One of the CD3epsilon Subunits within a T Cell Receptor Complex Lies in Close Proximity to the Cbeta FG Loop

By Yoseph Ghendler, Alex Smolyar, Hsiu-Ching Chang, and Ellis L. Reinherz

From the Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

    Abstract
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A recent crystal structure of the N15 alpha /beta -T cell receptor (TCR) in complex with an Fab derived from the H57 Cbeta -specific monoclonal antibody (mAb) shows the mAb fragment interacting with the elongated FG loop of the Cbeta domain. This loop creates one side wall of a cavity within the TCR Ti-alpha /beta constant region module (Calpha Cbeta ) while the CD and EF loops of the Calpha domain form another wall. The cavity size is sufficient to accommodate a single nonglycosylated Ig domain such as the CD3epsilon ectodomain. By using specific mAbs to mouse TCR-beta (H57) and CD3epsilon (2C11) subunits, we herein provide evidence that only one of the two CD3epsilon chains within the TCR complex is located in close proximity to the TCR Cbeta FG loop, in support of the above notion. Moreover, analysis of T cells isolated from transgenic mice expressing both human and mouse CD3epsilon genes shows that the heterologous human CD3epsilon component can replace the mouse CD3epsilon at this site. The location of one CD3epsilon subunit within the rigid constant domain module has implications for the mechanism of signal transduction throughout T cell development.

    Introduction
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Each TCR consists of a clonotypic TCR heterodimer (Ti-alpha /beta or Ti-gamma /delta subunits) in complex with the invariant CD3 chains (gamma , delta , epsilon , and zeta ). The disulfide-linked heterodimer represents the peptide-MHC ligand binding unit, thereby determining the ligand specificity of an individual T cell. In contrast, the CD3 polypeptides which are in noncovalent association with a given Ti heterodimer, mediate TCR-base signal transduction (for review see references 1). Although CD3-gamma and -delta are present in only one copy each (6), it appears that two copies of CD3epsilon and zeta  exist per TCR complex (9). The signaling function of the CD3 components involves a conserved motif, termed an immunoreceptor tyrosine-based activation motif (ITAM) present in one to three copies in the cytoplasmic domain of each CD3 subunit (12, 13). The various CD3 subunits exhibit different interactions with intracellular signaling factors and induce distinct patterns of cellular protein tyrosine phosphorylation upon activation (14). How peptide-MHC ligand binding to a Ti-alpha /beta or Ti-gamma /delta heterodimer subsequently initiates signaling via the CD3 molecules is currently unknown.

Aside from their role in signal transduction, the CD3 subunits are also required for cell surface expression of the TCR heterodimers on mature T lymphocytes (20, 21), as well as for pre-T cell receptor function on immature CD4-CD8- double negative (DN)1 thymocytes (22, 23). Thus, without CD3epsilon or -zeta subunit expression there is a marked decrease or absence of TCR molecules on the cell surface as shown by in vitro analysis (20, 21, 24). In addition, in genetically engineered mouse strains in which these CD3 components are deleted by homologous recombination, there is a developmental blockade of thymocytes at the DN stage (25). The CD3delta subunit, in contrast to CD3epsilon and -zeta chains, is required for TCR expression only at a later stage of thymic development. The absence of CD3delta in a knockout mouse specifically blocks the thymic selection processes mediating the transition from double positive (DP) (CD4+CD8+) to single positive (CD4+CD8- or CD4-CD8+) thymocytes (30).

