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The Journal of Immunology, 1998, 161: 2930-2937.
Copyright © 1998 by The American Association of Immunologists

TCR{alpha}ß Chains Associate with the Plasma Membrane Independently of CD3 and TCR{zeta} Chains in Murine Primary T Cells1

Jian Zhang2,*, Konstantin Salojin2,*, Jian-Xin Gao*, Mark Cameron*, Carsten Geisler§ and Terry L. Delovitch3,*,{dagger},{ddagger}

* Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, and Departments of {dagger} Microbiology and Immunology and {ddagger} Medicine, University of Western Ontario, London, Ontario, Canada; and § Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, Blegdamsvej, Copenhagen, Denmark


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TCR is a multisubunit complex composed of the clonotypic {alpha}/ß disulfide-linked heterodimer and noncovalently linked invariant CD3{gamma}{epsilon} and CD3{delta}{epsilon} and TCR{zeta} chains. Recent studies demonstrate that the surface expression of CD3 components can occur independently of the clonotypic TCR complexes in both thymocytes and splenic T cells. In this study, we report that free noncovalently associated TCR{alpha}ß heterodimers that exist independently of CD3 and TCR{zeta} chains are expressed on the cell surface of immature thymocytes and peripheral T cells, but not of T cell lines and T cell hybridomas. This suggests that the regulation of surface expression of TCR{alpha}ß heterodimers differs between primary T cells and T cell lines or T cell hybridomas. The isolation and biochemical characterization of surface clonotype-independent CD3 complexes and free membrane-associated TCR{alpha}ß complexes may provide a structural basis for the quantitative difference in amount of T cell proliferation stimulated by anti-CD3{epsilon} and anti-TCRß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The TCR is a multisubunit complex composed of the clonotypic {alpha}/ß disulfide-linked heterodimer and noncovalently invariant CD3{gamma}{delta}{epsilon} and TCR{zeta} chains. TCR{alpha}ß functions as to recognize Ag presented on MHC-encoded class I and II molecules on the surface of APCs (1, 2), whereas the major role of CD3 and TCR{zeta} chains is to mediate activation signals and to regulate the level of TCR expression (3, 4, 5, 6). All of the TCR chains are type I integral membrane proteins (7, 8, 9), and belong to the Ig superfamily, except for TCR{zeta} (10, 11, 12, 13).

Assembly of TCR complexes occurs within the endoplasmic reticulum (ER)4 and proceeds in a precisely ordered manner by: 1) formation of noncovalently associated pairs of CD3{gamma}{epsilon} and {delta}{epsilon} proteins; 2) association of individual clonotypic {alpha} and ß polypeptides with CD3{gamma}{epsilon} and {delta}{epsilon} pairs to form the intermediate TCR complexes; 3) rapid pairing of {alpha}{delta}{epsilon}- and ß{gamma}{epsilon}-chains and disulfide bonding of CD3-associated {alpha}ß proteins to yield the incomplete {alpha}ß{delta}{epsilon}{gamma}{epsilon} TCR complexes; and 4) association of TCR{zeta} homodimers to form the complete, fully assembled {alpha}ß{delta}{epsilon}{gamma}{epsilon}{zeta}{zeta} TCR complexes. All components of TCR complexes, with the exception of TCR{zeta}, are synthesized in excess and are susceptible to degradation in the ER, unless assembled with other chains. Whereas intermediate {alpha}ß{delta}{epsilon}{gamma}{epsilon} TCR complexes lacking TCR{zeta} are targeted to lysosomes for degradation, fully assembled {alpha}ß{delta}{epsilon}{gamma}{epsilon}{zeta}{zeta} TCR complexes are efficiently transported to the cell surface (14). Although recent studies demonstrate that the synthesis of complete TCR/CD3 complexes occurs in immature thymocytes (15, 16), we (17) and others (18, 19, 20) have observed that CD3{gamma}{epsilon} and {delta}{epsilon} as well as TCR{zeta} can associate with the plasma membrane in T cells independently of TCR{alpha}ß. In this study, we provide both biochemical and structural evidence that noncovalently associated TCR{alpha}ß chains can also exist on the plasma membrane independently of CD3 and TCR{zeta} chains in murine primary T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 (B6) and BALB/c (B/c) were purchased from The Jackson Laboratory (Bar Harbor, ME), maintained in the Animal Care Facility at University of Western Ontario (London, ON, Canada), and used at 6 to 10 wk of age.

Reagents and Abs

The 145-2C11 anti-CD3{epsilon} and the H57-597 anti-TCRß mAbs were purified by protein G affinity chromatography (Pharmacia Biotech, Uppsala, Sweden) of hybridoma supernatants (kindly supplied by Dr. J. Bluestone, University of Chicago, Chicago, IL, and Dr. R. Kubo, Cytel, La Jolla, CA, respectively). Rabbit antisera 387 to TCR{zeta} chains and mouse anti-TCR{zeta} mAb (6B10.2) were provided by Drs. L. E. Samelson (National Institutes of Health, Bethesda, MD) and A. Weiss (University of California, San Francisco, CA), respectively. Polyclonal Abs against TCR{alpha} (S-20), TCRß (A-19), and CD3{epsilon} (48-2B) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse anti-human CD3{epsilon} (UCHT1), mouse anti-human TCR{alpha}ß (T10B9.1A-31), and rat anti-mouse CD4 (RM4-5) mAbs were purchased from PharMingen (San Diego, CA). The polyclonal Abs against calnexin and calreticulin were obtained from StressGen Biotechnologies (Victoria, BC, Canada). The rabbit anti-hamster affinity-purified Ig, protein G, and ExAvidin-peroxidase (POD) were purchased from Sigma (Mississauga, ON, Canada). Horseradish peroxidase-conjugated goat anti-mouse IgG, rabbit anti-goat, and donkey anti-rabbit IgG were purchased from Amersham (Oakville, ON, Canada). Indo-1 AM was purchased from Molecular Probes (Eugene, OR).

