Molecular mechanisms in the TCR (TCR{alpha}ß–CD3{delta}{epsilon},{gamma}{epsilon}) interaction with {zeta}2 homodimers: clues from a `phenotypic revertant' clone

Eric P. G. Martin, Jacques Arnaud, Laeticia Alibaud, Cécile Gouaillard, Régine Llobera, Anne Huchenq-Champagne and Bent Rubin

Unité de Physiopathologie Cellulaire et Moléculaire, CNRS, ERS 1590, IFR 30 d'Immunologie Cellulaire et Moléculaire, CHU de Purpan, 31059 cedex 03 Toulouse, France

Correspondence to: B. Rubin


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The association between the TCR{alpha}ß–CD3{gamma}{epsilon}{delta}{epsilon} hexamers and {zeta}2 homodimers in the endoplasmic reticulum (ER) constitutes a key step in TCR assembly and export to the T cell surface. Incompletely assembled TCR–CD3 complexes are degraded in the ER or the lysosomes. A previously described Jurkat variant (J79) has a mutation at position 195 on the TCR C{alpha} domain causing a phenylalanine to valine exchange. This results in a lack of association between TCR{alpha}ß–CD3{gamma}{epsilon}{delta}{epsilon} hexamers and {zeta}2 homodimers. Two main hypotheses could explain this phenomenon in J79 cells: TCR–CD3 hexamers may be incapable of interacting with {zeta}2 due to a structural change in the TCR C{alpha} region; alternatively, TCR–CD3 hexamers may be incapable of interacting with {zeta}2 due to factors unrelated to either molecular complex. In order to assess these two possibilities, the TCR–CD3 membrane-negative J79 cells were treated with ethylmethylsulfonate and clones positive for TCR membrane expression were isolated. The characterization of the J79r58 phenotypic revertant cell line is the subject of this study. The main question was to assess the reason for the TCR re-expression. The TCR on J79r58 cells appears qualitatively and functionally equivalent to wild-type TCR complexes. Nucleotide sequence analysis confirmed the presence of the original mutation in the TCR C{alpha} region but failed to detect compensatory mutations in {alpha}, ß, {gamma}, {delta}, {epsilon} or {zeta} chains. Thus, mutated J79-TCR–CD3 complexes can interact with {zeta}2 homodimers. Possible mechanisms for the unsuccessful TCR–CD3 interaction with {zeta}2 homodimers are presented and discussed.

Keywords: endoplasmic reticulum, Jurkat cell, molecular chaperones, point mutation, TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The TCR{alpha}ß is a structurally variable disulfide-linked heterodimer associated with invariant components of the CD3 molecule ({gamma}, {delta}, {varepsilon}, {zeta}) on the T cell surface (1). While {alpha}ß dimers recognize peptide fragment antigens embedded in MHC molecules, the CD3 subunit transduces this recognition event into an intracellular signal, which activates the T cells (2,3). Membrane expression of TCR–CD3 complexes is tightly regulated, with only completely assembled complexes being transported through the secretory pathway to the cell surface (4). Lack of, or mutations in, any one of the chains is sufficient to stop surface expression by retaining partially assembled or unassembled TCR–CD3 components in the endoplasmic reticulum (ER) before targeting them for proteasomal or lysosomal degradation (510).

In thymocytes, normal T cells, T leukemia cells or hybridoma T cells, partial TCR–CD3 complexes are formed following a precisely regulated scheme (1113). CD3{gamma}{varepsilon} dimers associate with TCRß chains to form relatively stable molecules, whereas TCR{alpha} chains, which are rather labile, form stable complexes with CD3{delta}{varepsilon} dimers. Associations between TCR{alpha} and CD3{delta}{varepsilon} or TCRß and CD3{gamma}{varepsilon} appear to take place via the transmembrane (TM) or the extracellular regions respectively (1416). The interaction between TCRß–CD3{gamma}{varepsilon} and TCR{alpha}–CD3{delta}{varepsilon} complexes (TCR–CD3 trimers) is followed by the creation of disulfide linkages between TCR {alpha} and ß chains.

The limiting step in TCR assembly seems to be the interaction between correctly assembled TCR{alpha}ß–CD3{gamma}{varepsilon},{delta}{varepsilon} complexes (TCR–CD3 hexamers) and {zeta}2 homodimers (17,18): {zeta}2 homodimers do not associate with single TCR or CD3 chains or with partial complexes such as TCR{alpha}–CD3{delta}{varepsilon} or TCRß–CD3{gamma}{varepsilon} (11,18, 19). Thus, it would seem reasonable to assume that the interaction site of TCR{alpha}ß–CD3 hexamers for {zeta}2 homodimers requires the interaction of several molecular components (20).

Several TCR–CD3 membrane-negative variants of the human T cell leukemia line, Jurkat, have shown the importance of conserved amino acids in the Ig superfamily domain C1 (IgSF-C1) (16,18,19,21,22), in the connecting peptide (15) or in the TM region (13) of TCR C{alpha} or TCR Cß regions for the assembly of TCR{alpha}ß–CD3{gamma}{varepsilon},{delta}{varepsilon}/{zeta} complexes. The 3P11 cells have the second of the TCR Cß intrachain disulfide-forming cysteines replaced by a tyrosine (19). In the J79 cells, a phenylalanine is substituted by a valine in position 195 of the TCR C{alpha} region (TCR{alpha}FV) (18). A phenylalanine to valine exchange in the equivalent position of the TCR Cß region has the same effect. In all cases, the precise phenotype is: (i) disulfide-linked TCR{alpha}ß–CD3 hexamers are formed apparently normally, but they do not interact with {zeta}2 homodimers, (ii) the glycosylation of TCR{alpha}ß–CD3 hexamers is incomplete and the hexamers are not transported to the cis-Golgi, and (iii) the TCR ß chain is unusually stable (16,1820). Thus, one interpretation of the data would be that the mutations in the TCR C{alpha} or TCR Cß regions decrease the avidity of the hexamer–{zeta}2 homodimer interaction, i.e. the mutated amino acids are directly or indirectly (through steric interactions) implicated in the hexamer–{zeta}2 interaction (20, 22).

