cells compared with GFP^– nontransduced cells. As expected, the CD44/CD25 plot of GFP⁺DN cells in the CD3_{Q76S/Y78A/Y79A} transduction is similar to that of GFP^– DN cells. The subtle difference in the CD44/25 profiles of the GFP^– DN populations in CD3_{Q76S/Y78A/Y79A} transduced compared with CD3wt or CD3_C82S/C85S cultures may be due to CD25⁺ stromal cells whose maturation is affected by mature thymocytes and which, therefore, appear only in the GFP^– DN populations in CD3wt- or CD3_C82S/C85S-transduced cultures. Note that the percentages of CD44^–CD25⁺ and CD44^–CD25^– cells are increased in CD3_C82S/C85S-transduced DN cells compared with those in the CD3_{Q76S/Y78A/Y79A} transduction, but are less than those in CD3wt transduced cells. This was the case in each of three independent experiments performed. Relative to CD3wt, these findings suggest that introduction of CD3_C82S/C85S into CD3-deficient thymocytes does not fully support DN thymocyte development. We note that in neonatal CD3-deficient mice, thymocyte development is arrested at the DN3 stage (27). However, in this FTOC in vitro reconstitution system, development is less advanced than in vivo. Reconstitution of c^–/–RAG-2^–/– thymic lobes with C57BL/6 fetal thymocytes, for example, results in DN CD44/25 profiles similar to those of CD3wt-transduced CD3^–/– thymocytes (data not shown). Thus, the CD44/CD25 profile in CD3wt-transduced DN cells should be regarded as normal for this culture system.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Immature T cell development and pre-TCR expression in CD3–/– FTOC transduced with CD3 retroviral constructs. Double negative thymocytes in retrovirally transduced FTOC were further analyzed for CD44 and CD25 expression (a). The left three panels are analyses of GFP– DN cells and the right panels are GFP+ DN cells from CD3wt (upper), CD3Q76S/Y78A/Y79A (middle), and CD3C82S/C85S (lower) transductions. The percentages of cells in each quadrant are shown. Pre-TCR expression on DN cells was examined using anti-C mAb (H57), anti-CD3 mAb (2C11), and anti-CD3 heterosera (b). Because only small numbers of DN cells can be collected from each FTOC experiment, these experiments were done using cells from independent virus transductions. Results are representative of three independent transduction experiments for each condition. Each of the three vertical columns involves parallel analysis of the indicated retroviral transductions on a single day.

We also developed an anti-CD3 terminal peptide heteroantisera whose specificity was tested on CD3^+/+ vs CD3^–/– T cells by peptide blockade (data not shown). Transduction with CD3wt harboring retrovirus generated thymocytes that were positive for anti-CD3 heterosera in the DN population, while neither CD3_C82S/C85S nor CD3_{Q76S/Y78A/Y79A} transduction produced anti-CD3 heterosera-positive DN thymocytes. Given comparable H57 and 2C11 staining intensities, the reduced anti-CD3 peptide antisera reactivity with CD3_C82S/C85S relative to CD3wt-transduced DN thymocytes suggests that either the structure of the pre-TCR might be altered by the stalk mutation or that the variant otherwise modifies surface molecular interactions or glycosylation of the pre-TCR in cis to occlude the CD3 N terminus. Fig. 1b indicates that the position of the N-terminal CD3 segment is at a significant distance from the stalk region mutations in the CD3 heterodimer structural model.