Cell stimulation

Single cell suspensions of thymocytes were prepared by gently teasing cells from the thymic capsule and filtering over nylon mesh. Splenic T cells were isolated using T cell enrichment columns (R&D Systems, Minneapolis, MN) with a purity of >=98%, as assayed by FACS analysis of CD3 cell surface expression. CD4+CD8+ thymocytes were isolated by adherence to plastic plates coated with an anti-CD8{alpha} mAb (21), and were typically >96% CD4+CD8+. Jurkat {gamma}-negative (JGN) C19-8 cells, a CD3{gamma}-negative variant line of Jurkat T cells (22), and JGN{gamma}WT 14-5-16, a JGN T cell line transfected with wild-type CD3{gamma} (23), were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all purchased from Life Technologies, Grand Island, NY) at 37°C in 5% CO2. The DO-11.10 murine T cell hybridoma, which is I-Ad restricted and specific for the OVA 323–339 peptide (24), was obtained from Dr. B. Singh (University of Western Ontario, London, ON, Canada), and cultured in complete RPMI 1640, as above. Quiescent thymocytes were incubated with anti-TCR (H57-597) and anti-CD4 (RM4-5) on ice for 15 min and washed twice in RPMI 1640 medium. Cross-linking of mAbs at 37°C was accomplished using protein G (Sigma, St. Louis, MO) at a 4:1 w/w ratio for the times indicated.

Surface reexpression assay

Assay of surface reexpression was performed as previously reported (25). Briefly, thymocytes were resuspended at a concentration of 5 x 106/ml in PBS containing 100 µg/ml of pronase (70 U/mg) (Calbiochem, San Diego, CA) and incubated for 15 min at 37°C. Pronase-treated cells were resuspended at 106/ml in RPMI 1640 medium containing 10% FCS and cultured for 4 h at 37°C with Brefeldin A (BFA) (1 µg/ml) or cycloheximide (CHX) (10 µg/ml) (Sigma). After culture, viable cells were recovered by density-gradient centrifugation on lympholyte M (Cedarlane Laboratories, Hornby, ON, Canada) and then surface biotinylated.

Metabolic labeling and cell surface biotinylation

For metabolic labeling, cells were resuspended at 108 cells/ml in methionine-free RPMI 1640 medium containing 10% dialyzed FCS and incubated with 1.5 mCi [35S]methionine (Trans 35S label; ICN, Irvine, CA) for 1 h at 37°C. For chase experiments, cells were washed twice in chase medium (RPMI 1640 medium containing 10% FCS and excess cold methionine at 300 mg/ml), resuspended at their original concentration in fresh chase medium, and incubated at 37°C for the time period indicated. At each time point of pulse and chase, cell surface biotinylation were performed as reported (20), with minor modifications. Briefly, cells were washed three times in PBS and resuspended (107/ml) in PBS containing 0.1 mM CaCl2 and 1 mM MgCl2. Sulfo-N-hydroxysuccinimide-biotin (200 mg/ml; Pierce, Rockford, IL) dissolved in DMSO was added to the cell suspension to a final concentration of 0.5 mg/ml, and the cells were incubated for 1 h on ice. Biotinylated cells were washed three times in RPMI 1640 and lysed in 1% Brij 97 lysis buffer. The sulfonyl group of the sulfo-N-hydroxysuccinimide-biotin derivative confers a net negative charge on the molecule and prevents it from crossing the plasma membrane. This confines the biotinylation reaction to the exoplasmic face of the lipid bilayer and enables the cell surface biotinylation of lysine residues on membrane-associated proteins.

Immunoprecipitation, gel electrophoresis, and immunoblotting

Cells were solubilized in lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM iodoacetamide, 5 mM EDTA, 2 mM Na3VO4, 20 mg/ml aproptonin, 20 µg/ml leupeptin, 1 mM PMSF, and 10 mM NaF) containing 1% Brij 97 lysis buffer for 30 min on ice. Postnuclear supernatants of cell lysates from 1 to 2 x 107 cells were immunoprecipitated (2–16 h) with specific Abs or control isotype-matched preimmune Ig preadsorbed with 30 µl of protein A-Sepharose CL-4B (Pharmacia Biotech, Baie d’Urfe, PQ, Canada), protein A/G plus agarose (Santa Cruz Biotechnology), or streptavidin-agarose (Sigma). After incubation, the beads were washed three times in lysis buffer. Bound proteins were solubilized in 2x Laemmli sample buffer under reducing conditions, resolved by SDS-PAGE on 12 or 15% gels, and then transferred onto a polyvinylidene difluoride or nitrocellulose membrane. Immunoblotting was performed after first blocking the membranes with 5% nonfat dry milk in TBS-T (10 mM Tris, pH 7.6/150 mM NaCl/0.1% Tween-20) for 1 h at room temperature. To sequentially immunoblot a given membrane, the membrane was stripped of proteins for 30 min at 50°C in 62.5 mM Tris, pH 6.7, 2% SDS, and 0.1 M 2-ME, and was then further immunoblotted with the relevant Abs. The relative amounts of the proteins detected were quantitated by densitometry using a Molecular Imager (Bio-Rad, Hercules, CA).