The precise environment for the TCR–CD3 + {zeta}2 interaction is not known (23). The formation of TCR–CD3 trimers or hexamers is catalyzed or controlled by chaperones like BIP, calnexin, CD3{omega}, glucose-regulated protein 94 or protein disulfide isomerase in the ER (2431). The specificity of interactions between chaperones and polypeptide chains is not completely defined: chaperones react with heterogeneous protein structures or certain glycoside structures on glycoproteins (29,32). If the TCR–CD3 hexamer interaction with {zeta}2 homodimers is controlled by ER chaperones, catalytic enzymes or transporter molecules, mutations in such molecules could explain the defective phenotype of 3P11 or J79 cells: the amino acid exchanges might not influence the physical interaction between TCR–CD3 complexes with mutated TCR chains (TCRM–CD3) and {zeta}2 homodimers, but could play an important role in the interaction between TCR–CD3 hexamers and a molecule(s) permitting the subsequent association with {zeta}2 homodimers. This role could involve control of the hexamer structure, the transport of hexamers from one intracellular compartment to another or facilitating hexamer interaction with {zeta}2 homodimers.

In order to distinguish between these two possibilities, we generated phenotypic revertant clones from J79 cells by treatment with ethylmethylsulfonate (EMS). The data in the present paper characterize one such TCR–CD3 membrane-positive J79 phenotypic revertant cell line. The main question asked is whether the surface expression of TCR–CD3 complexes is due to a compensatory mutation in one of the TCR–CD3 chains or in a molecule implicated in the TCR–CD3 hexamer interaction with the {zeta}2 homodimers.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells, culture and activation.
Jurkat T cells (E6.1) and the membrane TCR–CD3-negative variant J79 (18,33) were cultured in 8% FCS/RPMI 1640–glutamax medium supplemented with PSN (penicillin/streptomycin/neomycin) antibiotics (Life Technologies, Grand Island, NY) and 5x10–5 M 2-mercaptoethanol. Since the isolation of J79 cells in 1988, they have been maintained in culture for several years and neither cell cloning at limited dilution (9 times) nor cell-sorting has revealed TCR–CD3 membrane-positive revertant cells. Treatment with EMS was performed as described previously (33 ). J79 cells were transfected with a TCR V{alpha}4 C{alpha} cDNA (34) in the pMH-neo vector (35) as described previously (19). LYON cells express TCR{gamma}{delta}–CD3 complexes at the surface membrane and they contain functionally competent, intracellular TCR ß chains (22).

E6.1, J79 and revertant cells were cultured in Albumax medium for 18 h in the presence of 1 ng/ml phorbol myristate acetate (PMA), or F101.01, OKT3, JOVI or anti-Vß8 mAb (100 ng/ml). After this incubation, the cells were washed and incubated with phycoerythrin (PE)-labeled anti-CD69 mAb. CD69 expression is given as percent CD69+ cells and mean fluorescence intensity (MFI). Internalization was measured by incubating the cells with F101.01, OKT3, JOVI or anti-Vß8 mAb for 18 h. After thorough washing, cells were incubated with PE–UCHT1 anti-CD3{varepsilon} mAb and analyzed for percent CD3+ cells and MFI (13,16). For mAb, see below.

mAb, flow cytometry and cell sorting
PE-labeled UCHT1 anti-CD3{varepsilon} mAb were obtained from Dakopatts (Glostrup, Denmark). The {alpha}F1 (anti-TCR C{alpha}) and ßF1 (anti-TCR Cß) mAb were purchased from T Cell Science (Cambridge, MA); mAb against CD3{gamma} (HMT-3.2), CD3{delta} (APA-1/2), CD3{varepsilon} (SP34), {zeta} (H146.968) and TCR Cß1 (JOVI) were obtained as culture supernatants from hybridomas provided by Drs Alarcon, Terhorst, Kubo and Owen respectively. OKT3 (anti-CD3{varepsilon}) hybridoma cells were obtained from ATCC (Rockville, MD). F101.01 (anti-TCR–CD3 mAb reacting against a conformational epitope on CD3{gamma}{varepsilon} and CD3{delta}{varepsilon} dimers in association with TCR chains) producing hybridoma cells (36) were kindly provided by Dr T. Plesner (Copenhagen, Denmark). PE-labeled anti-CD69 mAb and anti-Vß8 mAb were purchased from Immunotech (Marseille, France).

Cells were washed twice in ice-cold Dulbecco's phosphate buffered saline/0.3% BSA, and then incubated with PE-labeled mAb or first mAb for 25 min at 4°C followed by washing and an eventual second incubation with FITC-labeled F(ab')2 fragments of anti-mouse Ig (Dakopatts). For cell sorting, the mAb was dialyzed and sterile-filtered before incubation with the cells. Between 106 and 5x106 cells were sorted whenever necessary, and membrane CD3+ cells were grown in bulk cultures and cloned at limited dilution. Analyses and cell sorting were carried out on a Coulter Elite cytofluorometer equipped for cell sorting.

As described previously for membrane TCR–CD3-negative Jurkat variants (37), the phenotypic variants studied in the present paper were analyzed for surface expression of CD2, CD5, CD7, CD11A, CD28, CD45, CD54 or CD58 molecules using reagents from Becton Dickinson (Mountain View, CA): all 16 revertant clones had normal expression of these membrane markers.

In certain experiments, the cells were treated with saponin buffer in order to render them permeable for mAb and FITC-labeled secondary antibodies. This was particularly important in the quantitative measurements of {zeta} chain content of the different Jurkat clones.