Although the overall stoichiometry of the TCR complex is commonly given as TCR-alpha /beta -CD3gamma /delta /epsilon 2/zeta 2, there is no direct structural evidence to support this subunit composition. Recently, a three-dimensional structure of the N15 vesicular stomatitis virus-specific/H-2Kb-restricted alpha /beta -TCR (31) in complex with an Fab fragment from the H57 anti-mouse Cbeta -specific mAb (32) provided a clue with which to infer new details about the association between the TCR-alpha /beta heterodimer and CD3epsilon (33). We identified a cavity within the TCR-alpha /beta C module formed by the Cbeta FG loop, partially exposed Cbeta domain strands, and conserved glycans from both Calpha and Cbeta domains that can accommodate a single Ig-like domain. Based on size and charge considerations, we suggested that the cavity probably represents the CD3epsilon binding site. To determine whether there is a physical proximity between the Cbeta FG loop and the CD3epsilon chain, we performed a set of competition assays between the H57 and the CD3epsilon -specific 2C11 mAbs (34). The results of these experiments provide evidence that one of the two CD3epsilon subunits in a TCR complex is physically adjacent to the TCR-beta constant region FG loop.

    Materials and Methods
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transgenic Mice.

Transgenic mice expressing the human CD3epsilon gene (transgenic [tg] 600; reference 35) were provided by Dr. Cox Terhorst (Beth Israel Medical Center, Boston, MA) and are further referred to as hCD3epsilon tg. This transgenic mouse strain contains 10-12 copies of the human CD3epsilon transgene in a hemizygous mouse. Since T cell development is blocked in the homozygous mice of this strain, we used hCD3epsilon tg heterozygous mice for these studies. A littermate that does not contain the human CD3epsilon transgene was used as a control (wild-type, WT).

Molecular Modeling of the N15 TCR-H57 Fab Complex.

The molecular modeling was produced using the N15 TCR and H57 Fab complex crystal structure 3D coordinates (PDB code 1NFD; 33). The plot of the protein structure was created using the programs MOLSCRIPT (36) and RASTER3D (37).

Flow Cytometric Analysis.

The following mAbs were used: R-PE-labeled anti-mouse CD3epsilon (500A2; PharMingen, San Diego, CA), PE-labeled anti-mouse CD4 (H129.19; GIBCO BRL, Gaithersburg, MD), and Red613-labeled anti-mouse CD8 (53-6.7; GIBCO BRL). The mAbs hamster anti-mouse TCR-beta (H57-597; reference 32), hamster anti-mouse CD3epsilon (2C11-145; reference 34), and mouse anti-mouse Vbeta 8.1,2,3 (F23.1; reference 38) were purified and labeled with FITC using the FluoReporter® FITC protein-labeling kit (Molecular Probes, Eugene, OR). Generation and purification of 2C11 and H57 Fab fragments were performed by using the ImmunoPure Fab Preparation Kit (Pierce, Rockford, IL). The purity of the Fab fragments was confirmed by SDS-PAGE, which demonstrated no residual intact IgG. Splenocytes from nontransgenic mice (WT) or mice heterozygous for human CD3epsilon (hCD3epsilon tg) were triple stained as follows: cells were incubated with PE-labeled anti-CD4, Red613-labeled anti-CD8, and FITC-H57 or FITC-2C11 for 30 min at 4°C in PBS containing 2% FCS. For mAb competition assays, splenocytes were first incubated with unlabeled H57 or 2C11 mAbs at 10-fold molar excess of the FITC-labeled mAb for 30 min at 4°C, washed with PBS + 2% FCS, and triple stained as explained above. Flow cytometric analysis was performed on a FACScan (Becton Dickinson, San Jose, CA). Samples were gated on live cells based on forward and side scatter parameters. Data (10,000 events per sample) were collected in list mode using FACScan Research software and analyzed using LYSYS II software (Becton Dickinson). The mean fluorescence intensity (mFI) of the FITC-labeled H57 or FITC-labeled 2C11 labeled T cells (CD4+ or CD8+ splenocytes) was measured and the FITC mFI of non-T cell splenocytes (CD4- or CD8-; background) was deducted from this value in each sample.

    Results
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
A TCR Constant Domain Module Cavity as a Putative CD3epsilon Binding Site.