Immunoelectron microscopy

Splenic T cells from B6 mice were fixed with 3% paraformaldehyde in 0.1 mmol/L cacodylate buffer (pH 7.2) overnight, washed in cacodylate buffer, osmium tetroxide postfixed, gradually dehydrated in ethanol, and embedded in Epon/Araldite. Thin sections were cut and collected on nickel grids. Double-immunogold labeling was performed on the same thin sections of splenic T cells using gold particles of different sizes. One side of the tissue section was incubated with biotinylated anti-TCRß mAb and 20 nm colloidal gold/streptavidin-conjugated second Ab complexes. After the grid was dried, the second side of the section was incubated with anti-CD3{epsilon} mAb, followed by protein A-gold complexes formed with 10 nm gold particles. For controls, the primary mAbs were replaced by normal hamster IgG. After all labelings were completed, the grids were contrasted with ultranyl acetate and lead citrate.

Proliferation assay

Thymocytes or purified splenic T cells were cultured (106/ml) in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mM HEPES buffer, 1 mM Na2PO4, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 µM 2-ME for 72 h (thymocytes) or 48 h (splenic T cells) at 37°C in round-bottom 96-well plates (Nunc) precoated with H57-597 anti-TCRß or 145-2C11 anti-CD3{epsilon} mAb at concentrations of 1 to 100 µg/ml. [3H]Thymidine (1 mCi/well; Amersham) was added 24 h before the end of culture, and cultures were harvested using a Tomtec Harvester 96 cell harvester (Fisher Scientific, Ottawa, ON, Canada). The extent of cell proliferation was proportional to the amount of [3H]thymidine incorporation, which was determined using a Wallac 1450 Microbeta Plus beta counter (Fisher Scientific).

Ca2+ assay

Indo-1 loading and analysis of Ca2+ data were performed essentially as described (26). Briefly, DO-11.10 T cells (5 x 106/ml) were incubated with indo-1 (2 µg/ml) for 30 min at 37°C. Cells were washed in MEM medium containing HCO3-2 (4 mM) and buffered HEPES (25 mM). For [Ca2+]i measurements, aliquots (1 ml) of cells were sedimented and resuspended in 2 ml of a continuously stirred Na+ solution consisting of 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 20 mM sodium HEPES, pH 7.3. Cells were stimulated directly with anti-CD3 (50 µg/ml) or anti-TCRß (100 µg/ml) for 1 min at 37°C. The [Ca2+]i in indo-1-loaded cells was monitored using a dual wavelength fluorimeter (model RF-M2004; Photon Technology Internal, London, ON, Canada) at an excitation wavelength of 355 nm and emission wavelengths of 405 and 485 nm. Data were analyzed using Felix software (Photon Technology Internal) (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TCR{alpha}ß associates with the plasma membrane independently of CD3 and TCR{zeta} chains in immature and mature T cells

We examined whether TCR{alpha}ß chains are expressed on the cell surface of T cells only as part of complete clonotypic TCR/CD3 complexes, or whether they are also expressed on the plasma membrane of T cells independently of such complexes. To detect these surface complexes, thymocytes were surface biotinylated, and then serially immunoprecipitated with anti-CD3{epsilon} and anti-TCRß to recover and quantitate the distribution of all plasma membrane-bound TCR{alpha}ß. As shown in Figure 1GoA, anti-CD3{epsilon} immunoprecipitation removed virtually all CD3 components and TCR{zeta} chains as well as TCR{alpha}ß heterodimers, which were associated with CD3 components (Fig. 1GoA, lane 2). Subsequent immunoprecipitation with anti-TCRß mAb showed that TCR{alpha}ß heterodimers could still be seen on the plasma membrane of thymocytes (Fig. 1GoA, lane 3). This band did not cross-react with TCR{gamma}{delta} heterodimers that possess a similar m.w. to TCR{alpha}ß, because anti-TCR{gamma}{delta} immunoprecipitation did not yield any bands corresponding to TCR{gamma}{delta} chains (Fig. 1GoA, lane 1). The failure to detect the TCR{gamma}{delta} chains was not due to the quality of the anti-TCR{gamma}{delta} mAb used, since about 3% TCR{gamma}{delta}+ thymocytes were detected by flow cytometry (Fig. 1GoB). Rather, this result may be attributed to the difficulty of immunoprecipitating lysates derived from a low number of cells.