Biosynthetic and biotin labeling, immunoprecipitation, and SDS–PAGE
Metabolic labeling with [35S]methionine/cysteine of Jurkat T cells, solubilization (1% digitonin or 2% NP-40), immunoprecipitation with anti-TCR–CD3 mAb precoated onto Protein A–Sepharose, one-dimensional SDS–PAGE as well as deglycosylation with endoglycosidase H were performed as described previously (13,15,16). All immunoprecipitation experiments are performed under non-reducing conditions. Pulse–chase experiments were carried out as described before and the following mAb were used specifically for immunoprecipitation experiments: anti-calnexin (ref. 804-014-R100) from Alexis (San Diego, CA) or (AF8) (38) kindly provided by Dr M. Brenner (Boston, MA). Biotin labeling was performed according to the protocol described by Drs F. Lenfant (39) and T. Saito (40).

cDNA sequence analysis
Total RNA was prepared from 107 Jurkat cells by means of the guanidium isothiocyanate–cesium chloride method (41). The first-strand cDNA was synthesized with reverse transcriptase and oligo-dT primer. PCR amplification was done as described previously (13,15,16) with the primers described in Table 1Go. Amplified products from at least two different PCR were analyzed. A total of eight cDNA sequences were performed for each of apparently allelically excluded genes [TCR{alpha}, TCRß or CD3{gamma} (47)]; 12 cDNA sequence determinations were carried out with each of the non-allelically excluded genes (CD3{delta}, CD3{varepsilon} and {zeta}) (see Results).


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Table 1. Primers used for nucleotide sequence analysis
 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
General design of the experiments
The treatment of Jurkat cells with EMS was adjusted to induce single nucleotide exchanges preferentially. Thus, in J79 cells the first nucleotide, thymidine (T) in the Phe195 codon, i.e. TTC, was changed to a guanosine (G), GTC = valine (18). This nucleotide exchange seems to be the only TCR–CD3-related mutation, since TCR–CD3 structure and function is reconstituted upon transfection of J79 cells with wild-type TCR{alpha} cDNA (20). Furthermore, transfection of TCR{alpha}FV cDNA into a TCR{alpha} mRNA-negative Jurkat variant or into LYON cells generated intracellular TCR{alpha}FV chains, which form TCR{alpha}FVß–CD3 complexes but lead to no surface expression of TCR{alpha}ß–CD3 complexes (18,22).

The basic idea underlying the experiments in the present article was that induction in J79 cells of a novel mutation in any genes encoding a molecule controlling TCR–CD3->{zeta}2 association, in terms of either inter- or intramolecular interactions, could possibly compensate for the original mutation. Therefore, the J79 Jurkat variant cells were treated with EMS and analyzed by cytofluometry for surface expression of TCR–CD3 complexes. Eventual phenotypic revertant cells were cloned and analyzed with respect to TCR–CD3 assembly and TCR–CD3-mediated cell function. Finally, the compensatory mutation was chased by cDNA sequencing of TCR{alpha}, TCRß, {zeta}, CD3{delta}, CD3{gamma} or CD3{varepsilon} chains.

J79 phenotypic revertants (J79r58)
J79 cells were treated with EMS and analyzed for emergence of TCR–CD3 surface-expressing cells. As no TCR–CD3high surface-positive cells were detected, we tried to sort EMS-treated J79 cells that had the same mean fluorescence as control E6.1 cells when analyzed with PE–UCHT1 mAb (Fig. 1Go). A few thousand cells were sorted, expanded and cloned. Sixteen clones were obtained that expressed TCR–CD3 surface levels comparable to E6.1 cells (Figs 1 and 2GoGo) after a few weeks in culture. Such cells could in principle be E6.1 contaminants, back mutations of J79 TCR {alpha} chains (V->F195) or compensatory mutations. In order to distinguish among these possibilities, genomic DNA was produced from the phenotypic revertant clones and digested with HincII [J79 cell TCR{alpha} gene DNA contains an additional HincII site (18)] or BamHI (control). Southern blot analysis with 32P-labeled TCR C{alpha} probe demonstrated that all 16 revertant clones possessed the original FV195 mutation, a finding that was confirmed by nucleotide sequence analysis (see below). Seven cloning experiments with non-mutagenized J79 cells carried out during the last 10 years have not produced phenotypic revertant cells. Thus, phenotypic J79 revertant clones with surface membrane TCR–CD3 complexes were obtained and we subsequently attempted to identify putative compensatory mutation(s). It is unknown whether the 16 phenotypic revertant clones are all different or derive from a single precursor clone (see Fig. 2Go legend). The phenotypic revertant clone 58 (J79r58) was chosen for further analysis.



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Fig. 1. Selection of J79 phenotypic revertants by cell sorting and cloning at limiting dilution. The data show cytofluometrical analysis of TCR membrane-positive (mTCR+) Jurkat T cells (E6.1), of TCR membrane-negative (mTCR) variant J79 cells, of EMS-treated J79 cells, of sorted, EMS-treated J79 cells and subclones of sorted cells. In the cloning experiment, 48 clones were analyzed: 29 clones were mTCR+, three clones were weakly mTCR+ and 16 clones were mTCR. mTCR+ clones were tested three consecutive times during 2 months and 16 clones maintained the high TCR–CD3 membrane expression. Cell sorting of the variants presented here was performed 1 week after the limiting dilution step.