Mutated CD3 affects recognition of the paired CD3 by anti-CD3 mAb

The limited number of cells that can be harvested from FTOC precludes biochemical analysis. As an alternative system to investigate the effects of the CD3 mutations, we used a transient transfection system. A cDNA encoding a C-terminal FLAG-tagged wild-type or mutated CD3 molecule was transfected into COS-7 cells along with a cDNA encoding a C-terminal HA-tagged CD3wt molecule (Fig. 5a). After 48 h, COS-7 cells were collected and analyzed by intracellular staining or Western blotting for expression of the CD3 variants and CD3 using anti-FLAG and anti-HA Abs, respectively. Comparable levels of CD3wt or the CD3 variants were detected both by intracellular FACS analysis (Fig. 5b) and Western blotting (Fig. 5c, top). The wild-type and mutated CD3 molecules were detected equally well by our anti-CD3 heterosera in Western blotting as well as by anti-FLAG Ab in both FACS and Western blotting analysis (data not shown). Transfection of CD3 alone results in very low levels of CD3 expression, indicating that this protein is not stable in the absence of CD3 (data not shown). In contrast, the expression of CD3 is not affected by coexpression of CD3: nearly equal levels of CD3 are detected in CD3-only transfectants as well as CD3 plus CD3 cotransfectants upon probing with the anti-HA Ab (Fig. 5, b and c, top/left). However, the conformation-dependent anti-CD3 mAb, 2C11, failed to detect CD3 in the transfectants in the absence of CD3 (Fig. 5b). In addition, the conformation-dependent 17A2, 500A2, and YCD3–1 Abs also failed to detect CD3 by FACS analysis in the absence of CD3. By contrast, expression of CD3wt was detected by all of these anti-CD3 mAbs (2C11, 17A2, 500A2, and YCD3–1) when cotransfected with CD3wt, suggesting that the native conformation of CD3 cannot be achieved as a CD3 monomer or a CD3 homodimer, but only as a CD3 heterodimer (Fig. 5b and data not shown). In the cotransfectants expressing CD3 and CD3_{Q76S/Y78A/Y79A}, none of the anti-CD3 mAbs detect CD3 (Fig. 5b). In some experiments, 500A2 is slightly positive in the cotransfectants expressing CD3 and CD3_{Q76S/Y78A/Y79A} (data not shown). Note that when CD3 was cotransfected with CD3_C82S/C85S, reactivities with these mAbs were detected by FACS, but the fluorescence intensities of cells positive for the anti-CD3 mAbs measured as mean fluorescence intensity (MFI) values (upper right corner of each histogram) were lower, by 50% for 2C11 and 17A2 staining and by 30% for 500A2 staining compared with the fluorescent intensity in cotransfectants expressing CD3 and CD3wt (Fig. 5b). These experiments demonstrate that the triple mutations in the CD3 interface region and the CD3_C82S/C85S stalk mutant influence the formation of the CD3 epitope recognized by these anti-CD3 mAbs. It is surprising that although these anti-CD3 Ab epitopes are predicted to be in the head part of the CD3 ectodomain, the mutation of two cysteine residues in CD3 stalk region alters the binding pattern of certain anti-CD3 mAbs. How these cysteine residues in CD3 influence the conformation of CD3 in the cotransfectant is currently unknown.

View larger version (49K): [in this window] [in a new window]   FIGURE 5. Ab recognition of CD3 alone or in association with CD3wt or CD3 mutants in the CD3 heterodimer. a, Schematic outline of CD3wt, CD3wt, and mutant CD3 molecules with the indicated addition of cytoplasmic C-terminal tags used for COS-7 transfection. b, Two days following cotransfection of CD3wt and CD3wt or mutant cDNAs, COS-7 cells were intracellularly stained with anti-FLAG, anti-HA, or anti-CD3 mAbs (2C11, 17A2, 500A2) for FACS analysis. Percentages of positive cells are shown on the bar and MFI of positive cells are shown in the right upper corner of each histogram. c, Total lysates from transfected cells (upper row) were used for immunoprecipitation with anti-HA (middle row) or anti-CD3 mAb (2C11, lower row) followed by Western blotting with anti-FLAG (right panels) or anti-HA (left panels).

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Effect of single- or double-amino acid mutations in CD3 G -strand residues on CD3 heterodimer association. Three single mutants (Q76S, Y78A, Y79A) and two double mutants (Q76S/Y79A, Y78A/Y79A) tagged with FLAG were generated for COS-7 transfection. Two days after the cotransfection of FLAG-tagged CD3 and HA-tagged CD3, intracellular staining and FACS analyses were performed. MFI of cells positive for anti-HA, 2C11, 17A2, and 500A2 are shown.