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  		FIGURE 1. Both thymocytes and mature T cells express surface TCR{alpha}ß heterodimers that are not associated with CD3 components. TCR complexes were isolated from Brij 97 lysates of biotin surface-labeled total C57BL/6 (B6) thymocytes (A), purified B6 DP thymocytes (C), and B6 and BALB/c (B/c) splenic T cells (D), using mAbs reactive with either TCRß (57-597) or CD3{epsilon} (145-2C11), or control hamster IgG (HIgG). Cell lysates were immunoprecipitated with anti-CD3{epsilon} twice, and then sequentially with an anti-TCRß mAb. Immune complexes were resolved in SDS-PAGE gels, transferred to a nitrocellulose membrane, and visualized by ExAvidin-POD and chemoluminescence. TCR{gamma}{delta} surface expression by thymocytes was confirmed by flow cytometry (B). The presence of TCR{zeta} in TCR complexes of Brij 97 and digitonin thymocyte cell lysates was analyzed by SDS-PAGE and immunoblotting (E). The band indicated by an asterisk (*) may include the Fc{gamma}R. To identify TCR{alpha} and TCRß proteins, thymocytes or T cells were surface biotinylated and solubilized in 1% Brij 97 lysis buffer, and cell lysates were immunoprecipitated with anti-TCRß. Precipitates were boiled in 1% SDS to release bound proteins, Nonidet P-40 was added to a final concentration of 1%, and TCR{alpha} and TCRß chains were specifically recaptured with anti-TCR{alpha}, anti-TCRß, or both mAbs (F).

 
The fact that CD4+CD8+ double-positive (DP) thymocytes comprise about 85% of the T cells in a thymus raised the possibility that the results described above might be representative of only immature thymocytes. To test this possibility and to address whether the same is true in periperal T cells, the experiments presented in Figure 1Go, A and B, were repeated using purified DP thymocytes and splenic T cells. Free TCR{alpha}ß heterodimers were observed at the cell surface of both DP thymocytes and splenic T cells (Fig. 1Go, C and D). The position of migration TCR{zeta} was confirmed by anti-TCR{zeta} immunoblotting (Fig. 1GoC, lower panel). Since a previous report (19) that TCR{zeta} cannot be labeled by surface biotinylation conflicts with our observation in Figure 1GoC, we compared the sensitivity of detection of TCR{zeta} in cell lysates prepared with either the Brij 97 or digitonin detergents. We found that the Brij 97 lysis buffer, which we commonly use, is better at maintaining certain protein-protein interactions than the digitonin buffer, as evidenced by the greater abundance of TCR{zeta} in the Brij 97 than digitonin lysate (Fig. 1GoE). Note that the proteins migrating between the 6.5- and 16.5-kDa markers were also detected in normal rabbit serum control immunoprecipitates, and may include the Fc{gamma}R. To further determine whether TCR{alpha} or TCRß, or both, associate with the plasma membrane, thymocytes were surface biotinylated and lysed with 1% Brij 97. After depletion of CD3 chains by preclearing with anti-CD3{epsilon} mAbs, supernatants were immunoprecipitated with anti-TCRß. The precipitates were boiled in 1% SDS to release bound material, Nonidet P-40 detergent was added to counteract the SDS, and TCR{alpha} and TCRß proteins were recaptured by precipitation with anti-TCR{alpha} and anti-TCRß mAbs, respectively. Figure 1GoD shows that both TCR{alpha} and TCRß chains associate with the plasma membrane in the absence of any detectable CD3 and TCR{zeta} chains, suggesting that the free TCR{alpha} and TCRß may bind to each other noncovalently.

To obtain ultrastructural evidence in support of our biochemical data, we labeled ultrathin sections of splenic T cells with immunogold-conjugated anti-TCRß and anti-CD3{epsilon} mAbs and examined the cells by electron microscopy. Figure 2Go shows that there are three types of TCR/CD3 complexes at the cell surface of splenic T cells, which reflect TCR{alpha}ß (labeled with 20 nm gold particles) associated with CD3{epsilon} (labeled with 10 nm gold particles), free TCR{alpha}ß, and free CD3{epsilon}. Thus, TCR{alpha}ß heterodimers can be expressed on the cell surface independently of CD3 components in both immature and mature T cells. These CD3-independent TCR{alpha}ß proteins at the cell surface are referred to as free membrane-associated TCR{alpha}ß heterodimers.



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  		FIGURE 2. Immunoelectron micrographs of splenic T cells show three types of TCR/CD3 complexes on the plasma membrane. Thin sections of splenic T cells were labeled on one side with biotinylated anti-TCRß mAb and 20 nm colloidal gold/streptavidin-rabbit anti-hamster IgG, and on the other side with anti-CD3{epsilon} mAb and 10 nm protein A-gold. Sites of localization of free membrane-associated TCR{alpha}ß and CD3{epsilon} are indicated by an arrowhead and arrow, respectively. Sites of colocalization of TCR{alpha}ß and CD3{epsilon}, presumably representative of TCR/CD3 complexes, are indicated by an arrowhead and vertical bar.