 


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Fig. 2. External and internal expression of TCR–CD3 components in Jurkat mutant cell lines. (A) External labeling was performed with anti-Vß8 mAb followed by FITC-labeled anti-mouse Ig antibodies. Cells were E6.1, J79, J79r58 and J79r63; the latter phenotypic revertant clone was the only (out of 16) cell line which differed from the others with J79r58 as an example. J79r63 cells grow slower than the other cell lines, and data different from J79r58 and J79r58-like cells could be due to more than one mutation. Cytofluometric data were obtained at saturating mAb conditions. Titration experiments (varying amounts of mAb) demonstrated ~2 times more TCR–CD3 surface membrane complexes on J79r58 cells compared to E6.1 or J79r63 cells. However, this result varies slightly from experiment to experiment. Wild-type and phenotypic variant cells have approximately the same number of TCR–CD3 surface membrane complexes. (B and C) Internal labeling was carried out on saponin-treated cells. Raji B lymphoma cells served as negative control. The data in (B) were obtained with 450 µl of anti-{zeta} mAb (H146) supernatants. (C) Cells were first incubated with varying amounts (450, 150, 50, 50/3, 50/10, 50/30 and 50/100 µl) of H146 followed by saturating amounts of FITC-labeled anti-hamster Ig antibodies. Conclusion: there is a comparable amount of internal {zeta} chains in the three Jurkat cell lines.

 
TCR–CD3 surface structure and signaling of J79r58 cells
J79r58 cells have been grown in culture for several months without losing their high TCR–CD3 surface expression. The level of TCR–CD3 surface expression was determined with the PE–UCHT1 mAb directed against an epitope on the CD3{varepsilon} molecule. In order to determine whether there was any difference among anti-TCR–CD3 mAb-defined epitopes on TCR–CD3 complexes of E6.1 and J79r58 cells, cytofluorometric analyses were performed with all available mAb: anti-Vß8 (recognizing the TCR Vß domain on Jurkat cells), anti-Cß [JOVI mAb recognizing an epitope on Cß1 domains and not on Cß2 domains; this epitope is destroyed when the intrachain disulfide bond is absent (19)], F101.01 [defining epitopes on CD3{gamma}{varepsilon} and CD3{delta}{varepsilon} heterodimers, only when these are associated with TCR {alpha} or ß chains (36)] and OKT3 (anti-CD3{varepsilon}) mAb. Both E6.1 and J79r58 cell TCR–CD3 complexes expressed the different mAb-defined epitopes at similar levels (Fig. 3AGo). None of the mAb reacted with J79 cells (not shown).



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Fig. 3. The TCR–CD3 complex is structurally and functionally identical on both E6.1 and J79r58 cells. (A) E6.1 (grey bars) or J79r58 (open bars) cells were incubated with 10 ng/ml, 100 ng/ml, 1 µg/ml or 10 µg/ml of the individual mAb (OKT3 and W6.32 mAb only with 1 µg), washed and then incubated with saturating amounts of FITC-labeled anti-Ig Fab2 fragments. It can be seen that E6.1 and J79r58 cells express very similar amounts of the tested epitopes (W6.32 mAb is directed against a conserved epitope of MHC class I molecules). (B) E6.1 (grey bars) or J79r58 (open bars) cells were incubated with 10 ng/ml, 100 ng/ml, 1 µg/ml or 10 µg/ml of the indicated mAb at 4°C (OKT3 and W6.32 mAb only at 1 µg/ml), then washed and incubated with PE–UCHT1 mAb (600 ng/ml). The data represent the MFI obtained with the PE–UCHT1 mAb without competitor (0) or with competitive mAb. MFI without competitor was given the value 100% and the other experimental MFI values are expressed as percentage of this 100% value. It can be seen that only OKT3 and F101.01 mAb inhibit the interaction between TCR–CD3 and PE–UCHT1 mAb on E6.1 cells. The results obtained were very similar for E6.1 and J79r58 cells, indicating that the geometry of the tested epitopes is similar on the two cells. (C) E6.1 (grey bars), J79 (black bars) or J79r58 (open bars) cells were stimulated with PMA, PHA or different amounts of mAb for 24 h at 37°C as described in Methods. The cells were subsequently washed and analyzed with PE-labeled anti-CD69 mAb. The data represent the MFI obtained with the PE-labeled anti-CD69 mAb. The value obtained after PMA activation was defined as 100% and the other MFI values are expressed as 100% of this 100% value. The results indicate that TCR–CD3-mediated T cell activation is absent in J79 cells, in contrast to both E6.1 and J79r58 cells. PMA-induced T cell activation is similar in all three Jurkat T cell lines. It should be noted that TCR–CD3-mediated T cell activation induced by PHA is dependent on membrane expression of the TCR–CD3 complex. (D) E6.1 (grey bars) and J79r58 (open bars) cells were incubated with PMA or different concentrations of PHA or F101.01 mAb for 24 h at 37°C. The cells were then washed and incubated with PE–UCHT1 mAb. The data represent the MFI obtained with the PE–UCHT1 mAb on Jurkat cells incubated in culture medium alone (defined as 100%). The experimental MFI values are expressed as percentage of this 100% value. An aliquot of the cells was incubated with FITC-labeled anti-Ig Fab2 fragments; no labeling was observed, i.e. the lack of reaction of the UCHT1 mAb was not due to competitive blockage by the mAb used for inducing the internalization. It can be seen that both the recycling internalization pathway (induced by PMA) and the anti-TCR–CD3 mAb-induced internalization followed by the degradation pathway (47) is very similar in the two cell types.

 
Next, we asked whether the geometry between these epitopes was the same on TCR–CD3 complexes from E6.1 or J79r58 cells. The cells were preincubated with `cold' anti-Vß8, anti-Cß, F101.01 or OKT3 mAb, washed and then PE–UCHT1 mAb was added. The capacity of the four non-labeled mAb to inhibit TCR–CD3 labeling by PE–UCHT1 mAb was determined. The data in Fig. 3Go(B) demonstrate that for both cell clones only OKT3 and F101.01 mAb inhibited the interaction between TCR–CD3 complexes and PE–UCHT1 mAb. We next compared the ability of E6.1, J79 or J79r58 cells to transmit signals to the interior of the cell. In the first analysis, the three cell lines were induced to express the CD69 early activation molecule on the plasma membrane. PMA stimulates T cells independently of TCR–CD3 surface expression and PHA stimulates T cells with TCR–CD3 surface complexes, as do anti-Vß8, JOVI, F101.01 or OKT3 mAb. The data in Fig. 3Go(C) demonstrate that PMA induced CD69 expression in all three cell lines. In contrast, PHA and the mAb induced CD69 expression in a similar way in E6.1 and J79r58 cells, but not in J79 cells (Fig. 3CGo).