The three-dimensional structure of a complex between the N15 alpha /beta -TCR and the anti-TCR-beta chain mAb H57 Fab fragment resolved crystallographically to 2.8Å has revealed several interesting features about TCR structure relative to antibody structure. As shown in Fig. 1, there is an obvious difference between the ligand binding surface of the V domain modules of the two receptors (Valpha Vbeta versus VHVL), with a relatively flat antigen binding surface in the case of the TCR, versus a concave surface in the case of the Fab. The flatness of the N15 antigen binding surface and the concavity of the H57 Fab surface are complementary to the surface of their respective ligands, Kb and Cbeta . Perhaps the most striking difference between these immunoreceptors is the symmetry of the Fab molecule compared with asymmetry of the TCR molecule. This TCR asymmetry results from the unusual arrangement of the Calpha and Cbeta domains relative to one another. Moreover, as shown in Fig. 1, the H57 Fab binds to the 12-residue Cbeta FG loop conserved among TCR Cbeta domains from multiple species (39). This loop architecture is uniquely rigidified by a hydrophobic minicore and an internal hydrogen bonding network. In addition, we have noted that this loop creates one side wall of a cavity, while the CD and EF loops of the Calpha domain form the other side. The floor of the cave, which measures ~25Å in depth, ~20Å in height, and ~25Å in width, is presumably formed by the plasma membrane on the T cell surface. The murine CD3epsilon subunit, which is predicted to have an Ig fold (40), is nonglycosylated unlike CD3gamma and CD3delta , and could readily fit into a cavity of this size.


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Fig. 1.   Model of the N15 alpha /beta -TCR heterodimer complexed with the H57 anti-TCR Cbeta -specific mAb Fab fragment. Based on x ray coordinates and molecular modeling as described in Materials and Methods, a structure of the complex is shown. The TCR-alpha chain is in green, the TCR-beta chain is in red, the H57 Fab heavy chain is in gold, and H57 Fab light chain is in purple. The Cbeta FG loop, which is the epitope recognized by the mAb H57, is indicated. This loop creates one side wall of a cavity (whose center is marked by the Cbeta label) within the TCR Ti alpha /beta constant region while the CD (*) and EF (**) loops of the Calpha domain form the other side. The cavity size is sufficient to accommodate a single nonglycosylated Ig domain such as the CD3epsilon chain.

Nonreciprocal Cross-blocking of 2C11 and H57 mAbs.

To investigate our hypothesis that a CD3epsilon subunit might occupy this cavity within the C module and hence lie proximal to the Cbeta FG loop, a set of competition assays between the H57 and 2C11 mAbs were performed using direct immunofluorescence analysis by FACS®. 2C11 was previously shown to specifically bind to the mouse CD3epsilon subunit (34). As shown in Fig. 2 A, unlabeled competitor 2C11 or H57 was added before the addition of FITC-2C11, FITC-H57, or FITC-labeled anti-CD4, and log fluorescence was determined by quantitative FACS® analysis. As expected, unlabeled 2C11 blocks the subsequent binding of FITC-2C11 and, likewise, unlabeled H57 blocks the binding of FITC-H57. In contrast, neither unlabeled 2C11 nor H57 alters the binding of the unrelated FITC-H129.19. More importantly, note that under these conditions the anti-CD3epsilon mAb completely blocks the binding of FITC-H57 mAb, whereas H57 only partially reduces the mFI of FITC-2C11 binding to CD3epsilon on mouse splenic T cells. A second anti-CD3epsilon mAb, 500A2, also completely blocked FITC-H57 binding and, as with FITC-2C11, the binding of PE-500A2 was reduced by 50% when T cells were preincubated with unlabeled H57 (data not shown). Moreover, similar results were obtained by using 2C11 Fab or H57 Fab fragments for inhibition analysis. That the findings are not secondary to "general" steric blockade of the TCR by any mAb or Fab fragment binding to the complex is evident from the inability of Vbeta -specific antibodies to block FITC-H57 binding on TCR transgenic T lymphocytes (data not shown).