 
Jurkat T cells and T hybridoma cells do not express free membrane-associated TCR{alpha}ß complexes at the cell surface

Having found that TCR{alpha}ß chains can associate with the plasma membrane independently of CD3 subunits in murine primary T cells, we next investigated whether this also applies for T cell lines. Biochemical analyses were conducted using the JGN{gamma}WT 14-5-16 and CD3{gamma}-negative JGN C19-8 Jurkat human T cell lines as well as the DO-11.10 murine T cell hybridoma. After preclearing with anti-CD3{epsilon}, no TCR{alpha}ß chains were observed upon subsequent immunoprecipitation with anti-TCRß, indicating that free membrane-associated TCR{alpha}ß complexes are either absent or are present at below detectable levels in JGN{gamma}WT T cells (Fig. 3GoA). This result also suggests that CD3 subunits determine the surface expression of TCR complexes in Jurkat T cells, which is further supported by the absence of TCR{alpha}ß, CD3, and TCR{zeta} chains from the plasma membrane of JGN C19-8 and DO-11.10 T cells (Fig. 3Go, B and C). Thus, free membrane-associated TCR{alpha}ß heterodimers appear to exist only in primary T cells, but not in commonly studied T cell lines or T cell hybridomas, suggesting that primary T cells differ from T cell lines and T cell hybridomas in terms of their surface expression of TCR heterodimers.



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  		FIGURE 3. Human Jurkat T cells and murine T hybridoma cells do not express free TCR{alpha}ß complexes on the cell surface. JGN{gamma}WT 14-5-16 Jurkat T cells (A) and CD3{gamma}-negative variant JGN C19-8 T cells (B) were surface biotinylated, lysed, and precipitated first with anti-CD3{epsilon}, and then either anti-TCRß or anti-CD3{epsilon} mAb, or control mouse IgG (MIgG). DO-11.10 T hybridoma cells were biotinylated and serially immunoprecipitated with anti-CD3{epsilon} and anti-TCRß (C). Biotinylated proteins were detected as in Figure 1Go.

 
Free membrane-associated TCR{alpha}ß complexes are synthesized de novo

To examine whether free membrane-associated TCR{alpha}ß proteins are comprised of {alpha}- and ß-chains synthesized de novo, experiments were performed as outlined in Figure 4GoA. Thymocytes were metabolically labeled with [35S]methionine for 1 h, and then chased with an excess of cold methionine for 0.5, 1, 2, and 4 h. At each time point, thymocytes were surface biotinylated. Cells were lysed, precleared with anti-CD3{epsilon}, and immunoprecipitated with anti-TCRß. The anti-TCRß immunoprecipitates were boiled in 1% SDS to release TCR{alpha}ß proteins, and the released proteins were subsequently recaptured by streptavidin-agarose. Figure 4GoB shows that with longer chase times, the amounts of newly synthesized and membrane-associated free TCR{alpha}ß complexes (which bind to streptavidin-agarose and are [35S]methionine labeled) were gradually increased. Free TCR{alpha}ß complexes were detectable at 2 h of chase and were significantly increased by 4 h of chase. To confirm this finding, thymocytes were treated with either BFA, an inhibitor of vesicular transport (27), or CHX, a protein synthesis inhibitor, and then analyzed for the expression of free TCR{alpha}ß heterodimers. In the presence of either BFA or CHX, the expression of free TCR{alpha}ß heterodimers on the cell surface was effectively blocked (Fig. 4GoC), demonstrating that the escape of free TCR{alpha}ß proteins from the ER to the cell surface requires ongoing protein synthesis. Taken together, our data suggest that most free membrane-associated TCR{alpha}ß heterodimers are newly synthesized, and that it takes 2 h for newly synthesized free TCR{alpha}ß proteins within the ER to be transported to the plasma membrane. To explore whether the expression of free TCR{alpha}ß heterodimers on the surface is increased upon activation of T cells, thymocytes were stimulated with anti-TCRß plus anti-CD4 for 0, 2, and 4 h, lysed at each time point, precleared with anti-CD3{epsilon}, and then immunoprecipitated with anti-TCRß. The amount of free TCR{alpha}ß heterodimers on the plasma membrane did not change appreciably after stimulation (Fig. 4GoD).



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  		FIGURE 4. Free membrane-associated TCR{alpha}ß complexes are synthesized de novo in primary T cells. A, Schematic of the protocol of the pulse-chase experiment. B, Thymocytes from B6 mice were pulsed for 1 h with [35S]methionine and then chased for the periods indicated. At each time point of pulse and chase, cells were surface labeled with biotin and solubilized in 1% Brij 97 lysis buffer. Brij 97 lysates were precleared with anti-CD3{epsilon} and immunoprecipitated with anti-TCRß. Precipitated proteins were then released by boiling samples in 1% SDS, and released proteins were recaptured with streptavidin (SAV)-agarose. Biotinylated proteins that were also [35S]methionine labeled were analyzed by SDS-PAGE. C, Cells were otherwise treated with BFA or CHX, following which cells were surface labeled with biotin and lysed. D, Thymocytes were stimulated with anti-TCRß and anti-CD4 mAb for 0, 2, and 4 h at 37°C. Cell lysates were precleared with anti-CD3{epsilon} twice, precipitated with anti-TCRß mAb, and visualized as in Figure 1Go. The relative amounts of TCRß quantitated by densitometry are shown below the gel band.