Finally, TCR–CD3 complex internalization induced by anti-TCR reagents was investigated. As can be seen in Fig. 3Go(C), F101.01 mAb, PHA and PMA induce TCR–CD3 internalization to a similar extent in both E6.1 and J79r58 cells. Within the limits of the presented experiments, our results indicate that TCR–CD3 complexes on the surface membrane of E6.1 and J79r58 cells are similar regarding both tertiary structure and function.

Assembly of TCR–CD3 complexes in J79r58 cells
J79r58 cells express a TCR–CD3 complex that contains the J79-mutated TCR {alpha} chain. The main question is whether the TCR–CD3/{zeta} assembly and stoichiometry are the same in J79r58 cells as in E6.1 cells or whether partial TCR–CD3/{zeta} complexes are formed in J79r58 cells (45 ). Metabolic labeling of TCR–CD3 chains in J79 cells has shown that TCR–CD3 complexes do not associate with {zeta} chains. Moreover, TCR{alpha}ß heterodimers do not mature, indicating ER retention of incompletely processed TCR chains (they do not proceed beyond the high mannose form sensitive to endo-H glycosidase) and TCR ß chains are much more stable in J79 cells compared to E6.1 cells (20). Thus, E6.1, J79 and J79r58 cells were cultured for 90 min in the presence of 35S-labeled methionine/cysteine and soluble TCR–CD3 proteins from lysed cells were immunoprecipitated with mAb against the six polypeptide chains. The results in Fig. 4Go demonstrate that TCR {alpha}, ß and CD3 {gamma}, {delta}, {varepsilon} or {zeta} chains reacted normally with their respective mAb. However, {zeta} chains were co-precipitated with anti-TCR–CD3 mAb only in E6.1 or J79r58 cells, but not in J79 cells (note that the autoradiogram of J79r58 cells was obtained after 3 days of exposure, whereas the autoradiograms of E6.1 or J79 cells were obtained after 21 days). In addition, it can be seen that the high mol. wt J79-TCR{alpha}ß heterodimers are not present in J79r58 cells. Furthermore, a pulse–chase experiment shows normal maturation of TCR{alpha}ß heterodimers in J79r58 cells (Fig. 5Go; note that the exposure time for J79r58 cell immunoprecipitates was 4 days, and for E6.1 or J79 cell immunoprecipitates, the exposure time was 15 days). The results in Figs 4 and 5GoGo suggest that the TCR–CD3/{zeta} associations occur normally in J79r58 cells. In order to determine whether the TCR–CD3/{zeta} interactions in J79r58 cells take place quantitatively the same way as in E6.1 cells, [35S]methionine/cysteine-labeled TCR–CD3 proteins from E6.1, J79 and J79r58 cells were immunoprecipitated with different quantities of mAb directed against CD3{gamma}, CD3{delta}, CD3{varepsilon} chains or CD3{gamma}{varepsilon}–CD3{delta}{varepsilon} complexes. There appears to be no quantitative differences in TCR–CD3 assembly between E6.1 and J79r58 cells (not shown). Thus, the immunoprecipitation experiments did not elucidate whether the compensatory mutation has taken place in TCR or CD3 chains.



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Fig. 4. Metabolically labeled TCR–CD3 proteins immunoprecipitated from E6.1, J79 or J79r58 cells. Jurkat T cells were labeled for 90 min with [35S]methionine/cysteine, lysed in 1% digitonin and immunoprecipitated with 10 µl of a 50% suspension of Protein A–Sepharose beads saturated with different anti-TCR–CD3 mAb. Solubilized proteins were subjected to SDS–PAGE in a 10% polyacrylamide gel (non-reducing conditions). It can be seen that the immunoprecipitation patterns of E6.1 and J79r58 cells are similar, and very different from the pattern obtained with J79 cells (lack of {zeta} chain-co-precipitation and of maturation of TCR{alpha}ß heterodimers). NB: E6.1 and J79 films were exposed 3 weeks, whereas the J79r58 autoradiogram corresponds to a 3 day exposure.

 


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Fig. 5. Pulse–chase immunoprecipitation experiments with E6.1, J79 and J79r58 cells. Jurkat T cells were labeled for 30 min with [35S]methionine/cysteine, washed, and aliquots of cells were lysed in 1% digitonin after 0, 30 min, 1 h, 2 h and 4 h of culture in normal medium (tracks 1, 2, 3, 4 and 5 respectively). Immunoprecipitations were performed with 5 µl Protein A–Sepharose beads saturated with OKT3 mAb. Raji B lymphoma cells labeled metabolically for 30 min and subsequently lysed in 1% digitonin served as negative control (non-reducing conditions). Whereas both E6.1– and J79r58–TCR{alpha}ß heterodimers mature during the chase period of 4 h, the J79–TCR{alpha}ß heterodimers remain incompletely glycosylated and not associated with {zeta}2 homodimers.

 
Nucleotide sequence analysis of TCR–CD3/{zeta} chains in J79r58 cells
Karyotype analysis (Kuhlmann, unpublished) and analysis of cell cycling by cytometry have shown that Jurkat E6.1, J79 and J79r58 cells possess 46 chromosomes (2n) as compared to peripheral blood lymphocytes (also 2n) or to LYON TCR{gamma}{delta} (4n) cells (data not shown). At least two different RT-PCR were performed for each of the individual TCR–CD3 chains from total RNA of E6.1, J79 and J79r58 cells. Each resulting cDNA was cloned in the pCRII (Invitrogen) vector and prepared for nucleotide sequence analysis. The TCR {alpha} and ß chains undergo allelic exclusion: previous studies have shown that Jurkat cell RNA only hybridizes with V{alpha}1 (out of 24 TCR families) and Vß8 (out of 21 TCR families) cDNA probes (46). Moreover, Jurkat cells have been described to only express one allele of the CD3 {gamma} chain (47). Therefore, eight cDNA sequence determinations (four sequences for each PCR product) seemed reasonable to ascertain the nucleotide sequence of TCR {alpha}, TCR ß and CD3 {gamma} chains. When two alleles are expressed (in the case of CD3 {varepsilon}, {delta} and {zeta} chains), 12 sequences were analyzed (six for each PCR product) giving a probability of 1–2(0.5)12 > 99% to have analyzed two equally expressed alleles.