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Fig. 2.   The anti-CD3epsilon mAb (2C11) blocks the binding of the anti- TCR-beta mAb (H57). (A) Splenocytes (106 per sample) were incubated either with no antibody or with 10 µg/ml unlabeled mAbs 2C11 or H57 for 30 min, then washed and reincubated with 1 µg/ml of the FITC-labeled mAbs 2C11 or H57 or anti-mouse CD4 mAb. After washing, the cells were analyzed on a FACScan as described in Materials and Methods. Each unlabeled mAb blocks the identical FITC-labeled antibody. The unlabeled 2C11 blocks FITC-H57 (center histogram), whereas H57 only partially blocks the binding of FITC-2C11 to CD3epsilon (lower left histogram). Neither 2C11 nor H57 block the FITC-labeled anti-CD4 mAb (right column). (B) The same competition experiment was performed as described above but after the preincubation with the unlabeled competitor mAb the splenocytes were triple-stained with FITC-H57 or FITC-2C11 and both PE-anti-CD4 and Red613-anti-CD8. Histograms show the FITC-H57 or FITC-2C11 staining of T cells (after gating for CD4+ and CD8+ cells) in the presence or absence (Non) of the unlabeled competitor mAbs. The mFI of each curve is indicated in parenthesis. Only CD4+ data is shown.

Only One of the Two CD3epsilon Subunits Is Proximal to the Cbeta FG Loop.

To quantify the mFI reduction in FITC-2C11 resulting from unlabeled H57 preincubation in individual CD4 and CD8 T cell subsets, we performed the analogous competition experiments by three-color analysis, in addition using PE-labeled anti-mouse CD4 and Red613-labeled anti-mouse CD8. We collected data comparable to that shown in Fig. 2 A but on individual splenic CD4+ and CD8+ T lymphocytes. As shown in Fig. 2 B (left) for CD4+ T cells, although unlabeled 2C11 completely blocked the binding of FITC-2C11 to the mouse T cells, the unlabeled H57 mAb reduced the binding of the FITC-2C11 by ~50% (mFI, from 402 to 183). On the other hand, unlabeled 2C11 almost completely blocked the binding of FITC-H57 (mFI, from 398 to 45; Fig. 2 B, right). Identical results were obtained when CD8+ T cells subset were examined. Three conclusions may be drawn from these data. First, one CD3epsilon subunit is close to the H57 mAb binding site on the Cbeta FG loop. Second, given that there are two CD3epsilon components per TCR (10, 11) and the H57 mAb can only block 50% of the FITC-2C11 binding, the second CD3epsilon subunit must exist at a distance from the Cbeta FG loop. Third, this postulated TCR subunit arrangement is found in both CD4+ and CD8+ T cell subpopulations.

The Human CD3epsilon Subunit as well as the Mouse CD3epsilon Subunit Associates with the Calpha Cbeta Module.

To test whether replacement of the murine CD3epsilon chain with the human CD3epsilon subunit might alter the binding affinity of the H57 mAb to the Cbeta FG epitope, we used a well-characterized transgenic mouse strain (tg 600) engineered to express the human CD3epsilon component (reference 35; here referred to as hCD3epsilon tg). Splenocytes from littermates that do not express the human CD3epsilon (WT), as well as a heterozygous hCD3epsilon tg mouse, were stained with directly labeled FITC-2C11 mAb at concentrations ranging from 5 × 10-6-10-10 M. As shown in Fig. 3 A, the hCD3epsilon tg mouse expressed only ~50% of the normal cell surface level of mouse CD3epsilon . However, 2C11 binding affinity to the mouse CD3epsilon subunit is not altered by the presence of the human CD3epsilon component (Kd ~10-8 M). The capacity of the unlabeled 2C11 mAb to block binding of FITC-H57 to heterozygous hCD3epsilon tg T cells was also examined. As shown in Fig. 3 B, the same amount of unlabeled 2C11 that blocks FITC-H57 binding to the WT mouse T cells only partially blocks the FITC-H57 binding to hCD3epsilon tg T cells. This suggests that the human CD3epsilon subunit can replace the mouse CD3epsilon subunit in agreement with the finding of others (10, 41), and that it can occupy the cavity within the mouse TCR Cbeta Calpha module. Fig. 3 B also demonstrates that the incubation of the mouse splenocytes with the 2C11 mAb does not block the binding of H57 to the TCR-beta chain by downmodulation of the TCR complex from the T cell surface (42, 43). Thus, unlabeled 2C11 preincubation does not change the binding of the anti Vbeta 8 mAb, FITC-F23.1, to T cells.