 
Free membrane-associated TCR{alpha}ß complexes associate with calnexin on the plasma membrane

Recent studies indicate that the related molecular chaperones, calnexin and calreticulin, differentially associate with nascent TCR proteins within the ER, and that many, but not all, resident ER proteins can escape from the ER and reach the cell surface in immature thymocytes (28, 29, 30, 31). We investigated whether the same phenomenon occurs with free membrane-associated TCR{alpha}ß heterodimers. Lysates of surface-biotinylated thymocytes were either immunoprecipitated with anti-calnexin or anti-calreticulin polyclonal Abs or otherwise serially immunoprecipitated with anti-CD3{epsilon} and anti-TCRß mAbs, and then immunoblotted with anti-calnexin and anti-calreticulin, respectively. Both calnexin and calreticulin were found to be associated with the plasma membrane (Fig. 5GoA). Interestingly, calnexin, but not calreticulin, associated with TCR{alpha}ß/CD3 complexes and free TCR{alpha}ß complexes on the cell surface (Fig. 5GoA, lanes 1 and 2; Fig. 5Go, B and C, upper panel). However, association of CD3 or free TCR{alpha}ß with calreticulin may occur within the ER, since reprobing the same membrane with anti-calreticulin showed that calreticulin is present in anti-CD3{epsilon} and subsequent anti-TCRß immunoprecipitates (Fig. 5GoC, lower panel). These data suggest that calnexin can escape from the ER and be transported to the plasma membrane with either the clonotype-independent CD3 complexes or free TCR{alpha}ß heterodimers.



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  		FIGURE 5. Calnexin, but not calreticulin, associates with free TCR{alpha}ß proteins on the cell surface. Thymocytes from B6 mice were surface biotinylated and lysed. A, Cell lysates were immunoprecipitated with polyclonal rabbit Abs against calreticulin (CRT) or calnexin (CNX), or control normal rabbit IgG (RIgG). B and C, Lysates were serially immunoprecipitated with anti-CD3{epsilon} and anti-TCRß mAbs, and immunoblotted with anti-CRT or anti-CNX. D, B6 thymocytes were biotinylated, lysed, and immunoprecipitated with anti-calnexin and anti-TCR{zeta}, respectively. Biotinylated proteins associated with the immunoprecipitates were visualized as in Figure 1Go. The membrane was stripped, and reprobed with anti-TCR{zeta} and anti-calnexin, respectively.

 
To determine whether TCR{zeta} and calnexin associate on the plasma membrane, thymocytes were surface biotinylated, lysed, and immunoprecipitated with either anti-TCR{zeta} or anti-calnexin, and membrane-bound proteins were visualized by ExAvidin-POD and chemoluminescence. Consistent with the result shown in Figure 5GoA, calnexin was found to be associated with TCR{zeta} on the plasma membrane (Fig. 5GoD, upper panel). The presence of calnexin and TCR{zeta} in anti-calnexin and anti-TCR{zeta} immunoprecipitates was confirmed by anti-TCR{zeta} and anti-calnexin immunoblotting (Fig. 5GoD, middle and lower panels).

Differential T cell proliferation induced by anti-CD3{epsilon} and anti-TCRß stimulation

Our data suggest that, in murine primary T cells, there are at least three types of TCR/CD3 complexes on the cell surface, namely clonotypic TCR/CD3 complexes, clonotype-independent CD3 components, and free membrane-associated TCR{alpha}ß heterodimers. To compare the relative strength of signals transmitted by TCRß stimulation and CD3{epsilon} stimulation to downstream events that lead to T cell proliferation, we assayed thymocyte and splenic T cell proliferation induced by anti-CD3{epsilon} and anti-TCRß. Significantly higher levels of proliferation were observed in thymocytes and splenic T cells after stimulation with anti-CD3{epsilon} than with anti-TCRß, with the difference being more evident in thymocytes than splenic T cells (Fig. 6Go, A and B). To examine whether this difference is due to the low expression of TCR{alpha}ß heterodimers and high expression of clonotype-independent CD3 complexes on the plasma membrane of thymocytes, equal numbers of thymocytes and splenic T cells (107) were surface biotinylated, lysed, and serially immunoprecipitated with anti-TCRß and anti-CD3{epsilon}. TCR{alpha}ß/CD3 complexes, including clonotypic TCR{alpha}ß complexes and free TCR{alpha}ß heterodimers (detected by anti-TCRß immunoprecipitation) and clonotype-independent CD3 complexes (detected by subsequent immunoprecipitation with anti-CD3{epsilon}), were visualized by ExAvidin-POD and chemoluminescence. TCR{alpha}ß expression was found to be lower in thymocytes than in splenic T cells, but the expression of clonotype-independent CD3 complexes is higher in thymocytes than in splenic T cells (Fig. 6GoC).



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  		FIGURE 6. Ligation of CD3{epsilon} or TCRß induces differential T cell proliferation. A and B, Thymocytes and splenic T cells (2 x 105/well) were cultured in triplicate wells in the presence of plate-bound anti-CD3{epsilon} (145-2C11 mAb) or anti-TCRß (H57-597 mAb) at varying concentrations (1–100 µg/ml) for 72 h (thymocytes) and 48 h (splenic T cells). Cell proliferation was determined by [3H]thymidine incorporation. C, Thymocytes (THY) and splenic T cells (SP-T) (107) were surface biotinylated, lysed, and immunoprecipitated first with anti-TCRß and then with anti-CD3{epsilon}. Biotinylated proteins were detected as in Figure 1Go.