Nucleotide sequence analysis of V{alpha}, C{alpha}, Vß and Cß demonstrated that there were no compensatory mutations in these genes; however, the original phenylalanine->valine mutation was found in all C{alpha} sequences. The same result was obtained from sequence analysis of CD3{gamma}, CD3{delta}, CD3{varepsilon} and {zeta} cDNA in either J79 or J79r58 cells (not shown). These data suggest that the compensatory mutation has not occurred in the individual TCR–CD3 chains but possibly in an as yet unidentified molecule involved in the TCR–CD3->{zeta}2 homodimer interaction. Thus, TCR–CD3 complexes with a phenylalanine-> valine mutated TCR {alpha} chain can interact with {zeta}2 homodimers and form a functional TCR

Chaperone function in J79r58 cells
ER chaperones control proper folding and egress of newly synthesized membrane proteins (29,32,43). Three chaperones have been reported to transiently interact with TCR–CD3 subunits in T cells: BIP (25), CD3{omega} (24,27) and calnexin (26,28,30,31). As the compensatory mutation in J79r58 cells may have occured in a chaperone-like molecule, we attempted to compare chaperone behavior in J79r58 cells with that of J79 or E6.1 cells. BIP has been implicated in the assembly of TCR{alpha} chains with CD3{delta}{varepsilon} heterodimers (25). Since this interaction appears to take place normally in J79 cells, we did not pursue BIP as a possible target for the compensatory mutation.

It has been shown that the CD3{omega} interaction with CD3 complexes appears similar in E6.1 and J79 cells as judged by co-precipitation experiments achieved with anti-CD3{varepsilon} mAb (33). Several studies indicate that CD3{omega} interacts primarily with CD3{delta}{varepsilon} heterodimers (12,24,27). Therefore, J79r58 cells (with E6.1 and J79 cells as controls) were labeled with [35S]methionine/cysteine and chased for 30 or 90 min. Individual lysates were immunoprecipitated with anti-CD3{delta} (not shown) or anti-CD3{varepsilon} mAb (Fig. 6Go). OKT3 anti-CD3{varepsilon} mAb (and less strongly APA-1/2 anti-CD3{delta} mAb) co-precipitate CD3{omega} molecules labeled with [35S]methionine/cysteine for 20 min; the association of CD3{omega} with CD3{varepsilon} molecules or CD3{varepsilon}-containing complexes was stable for ~30 min (Fig. 6Go). Most importantly, these results were very reproducible. One study has indicated that CD3{omega} can physically associate with TCR–CD3 hexamers and actually be exchanged with {zeta}2 homodimers (27). Our results from experiments with normal or PMA-stimulated Jurkat cells solubilized in digitonin, NP-40 or Triton-X100 were unable to demonstrate any association between TCR–CD3 hexamers and CD3{omega}. However, CD3{omega} is associated to a similar extent to CD3{delta}{varepsilon} heterodimers or CD3{varepsilon} homodimers in E6.1, J79 or J79r58 cells (in preparation).



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Fig. 6. Co-precipitation of CD3{omega} by anti-CD3 mAb in a pulse–chase experiment with E6.1, J79 and J79r58 cells. Jurkat T cells were labeled with [35S]methionine/cysteine for 20 min at 37°C. The cells were then cultured for 0, 30 or 90 min in normal culture medium before lysis in 2% NP-40. Lysates were immunoprecipitated with anti-CD3{varepsilon} mAb as described above (non-reducing conditions). CD3{omega} is co-precipitated with TCR–CD3 proteins and stays associated with them for similar periods of time in all three cell lines.

 
Calnexin is essential for TCR–CD3 trimer formation (26,28,30,31), i.e. early in the TCR–CD3 assembly pathway. Immunoprecipitation of biosynthetically labeled lysates from E6.1, J79 and J79r58 cells with two different anti-calnexin mAb, with {alpha}F1 anti-TCR C{alpha} mAb and with OKT3 anti-CD3{varepsilon} mAb, showed little if any co-precipitation of TCR components with anti-calnexin mAb; and when present, similar results were obtained with lysates from all three cells (not shown). The conclusion from these data is that in J79 or J79r58 cells nothing seems abnormal in the initial assembly interactions leading to formation of TCR–CD3 trimers or hexamers. Actually, in all three cells, disulfide-linked TCR–CD3 hexamers are normally formed (Figs 4 and 5GoGo); the major difference being that in J79 cells the hexamers remain in the ER in an incompletely glycosylated form, whereas J79r58 hexamers associate with {zeta}2 homodimers and are transported to the plasma membrane.

It has been shown previously that {zeta}2 homodimers can be expressed on the T cell surface membrane in an autonomous way; in addition, it has been suggested that the {zeta}2 molecules may serve as transporters of complete TCR–CD3 complexes (in addition to their function in signal transduction) (40). Quantitative cytofluometric analyses using anti-{zeta} mAb demonstrated that the concentration of intracellular {zeta} chains was very similar in the three cells (Fig. 2CGo). Furthermore, the data in Fig. 7Go show clearly that all three Jurkat T cell lines express similar amounts of TCRß and {zeta}2 molecules. In order to ascertain whether TCR–CD3 hexamer->{zeta}2 homodimer interaction takes place in a similar way in E6.1 or J79r58 cells, these cells were surface biotinylated and a pulse–chase experiment was carried out (Fig. 8Go). It can be seen that in all three cell lines, {zeta}2 homodimers are well labeled at time zero and the biotinylated {zeta}2 molecules disappear from the cell surface following 2 h of in vitro culture. In addition, relabeling with biotin after 2 h of culture shows the same quantity of {zeta}2 homodimers in E6.1, J79 and J79r58 cells. Thus, the transport function of {zeta} 2 molecules seems to work normally in the three Jurkat cell lines.