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Fig. 3.   Human CD3epsilon can replace the mouse CD3epsilon within the rigid TCR-alpha /beta constant domain module. (A) Splenocytes from a nontransgenic mouse (WT) or a mouse heterozygous for human CD3epsilon (hCD3epsilon tg) were triple stained with the indicated molar concentration of FITC-2C11 as well as with PE-anti-CD4 and Red613-anti-CD8. The mFI of the FITC-2C11 labeled T cells was calculated as explained in Materials and Methods. Note that although the hCD3epsilon tg T cells express only half of the WT CD3epsilon , the 2C11 binding affinity to the mouse CD3epsilon is not altered by the presence of the human CD3epsilon subunit. 2C11 preincubation completely blocks FITC-2C11 binding in WT mice. (B) Splenocytes from WT or hCD3epsilon tg mice were preincubated with no antibody or with the unlabeled 2C11, washed and then reincubated with the FITC-labeled mAb H57 or the anti-Vbeta 8 (F23.1) as explained in the legend of Fig. 1. Although the total copy number of the mouse CD3epsilon subunits in hCD3epsilon tg T cells is half of that in WT animals, 2C11 mAb only partially blocks the FITC-H57 binding in the hCD3epsilon tg mouse (lower center histogram). Note that preincubation of the T cells with 2C11 had no effect on the TCR expression level since the anti-Vbeta 8 mAb binds to a subset of T cells expressing Vbeta 8 as well with or without 2C11 competition (right).

The Binding of the H57 mAb to the Cbeta FG Loop Is Unaffected by Human Versus Mouse Origin of the CD3epsilon Component.

In contrast to the results comparing mouse CD3epsilon in WT and hCD3epsilon tg mice where the level of 2C11 expression is ~50% on the latter (Fig. 3 A), H57 reactivity is equivalent in both (Fig. 4).This implies that the affinity of H57 mAb to the Cbeta FG loop is not altered by the presence of the human CD3epsilon component. Had human CD3epsilon altered the affinity of H57, then the curves of mFI at different molar concentrations of FITC-H57 would have shown a difference in the two mouse strains. Unlabeled 2C11 competitor mAb shifts the binding curve by ~2 logs, at concentrations of FITC-H57 below 10-6 M. At the highest concentration of FITC-H57 (10-6 M), the labeled antibody binds similarly in the presence or absence of unlabeled competitor 2C11 mAb. These finding are consistent with the notion that the epitopes defined by H57 and 2C11 mAbs are distinct but nevertheless partially overlapping.


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Fig. 4.   Human CD3epsilon does not alter the affinity of H57 for its epitope. Splenocytes from WT or hCD3epsilon tg mice were triple stained with the indicated molar concentration of FITC-H57 and with the labeled anti-CD4 and anti-CD8 with or without preincubation with an unlabeled competitor mAb (designated after the named mouse strain). The mFI of FITC-H57 binding to the T cells (CD4+ or CD8+ cells) was calculated for each sample as explained in Materials and Methods. Results are given for CD4+ T cells and identical data was observed for CD8+ T cells.

    Discussion
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The crystal structure of the complex between the N15 TCR and an Fab fragment of the anti-Cbeta mAb H57 led us to the hypothesis that one CD3epsilon may physically associate with the H57 mAb epitope on Cbeta (33). In this paper we demonstrate that one of the two CD3epsilon subunits of the TCR lies adjacent to the Cbeta FG loop.