Altered heterodimer formation between CD3 and mutated CD3

As shown by Western blot analysis using anti-HA and anti-FLAG Abs, in total lysates of COS-7 cell transfectants there is comparable expression of CD3 in all and equivalent CD3 amounts in the three cell cultures transfected with CD3wt, CD3_{Q76S/Y78A/Y79A}, or CD3_C82S/C85S (Fig. 5c, top panels). The physical association between CD3wt and CD3 variants was assessed by immunoprecipitation with anti-HA followed by Western blotting with anti-FLAG Ab. Although comparable levels of CD3wt were obtained after the anti-HA Ab immunoprecipitation and anti-HA Western blotting (Fig. 5c, middle left panel), very different amounts of CD3wt, CD3_{Q76S/Y78A/Y79A}, and CD3_C82S/C85S coimmunoprecipitated with CD3 as detected by anti-FLAG Western blotting (Fig. 5c, middle right panel). The association was lower between CD3 and CD3_{Q76S/Y78A/Y79A} relative to CD3wt subunits (reduced to 44% ± 14, n = 4, according to densitometry scans). In contrast, very little CD3_C82S/C85S coprecipitated with CD3 (reduced to 8.8% ± 6.6, n = 4) despite equivalent cellular levels of this CD3 mutant in total lysates. These data suggest that the association between CD3 and the CD3_C82S/C85S mutant is greatly reduced compared with that of CD3 plus CD3wt or that of CD3 plus CD3_{Q76S/Y78A/Y79A} combinations under these conditions. This current result with CD3_C82S/C85S is somewhat unexpected in view of a prior study where the two comparable cysteine residues in the stalk region of CD3 were substituted by serine (C80S/C83S) and only a modest impairment of the association with CD3wt was reported (23). Nonetheless, the earlier observation was reconfirmed (data not shown) and suggests that the cysteine stalk residue mutations in each CD3 subunit do not have equivalent impact on CD3 heterodimer formation (see Discussion).

Immunoprecipitation with 2C11 followed by Western blotting with anti-HA yielded a strong signal only in the presence of CD3wt. A low level of CD3 is immunoprecipitated in the presence of CD3_C82S/C85S and even slightly less CD3 is immunoprecipitated by 2C11 in the presence of CD3_{Q76S/Y78A/Y79A} as shown in Fig. 5c (left bottom panel). This result is consistent with the cellular FACS analysis showing that 2C11 binds weakly to CD3 in CD3_C82S/C85S cotransfectants compared with CD3wt cotransfectants, but does not bind at all to CD3_{Q76S/Y78A/Y79A} cotransfectants. Because of the diminished association between CD3 and CD3_C82S/C85S and impaired recognition of that heterodimer by 2C11, the amount of CD3_C82S/C85S after the immunoprecipitation with 2C11 might be below detectable levels. Therefore, only CD3wt protein is detected in Western blots by anti-FLAG after 2C11 immunoprecipitation. These experiments suggest that precise pairing between the G-strands of CD3 and CD3 is necessary to maintain the native conformation of CD3 in the CD3 heterodimer. The conserved motif in the CD3 stalk region affects the conformation of CD3, perhaps additionally contributing to the association of the CD3 heterodimer.

CD3_C82H or CD3_C85H mutations do not restore CD3 heterodimer association

The conserved RxCxxCxE motifs in the stalk regions of both CD3 and CD3 chains are immediately distal to their corresponding G -strand and proximal to the TM segment of each subunit. Consequently, the four cysteine stalk residues in the CD3 heterodimer may be close enough to form a metal-coordinated cluster, possibly a zinc binding site similar to the Cys2-Cys2 motifs found in DNA-binding proteins (32, 33). To examine whether metal coordination by the four cysteines might play a role in the CD3 heterodimer association, we generated additional FLAG-tagged CD3 mutants in which the cysteines were individually changed to histidine (C82H and C85H). Replacement of either CD3 cysteine with histidine should preserve zinc binding but remove a sulfur atom that may serve a critical structural function, including disulfide bond formation, for example.