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Fig. 7. Jurkat T cells were labeled for 90 min with [35S]methionine/cysteine, washed and chased for 1 h; lysis was performed in 1% digitonin. Immunoprecipitations were performed with Protein G–Sepharose beads precoated with either anti-Vß8 or anti-{zeta} mAb. SDS–PAGE was carried out in 10% polyacrylamide gels under non-reducing conditions as described in Methods. E6.1, J79 and J79r58 cells contain similar quantities of TCRß/{zeta} chains.

 


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Fig. 8. Turnover of TCR–CD3/{zeta} proteins on the surface of E6.1, J79 or J79r58 cells. Wild-type, mutant and revertant Jurkat T cells were biotin labeled (1), cultured for 2 h (2) and then relabeled with biotin (3). Cell lysates (1% digitonin) were immunoprecipitated with either anti-CD3{varepsilon} mAb or with anti-{zeta} mAb and proteins were separated in a 10% polyacrylamide gel (non-reducing conditions). Raji cells served as negative control (4). E6.1 cells were labeled with biotin and the cell lysate was immunoprecipitated with an anti-Lck antiserum (A. Alcover) (5). {zeta}2 homodimers recycle in a similar manner in wild-type and in revertant cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Newly synthesized membrane proteins fold rapidly upon entry into the ER, where they assemble with other polypeptides in order to acquire a correct quaternary structure. The ER provides an oxidizing environment that favors formation of disulfide bonds. In addition, a number of molecules that facilitate the folding and subunit assembly are present in the ER. Such molecules include soluble chaperones like BIP, protein disulfide isomerase and glucose-regulated protein 94, and chaperones that are integral components of the ER membrane (e.g. calnexin) (32). Chaperones may have at least three functions: (i) quality control over protein folding, e.g. retention of proteins that are incorrectly folded or delayed in acquiring their native structure, which might prolong their retention in the ER, (ii) a provision of increased energy for protein–protein interactions, i.e. a kind of enzyme activity, and (iii) a transporter function, i.e. increasing the concentration of interacting components in a particular subcompartment of the ER (4,25,27,2931). Calnexin and calreticulin possess a lectin-like domain, that has specificity for oligosaccharide moieties characteristic of incompletely folded glycoproteins (26,30,31,32,43). Other chaperones, while interacting with non-assembled proteins, dissociate rapidly as new polypeptides assemble to the complex (BIP and CD3{omega}) (24,25). In TCR–CD3 assembly, BIP, calnexin, calreticulin, CD3{omega} and many others appear to play important roles in TCR–CD3 trimer and hexamer formation (30,31,32,38,43,4850).

The data presented in this paper bear on the mechanism of assembly and membrane expression in phenotypic revertant cells derived from a TCR–CD3 membrane-negative variant J79 (20). This TCR–CD3 surface membrane-negative Jurkat T cell variant has a phenylalanine->valine exchange in position 195 of the external TCR C {alpha} region; this defect causes production of partially glycosylated TCR{alpha}ß–CD3 hexamers that do not interact with {zeta}2 homodimers (20). Many experiments have indicated that the Phe195 of TCR C{alpha} and the equivalent phenylalanine in TCR Cß regions are involved in a molecular interaction site for {zeta}2 homodimers (18,20,21). However, the crystal structure of the TCR{alpha}ß heterodimers (51,52,53) show that the phenylalanine in question is situated in a hydrophobic pocket facing the TCR V region rather than the plasma membrane. Since {zeta} chains have only nine external amino acids, these observations made the hypothesis of a mutated interaction site for {zeta}2 on the C{alpha}/Cß region unlikely. It appears now clear that TCR{alpha}FVß–CD3 hexamers can interact with {zeta}2 homodimers. Moreover, those mutant TCR{alpha}FVß– CD3/{zeta}2 complexes on the J79r58 cell surface have apparently the same structure, epitope expression or geometry and signaling function capacities as TCR–CD3 complexes on wild-type Jurkat T cells. Most importantly, the compensatory mutation permitting TCRM–CD3 membrane expression on J79r58 cells did not occur in any of the TCR–CD3 chains: {alpha}, ß, {gamma}, {delta}, {varepsilon} or {zeta}. Thus, with our present state of knowledge, it is likely that the compensatory mutation has occurred in a chaperone-like molecule(s), which normally controls the TCR–CD3 hexamer interaction with {zeta}2 homodimers. The J79 phenylalanine->valine mutation causes lack of or inefficient interaction between TCRM–CD3 hexamers and the hypothesized chaperone, rather than between TCRM–CD3 hexamers and {zeta} 2 homodimers.

Both calnexin, calreticulin and CD3{omega} have been implicated in prolonged interactions with TCR–CD3 complexes. Thus, calnexin seems to interact transiently with all TCR–CD3 components except {zeta} chains (12,26,28,30,31), whereas calreticulin reacts transiently only with TCR{alpha} or TCRß chains (30,31,32). Our analysis of calnexin association with TCR–CD3 components in E6.1, J79 or J79r58 cells showed no significant differences; in contrast, calnexin is indeed strongly associated with TCR ß chains in the TCR {alpha} chain-negative JR3.11 variant cells (16). These data indicate that calnexin is not involved in the defective TCRM–CD3->{zeta}2 interaction in J79 cells.