The overall shape of the TCR C domain module is remarkably asymmetric (Fig. 1). The Cbeta domain bends more acutely towards the Vbeta domain compared with the angle formed between the Calpha and Valpha domains. Cbeta also has an unusually long and well-structured FG loop (the H57 mAb epitope) projecting down from the Vbeta -Cbeta interface in all TCRs studied to date (33, 44, 45). Although half of the Cbeta domain's ABED sheet is surface exposed, it does not make contact with the Calpha domain. As described in detail by Wang et al. (33), this asymmetry creates a cave-like structure or cavity below the beta  chain sufficient in size to accommodate a single Ig domain. The partially exposed ABED beta  sheet of the Cbeta domain forms an extensive ceiling. The CD and EF loops of the Calpha domain, along with the glycans attached to Calpha N185, Calpha N121, and Cbeta N186 form one side wall, and the FG loop of Cbeta and the glycan emanating from Cbeta N236 form the other side wall of the cavity. The glycans project outward and do not obstruct the cavity. The floor of the cave is presumably formed by the plasma membrane on the T cell surface (33). It is noteworthy that the accessible surface at the alpha /beta -TCR cavity contains multiple basic residues.

The murine CD3epsilon subunit consists of 87 residues in the extracellular segment, and has Ig-like characteristics suggesting that this segment can readily fold into a small Ig domain (40). CD3epsilon is the only CD3 subunit with a nonglycosylated Ig-like ectodomain in the TCR complex of man and mouse (40). Moreover, the CD3epsilon subunit has twice as many acidic residues as basic ones and is therefore negatively charged (pI = 4.5, whereas the predicted pIs of the CD3gamma and CD3delta extracellular domains are more basic, being 8.76 and 5.82, respectively). The charge complementation between the acidic residues of the CD3epsilon subunit and the basic cavity also argues in favor of the CD3epsilon subunit occupying this site. The initial hypothesis regarding the proximity of CD3epsilon and the Cbeta FG loop was confirmed here by a set of competition assays in which the 2C11 and H57 mAbs, which bind to epitopes on CD3epsilon and TCR-beta , respectively, were able to alter each other's binding capacity. We further showed that although there are two CD3epsilon subunits per TCR-beta subunit, only one of the CD3epsilon components is in close proximity to the Cbeta FG loop. Given that the gamma /delta -TCR includes CD3 components (46) and is predicted to contain an equivalent insertion in the corresponding constant domain loop (47) analogous to the Cbeta FG loop, we can reasonably predict that a cavity with a comparable arrangement for CD3epsilon exists in gamma /delta T cells.

If the TCR C module cavity associates with one CD3epsilon , where is the second CD3epsilon located? In vitro translation studies have shown that when CD3epsilon is translated alone it tends to form disulfide-linked oligomers. However, using more physiological TCR-alpha /beta -CD3 expression conditions, namely simultaneous cotranslation of all the TCR subunits, the cotranslation of CD3gamma or CD3delta was sufficient to keep CD3epsilon in a monomeric state (48). Both CD3gamma and CD3delta compete for binding to CD3epsilon (49). In addition, it was shown that the CD3delta /epsilon pair associates with TCR-alpha , whereas the CD3gamma /epsilon pair associates with TCR-beta . This association takes place upon formation of an intrachain disulfide bridge between TCR-alpha and -beta (10). Additional studies have shown that CD3epsilon and TCR-beta pair via their ectodomains, whereas the association of the other CD3 components with TCR-alpha and -beta is largely mediated by interactions within their transmembrane regions. The proximity of TCR-beta and CD3gamma has been suggested by cross-linking experiments in humans (50) and mice (7). Based on these findings, it is likely that the CD3epsilon subunit that is physically associated with the Cbeta FG loop is paired with CD3gamma and that the second CD3epsilon , which pairs with the extracellular domain of the CD3delta subunit, associates via its transmembrane domain with the TCR-alpha (51). The ability of the CD3epsilon /gamma dimer-specific mAb 7D6 (52) to partially inhibit FITC-H57 binding to T cells is also consistent with this view (data not shown).