As shown in Fig. 7a, immunoprecipitation of the indicated transfected COS-7 cell lysates with anti-HA followed by Western blotting with anti-FLAG showed very reduced association between CD3 and CD3_C82H or CD3_C85H, although higher than the association observed between CD3 and CD3_C82S/C85S (CD3_C82S/C85S, 4.3%; CD3_C85H, 22%; CD3_C82H, 26%, as measured by densitometry scanning relative to CD3wt). Fig. 7b shows the MFI of intracellular 2C11 staining in these same transfectants. Compared with CD3wt, when CD3_C82S/C85S, CD3_C82H, or CD3_C85H are cotransfected with CD3wt, 2C11 fluorescence is reduced to <50%. Collectively, the results suggest that the four cysteine residues in the stalk regions of CD3 do not coordinate zinc or other divalent cations in a structurally critical manner.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Analysis of single CD3 stalk region mutations involving C82 and C85. a, Analysis of COS-7 transfectants as in Fig. 5c but using additional mutants CD3C82H and CD3C85H. b, MFI of cells positive for anti-HA, 2C11, 17A2, and 500A2 intracellular staining after transfection with the indicated mutants. Intracellular staining was performed as in Fig. 5b.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

To date, there have been no direct investigations of the functional significance of these elements. In the present set of studies, therefore, we use site-directed mutagenesis guided by NMR-based structural information to begin to assess the importance of the paired G-strands and conserved cysteines within the CD3 stalk regions. We have focused efforts on CD3 for two reasons: 1) the importance of CD3 in both TCR and pre-TCR function (27, 34) and 2) in an effort to complement earlier biochemical investigation of CD3 residues in the murine CD3 heterodimer (23). We chose to use an FTOC system with CD3^–/– lymphoid progenitors retrovirally transduced with CD3wt or CD3 variants and then assessed thymocyte development in the lymphopenic c^–/– RAG-2^–/– fetal thymic stromal environment.

Earlier studies of CD3^–/– mice documented that both TCR and TCR lineages failed to develop in these animals, with the number of cells in the thymus reduced to <1% of normal mice (27). The developmental arrest was primarily at the DN stage so that few DP or SP thymocytes were detected. The defect in DN thymocytes proved that the CD3 subunit is essential for murine pre-TCR function. Therefore, as expected, vector-only transduced CD3^–/– thymocytes were largely blocked before the DP stage and after DN2 in the current study. CD3wt transduction rescued thymocyte development, leading to the appearance of DP and SP thymocytes with greater levels of TCR, as detected by H57 mAb. In contrast, the triple CD3 G -strand mutant, CD3_{Q76S/Y78A/Y79A}, failed to support any rescue of surface pre-TCR or TCR expression while the CD3 stalk mutant, CD3_C82S/C85S, restored DN development and partially rescued both DP and SP subset differentiation. We interpret these results to suggest that the G -strand mutations lead to poor pre-TCR and TCR assembly and surface expression. In contrast, the mutations of the two CD3 stalk cysteine residues to serines have a significant effect. Pre-TCR function is diminished such that pre-TCR-mediated proliferation is attenuated compared with that of CD3wt-transduced thymocytes, giving rise to fewer GFP⁺ thymocytes in FTOC (Fig. 3). As an additional consequence, DP thymocyte numbers are reduced as are SP thymocyte numbers. These data suggest that both pre-TCR and TCR functions are affected. Although we did not directly evaluate TCR-expressing cells, given that CD3 is the only heterodimer associated with this TCR (35), one would expect this lineage to be altered as well, consistent with findings in CD3^–/– mice noted previously (27, 34).

There is not a direct correlation between CD3 and CD3 subunit association to form stable CD3 heterodimers in COS-7 cells and surface receptor expression on T lymphoid cells as shown by this set of mutations. For example, CD3_{Q76S/Y78A/Y79A} heterodimer formation with CD3 in COS-7 transient transfection and immunoprecipitation analysis is greater than that of CD3_C82S/C85S. Yet, pre-TCR and TCR surface expression in thymic development supported by the former is negligible (or nonexistent), while CD3_C82S/C85S fosters expression of both receptors. This "paradox" may be a consequence of CD3 G -strand mutations perturbing less the isolated CD3 heterodimer formation per se and more the pre-TCR and TCR complex assembly relative to the CD3 stalk region mutations. Alternatively, weakened CD3 heterodimer formation resulting from incorporation of CD3_C82S/C85S may be compensated by the other subunits of the receptor complex. There are two clusters of transmembrane helices in the TCR, namely, the three CD3-CD3-TCR segments and the five CD3-CD3-TCR-CD3-CD3 segments that presumably are centered beneath the G -strand-paired CD3 heterodimers (24, 36). These associations may stabilize the weakened CD3 stalk mutations. Such possibilities are not mutually exclusive.