Most data available suggest that CD3{omega} plays a role early in the TCR–CD3 assembly pathway, in particular in CD3 heterodimer and TCR–CD3 trimer formation (12,24,27,49,50). However, one study suggests that CD3{omega} may stay associated with TCR–CD3 complexes until the interaction with {zeta}2 homodimers; in addition, it has been suggested that the TCR–CD3->{zeta}2 interaction may dissociate CD3{omega} from the TCR–CD3 complexes (27). Therefore, we analyzed possible differences in the interaction between CD3{omega} and TCR–CD3 components among E6.1, J79 or J79r58 cells. The results of these experiments carried out in different detergents showed no evidence of differential CD3{omega} interactions with TCR–CD3 components from E6.1, J79 or J79r58 cells. The suggested CD3{omega} displacement by {zeta}2 homodimers could not be demonstrated despite several attempts. On the contrary, CD3{omega} seems to be mostly involved in the control of CD3{delta}->CD3{varepsilon} and TCR{alpha}->CD3{delta}{varepsilon} interactions like BIP (25). As these interactions seem normal in J79 cells, it could be expected that CD3{omega} is not a possible candidate for the mutated chaperone in J79r58 cells. Nevertheless, the above described experiments were carried out due to the data published by Neisig et al. (27) and due to the observation that in J79r58 cell lysates, anti-CD3{varepsilon} mAb co-precipitates more CD3{omega} compared to wild-type Jurkat T cells (not shown).

The {zeta} chain is an important molecule for TCR–CD3 membrane expression, for signal transduction and for thymocyte differentiation (2,17,54,55). The amount of intracellular {zeta} chains seems to be an essential control point for TCR–CD3 complex activities. Intracellular cytometry analysis on saponin-treated E6.1, J79 or J79r58 cells, as well as quantitative immunoprecipitation studies, showed similar amounts of {zeta} chains in all three cell lines. The concentration of {zeta} chains appears to be regulated by two mechanisms: limited {zeta} chain biosynthesis (17,54) and {zeta} chain recycling from the surface membrane (40,56). Thus, whereas TCR–CD3 complexes seem stable at the T cell surface, {zeta} chains have a half-life of <2 h at the surface membrane. However, dissociation of biotinylated {zeta} chains from TCR–CD3 complexes on E6.1 or J79r58 cells was similar (Fig. 8Go). Therefore, TCR–CD3 hexamers in J79r58 cells appear to assemble and to be transported by {zeta}2 homodimers to the cell surface in the same way as in E6.1 cells. In addition, it seems likely that {zeta}2 homodimers recycle normally in J79 cells compared to E6.1 or J79r58 cells. This means that {zeta}2 homodimers are expressed autonomously at the T cell surface even without TCR–CD3 complexes (40).

The conclusion from the experimental data with J79r58 cells is (i) TCR{alpha}FVß–CD3 complexes can interact physically with {zeta}2 homodimers, (ii) the quantity and recycling function of {zeta} chains is normal, and (iii) TCR{alpha}FVß–CD3/{zeta} complexes on the cell surface are functional. The J79-TCR–CD3 function can be reconstituted by transfection with wild-type TCR{alpha} cDNA, and {zeta} chain turnover and function is normal in J79 cells.

Thus, without changing the cDNA sequences of any of the {alpha}, ß, {gamma}, {delta}, {varepsilon} or {zeta} transcripts in J79r58 cells, these cells express functional TCR–CD3 complexes at the cell surface membrane. It would appear logical then to suggest that TCR{alpha}FVß–CD3 complexes and {zeta}2 homodimers can interact physically in J79 cells but they never get a chance to do so, because the two partners are present in two different intracellular compartments. Preliminary confocal and electron microscopy experiments indicate that TCR–CD3 hexamers and {zeta} chains are separate in J79 cells; in contrast, TCR–CD3 hexamers and {zeta} chains overlap in E6.1 cells, J79r58 cells and J79 cells transfected with TCR{alpha} cDNA (in preparation). Therefore, we suggest that the compensatory mutation in J79r58 cells has happened in an intracellular molecule that (i) controls the tertiary structure of TCR–CD3 complexes before interaction with {zeta} chains, (ii) catalyzes the TCR–CD3->{zeta}2 homodimer interaction or (iii) transports TCR–CD3 complexes to the {zeta}2 recycling compartment.

In summary, we have described a phenotypic revertant T cell clone J79r58, which has a compensatory mutation in a molecule behaving as an intracellular chaperone. This new chaperone-like function, which catalyzes the TCR–CD3->{zeta}2 homodimer interaction, could not be attributed to calnexin, to CD3{omega} or to {zeta}2 homodimer recycling. We are now attempting to clone the mutated chaperone cDNA by transfection of an expression cDNA library from J79r58 cells into J79 variant Jurkat T cells.


    Acknowledgments
 
The collaboration of Drs F. Lenfant, M. Brenner and J. Kuhlmann during parts of this work is gratefully acknowledged. Many thanks are sent to Dr W. R. Clark (Los Angeles) for comments to and for reading the manuscript. We want to express our gratitude for help with cell sorting from H. Brun and G. Cassar. Finally, we thank our colleagues Drs B. Alarcon, M. Brenner, R. Kubo, M. Owen, T. Plesner and C. Terhorst for providing mAb or mAb-producing hybridomas. The present work was supported by the CNRS, l'Université Paul Sabatier, l'ARC (no. 6223), la Ligue Régionale contre le Cancer (Toulouse) and Immunotech (Marseille).


    Abbreviations
 
Cconstant region
EMSethylmethylsulfonate
ERendoplasmic reticulum
FVphenylalanine->valine exchange
MFImean fluorescence intensity
PBLperipheral blood lymphocyte
PEphycoerythrin
PHAphytohemagglutinin A
PMAphorbol myristate acetate
TMtransmembrane
TCRMTCR heterodimer with either TCR {alpha} or ß chains mutated in the TCR C{alpha}-Phe195 equivalent position
Vvariable region

    Notes
 
Transmitting editor: M. Reth

Received 4 November 1998, accepted 9 March 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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