An interesting feature of the potential interaction of CD3epsilon within the TCR C domain module relates to certain critical Calpha residues that are preserved in pre-Talpha (pTalpha ), a 30-kD glycoprotein whose expression is restricted to early CD4-CD8- DN T lineage cells (53). The pre-TCR, a disulfide-linked heterodimer of a functionally rearranged TCR-beta chain and the pTalpha chain, noncovalently associated with the CD3 components is expressed in DN immature thymocytes (56). During development, the DN to DP thymocyte transition is induced by an as yet unknown ligand that binds to the pre-TCR, resulting in expression of mature type alpha /beta TCR heterodimers on DP thymocytes (57- 59). Although there is only 12% identity between pTalpha and Calpha in humans and mice (60), the Calpha residues involving important polar interactions with the Cbeta domain are all conserved in the pTalpha sequence. Given that conserved structural elements account for close to half of the pTalpha -Calpha amino acid sequence identities, it is very obvious that the heterodimer interface of pTalpha -Cbeta will be extremely similar to that of Calpha -Cbeta . With this in mind and since genetic disruption of the CD3epsilon gene results in T cell development blockage at the DN stage, it is likely that the CD3epsilon subunit is accommodated in the pre-TCR module in a manner similar to its mode in the mature alpha /beta -TCR.

One can imagine that certain mouse CD3epsilon residues contribute to the contacts made between H57 and the TCR. In this regard, the total buried molecular surface area at the N15 TCR-H57 Fab interaction is 1460A2 (720A2 for N15 and 740A2 for H57) (33), consistent with the range of values observed for other intact protein antigen-Fab interactions (61). However, one unusual feature of the N15- H57 interaction is the predominance of contacts made by the light chain (430A2 buried surface) rather than the heavy chain (310A2 buried surface) (33). This unusual characteristic led us to postulate that the mAb H57 heavy chain may interact primarily with TCR Cbeta but in addition with some CD3epsilon residues. In agreement with that notion is the large rotational mobility (~20°) of the Fab relative to the TCR Cbeta FG loop observed in the two independent copies of the TCR-Fab complexes in the asymmetric unit of the crystal (33). Nevertheless, analysis of the hemizygous hCD3epsilon tg mice revealed that although those T cells contain only half the surface copy number of the mouse CD3epsilon , H57 affinity is unaltered. This result suggests that the assembly of the TCR complex is not affected by the expression of the human CD3epsilon , and that the extracellular domain of the human CD3epsilon subunit can fit into the mouse TCR C domain module cavity (Fig. 3 B). Given that the extracellular domain of the mouse and human CD3epsilon share 53% identity in their amino acid sequence, it is possible that any putative CD3epsilon residues that interact with the H57 heavy chain are conserved among mouse and human CD3epsilon .

Although the precise functional role of the FG loop in Cbeta is far from clear, it is likely to have an important impact on signaling and/or TCR assembly and structure. The unique FG loop is conserved among sequences of mouse, rat, human, and rabbit TCRs (39). Although absent in TCRs sequences from some other species, it is replaced by a potential glycosylated addition site whose glycan may serve a similar structural function (33). Undoubtedly this loop influences the mobility and disposition of Vbeta relative to Cbeta domains. Although the minihydrophobic patch of this loop as resolved from the crystal structure fixes the overall loop orientation relative to the Vbeta and Cbeta domains, local movements are permitted (33). How, if at all, peptide-MHC interactions might transfer information to the CD3 signaling subunits by affecting the FG Cbeta loop remains to be tested experimentally.

    Footnotes

Address correspondence to Ellis L. Reinherz, Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA 02115. Phone: 617-632-3412; Fax: 617-632-3351; E-mail: ellis_reinherz{at}dfci.harvard.edu

Received for publication 22 December 1997 and in revised form 5 March 1998.

We thank Drs. Linda K. Clayton and Raute Sunder-Plassmann for helpful comments on the manuscript and Dr. Cox Terhorst for generous provision of the human CD3epsilon transgenic mice.

This work was supported by National Institute of Health grants AI-19807 (to E.L. Reinherz) and AI-39098 (to H.-C. Chang).

Abbreviations used in this paper DN, double negative; DP, double positive; mFI, mean fluorescence intensity; pTalpha , pre-Talpha ; tg, transgenic; WT, wild-type.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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