Our earlier analysis with a CD3 G -strand variant, termed CD3_tm, contained mutations analogous to those in CD3_{Q76S/Y78A/Y79A}. Each were aimed at disrupting the interdigitating interface side chains from their respective subunits (23). The biochemical consequences on CD3 heterodimer subunit pairing are similar. However, the CD3 stalk mutant CD3_C– subunit harboring C80S/C83S mutations, comparable to the cysteine mutations in CD3_C82S/C85S, had a less pronounced effect on CD3 heterodimer formation than the CD3_C82S/C85S. These results collectively indicate that the effect of disruption of free thiols or an intrachain disulfide on heterodimer formation is not equivalent for each subunit in CD3. Notwithstanding, anti-CD3 mAb reactivity with mutant CD3 heterodimers is dramatically diminished by mutation of the pair of cysteines in the stalk of either subunit of CD3.

What are the structural implications of the important role of CD3 C82 and C85 residues as revealed by the current functional mutation analysis? Given that the two cysteines are adjacent to the TM helix (Fig. 1b) and in view of a recent study showing that a CxxC motif is found at the N termini of helices stabilizing helical structures, this juxtaposition is noteworthy (37). Assuming an intrachain disulfide is formed in each stalk region, one possibility is that the CD3_TM helix is stabilized and perhaps even extended as an elongated helix above the plane of the cell membrane. Alternatively, this CxxC motif may support a tight turn (38). In either case, the disposition of the CD3 ectodomain relative to the cell membrane may be affected, attenuating signaling and altering pre-TCR and TCR quaternary structure if a disulfide bond is removed. That the N-terminal segment of CD3 is no longer accessible to anti-CD3 Ab binding in CD3_C82S/C85S (Fig. 4b) is consistent with this view, although the exclusion of free CD3 heterodimers from surface expression as an alternative explanation must be considered for the CD3_C82S/C85S observation on DN thymocytes.

In contrast, it is also possible that free sulfhydryls are important for stalk function, since as yet no unequivocal evidence for an intrachain disulfide bond has been provided. Whether physiologic modification of the redox state of the CD3 heterodimer is regulated during development or T cell activation is a matter of speculation at this time. Given that TCR cross-linking on murine and human T lymphocytes generates hydrogen peroxide and superoxide ions (39, 40) and that oxidative stress from macrophages alters the native CD3 association with the TCR (41), it is possible that reduction vs oxidation of CD3 stalk cysteines is critical for TCR quaternary structure, subunit composition, and functional responsiveness. The CD3_C82S/C85S fails to optimize T cell signaling in development, although overcoming some of the developmental blockade in CD3^–/– FTOC. This mutant has neither free sulfhydryls nor oxidized cysteines, making it unclear as to whether the inability to achieve functional reconstitution to the level of that with CD3wt is due to the missing SH groups or a putative intrachain disulfide constraint. Dynamic conversion between oxidized and reduced forms may be important for TCR triggering and down-regulation under physiologic circumstances. That CD3_C82H or CD3_C85H mutations fail to restore CD3 heterodimer formation (Fig. 7) argues strongly against a tetracysteine metal coordination site in the membrane proximal segment of CD3 dimers. We assume that these structural considerations are applicable to CD3 and CD3 subunits as well.

Since this work was completed, two studies have appeared investigating the impact of single-stalk region mutants in human CD3. The first involved hCD3-deficient patient T cells as recipients for retroviral transduction while the second exploited an in vitro transcription/translation system (42, 43). Unlike in the mouse system, the introduction of hCD3_C82S or hCD3_C85S failed to support association with CD3 or T cell surface expression (42). These data are consistent with earlier studies showing the striking effect of cysteine mutations of hCD3 stalk residues on both hCD3 and hCD3 in heterodimer formation (44). Although in the in vitro system, CD3, CD3, or CD3