 
To further investigate whether free membrane-associated TCR{alpha}ß heterodimers signal in T cells, we analyzed Ca2+ flux induced by anti-CD3{epsilon} and anti-TCRß mAbs in DO-11.10 T cells. Although the basal levels of Ca2+ concentration induced by anti-CD3{epsilon} and anti-TCRß stimulation differed slightly, the extent of increase in Ca2+ responses to these two mAbs was quite similar (Fig. 7GoA, upper and lower panels). Note that in DO-11.10 T cells, plasma membrane-associated clonotype-independent CD3 complexes are not detectable (Fig. 7GoB). These data provide additional support for the notion that free membrane-associated TCR{alpha}ß heterodimers are functional in T cell activation.



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  		FIGURE 7. A, Anti-TCRß and anti-CD3{epsilon} stimulation induce a similar Ca2+ flux in DO-11.10 T cells. DO-11.10 T cells were loaded with indo-1 (2 µg/ml), suspended in a Na+ solution, and stimulated by an anti-CD3{epsilon} (50 µg/ml) or anti-TCRß (100 µg/ml) mAb. [Ca2+]i was monitored by a dual wavelength fluorimeter. B, DO-11.10 T hybridoma cells were biotinylated and serially immunoprecipitated with anti-TCRß and anti-CD3{epsilon}. Biotinylated proteins were detected as in Figure 1Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate that both thymocytes and peripheral T cells express noncovalently associated TCR{alpha}ß heterodimers independently of CD3 components at the cell surface, which we refer to as free membrane-associated TCR{alpha}ß heterodimers. These free TCR{alpha}ß heterodimers are synthesized de novo in primary T cells, and are associated with the ER-resident protein calnexin. These free TCR{alpha}ß heterodimers do not contain CD3 components, and therefore differ structurally from the TCR{alpha}ß heterodimers present in clonotypic TCR/CD3 complexes. It is noteworthy that these free membrane-associated TCR{alpha}ß heterodimers are not expressed in commonly analyzed T cell lines, such as human Jurkat T cells and murine T cell hybridomas.

The biochemical characterization of free membrane-associated TCR{alpha}ß complexes required the analysis of surface-labeled membrane proteins. We used the strategy of surface biotinylation combined with serial immunoprecipitation to identify free TCR{alpha}ß proteins on the cell membrane. The possibility that internal proteins were also biotinylated during the surface-labeling reaction was excluded for four main reasons. First, surface staining detected by the use of streptavidin-Texas Red coupled with fluorescence microscopy revealed the absence of any intracellular staining. Second, the labeling of Lck, Fyn, and ZAP-70 was not observed in these cells after surface biotinylation. Third, an inhibitor of vesicular transport, BFA, completely blocked the expression of free TCR{alpha}ß heterodimers on the cell surface. Fourth, these free membrane-associated TCR{alpha}ß heterodimers were undetectable in both JGN{gamma}WT 14-5-16 Jurkat T cells and DO-11.10 murine T hybridoma cells. It is important to note that these free TCR{alpha}ß heterodimers on the plasma membrane are unlikely to be equivalent to TCRß-pre-TCR{alpha} complexes on the cell surface in immature thymocytes. The 33-kDa pre-TCR{alpha} protein is smaller than TCR{alpha}, and the free TCR{alpha}ß heterodimers we observed are also present on the surface of mature T cells. It is also unlikely that free membrane-associated TCR{alpha}ß heterodimers result from the dissociation of TCR{alpha}ß from CD3 chains in TCR{alpha}ß/CD3 complexes after cell lysis, as no free membrane-associated TCR{alpha}ß heterodimers were observed in the Jurkat human T cell lines and murine T cell hybridomas. In addition, ultrastructural evidence for three types of TCR/CD3 complexes on the surface of splenic T cells was obtained, which confirms our biochemical evidence and extends our previous findings (17).

Previously, we and others found clonotype-independent CD3 and TCR{zeta} proteins on the plasma membrane of both thymocytes and splenic T cells (17, 18, 19, 20). In this study, we demonstrate that free membrane-associated TCR{alpha}ß heterodimers are also expressed on both thymocytes and splenic T cells. Both thymocytes and peripheral T cells proliferate more vigorously in response to stimulation by anti-CD3{epsilon} than anti-TCRß. This finding raises the possibility that this result is attributable to different relative affinities of the anti-CD3{epsilon} and anti-TCRß mAbs for their respective TCR ligands. However, our ultrastructural and biochemical data argue against the affinity-related explanation for the proliferation differences induced by anti-CD3{epsilon} and anti-TCR{alpha}ß. Interestingly, this difference is more evident in thymocytes than in splenic T cells, and correlates with low TCR{alpha}ß expression and high expression of clonotype-independent CD3 complexes on the plasma membrane of thymocytes. Moreover, anti-TCRß and anti-CD3{epsilon} stimulation elicits similar Ca2+ responses in DO-11.10 T cells, which do not express clonotype-independent CD3 complexes on their surface. Figure 8Go presents a model that illustrates a potential mechanism of how these two mAbs differentially stimulate the proliferation of T cells. In this model, anti-CD3{epsilon} stimulation may not only act on clonotypic TCR/CD3 complexes, but also on clonotype-independent CD3 complexes on the plasma membrane, and thereby elicits two types of downstream signals for proliferation. In contrast, TCRß ligation may stimulate only clonotypic TCR/CD3 complexes and not free membrane-associated TCR{alpha}ß heterodimers, since the latter free TCR{alpha}ß heterodimers are not associated with any CD3 signaling components at the cell surface. Thus, signals initiating from free membrane-associated TCR{alpha}ß heterodimers cannot be transmitted downstream for proliferation.



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  		FIGURE 8. Model of differential T cell activation by anti-CD3{epsilon} and anti-TCRß mAbs. In this model, anti-CD3{epsilon} stimulation not only stimulates clonotypic TCR/CD3 complexes, but also clonotype-independent CD3 complexes on the plasma membrane, which elicits two signals to downstream events. In contrast, anti-TCRß stimulates only intact TCR/CD3 complexes, but not free membrane-associated TCR{alpha}ß heterodimers, since there are no CD3 signaling components associated with free noncovalently linked TCR{alpha}ß heterodimers at the cell surface. Thus, signals initiating from free membrane-associated TCR{alpha}ß heterodimers cannot be transmitted downstream.

 
The molecular chaperones, calnexin and calreticulin, are nonglycosylated resident ER transmembrane and lumenal proteins that associate with numerous oligomeric protein complexes within the ER and recognize partially trimmed, monoglycosylated glycan chains on newly synthesized glycoproteins (30). Calnexin associates with both clonotypic TCR{alpha} and TCRß proteins and invariant CD3{gamma}, CD3{delta}, and CD3{epsilon} chains, while calreticulin assembly is restricted to clonotypic, Ag-reactive TCR{alpha} and TCRß proteins (29, 30, 31). Moreover, clonotype-independent CD3 complexes have been found to associate with calnexin on the plasma membrane in immature DP thymocytes (18, 19). Calnexin and many other ER-resident proteins, including calreticulin, can escape from the ER of immature thymocytes to be expressed on the plasma membrane, indicating that ER retention is incomplete in these cells (25). Our data show that calnexin, but not calreticulin, is associated with free TCR{alpha}ß heterodimers on the surface of thymocytes. The reason may be ascribed in part to the more stable association of TCRß with calnexin than calreticulin (30). In addition, we found that calnexin is associated with the TCR/CD3 complexes on the cell surface, which may be mediated by the association of CD3 components with calnexin (19). Although calreticulin is not associated with free TCR{alpha}ß heterodimers on the plasma membrane, results from anti-calreticulin immunoblotting of anti-TCRß immunoprecipitates precleared of CD3{epsilon} suggest that calreticulin associates with the TCR/CD3 complex and the free TCR{alpha}ß heterodimers within the ER, consistent with a previous report (30). Recent studies suggest that association of nascent glycoproteins with calnexin proceeds in a two-step fashion involving the initial binding of monoglycosylated glycans by calnexin, followed by protein-protein interactions that stabilize these associations (28, 31). Conceivably, the association of calnexin with free TCR{alpha}ß heterodimers may mask their retention sequences and result in this complex escaping from the ER and reaching the cell surface.

In conclusion, this study is the first to demonstrate that free noncovalently linked TCR{alpha}ß heterodimers, which do not consist of either CD3 or TCR{zeta} chains, can associate with the plasma membrane in both immature and mature murine T cells. We show that this is not the case for transformed human Jurkat T cell lines and murine T hybridoma cells. This indicates that primary T cells differ from T cell lines in their expression of free TCR{alpha}ß heterodimers on the cell surface. Free TCR{alpha}ß can escape from the ER in association with calnexin, demonstrating that ER retention in murine primary T cells is incomplete. Further experimentation is required to identify the functional relevance of these free membrane-associated TCR{alpha}ß heterodimers.


   Acknowledgments
 
We thank Drs. L. Samelson and A. Weiss for their generous donation of reagents, Dr. B. Singh for providing the DO-11.10 T cell hybridoma, Dr. J. Dixon for his assistance with the Ca2+ assay, Ms. V. Martinez for her technical assistance with the immunoelectron-microscopy studies, Dr. S. Kaga for his valuable suggestions, all members of our laboratory for their valuable advice and encouragement, and Ms. A. Leaist for her expert and cheerful assistance with the preparation of this manuscript.


   Footnotes
 
1 This work was supported by a grant from the Juvenile Diabetes Foundation International (JDFI) and the Vern Bruder grant from Canadian Diabetes Association (to T.L.D.). J.Z. and K.S. were recipients of JDFI postdoctoral fellowships. Back

2 J.Z. and K.S. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Terry L. Delovitch, Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, 1400 Western Road, London, Ontario N6G 2V4, Canada. E-mail address: Back

4 Abbreviations used in this paper: ER, endoplasmic reticulum; BFA, Brefeldin A; [Ca2+]i, intracellular Ca2+; CHX, cycloheximide; DP, double-positive; JGN, Jurkat {gamma}-negative; POD, peroxidase; WT, wild-type. Back

Received for publication December 8, 1997. Accepted for publication May 12, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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