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Original Article |
Chain: Potential Role in the Regulation of Cell Surface Pre-Tcr Expression Levels
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.34-91-397-808734-91-397-8076
mtoribio{at}cbm.uam.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(pT) and associated with CD3/
components, is a critical regulator of T cell development. For unknown reasons, extremely low pre-TCR levels reach the plasma membrane of pre-T cells. By transfecting chimeric TCR-–pT proteins into pre-T and mature T cell lines, we show here that the low surface expression of the human pre-TCR is pT chain dependent. Particularly, the cytoplasmic domain of pT is sufficient to reduce surface expression of a conventional TCR-/β to pre-TCR expression levels. Such reduced expression cannot be attributed to qualitative differences in the biochemical composition of the CD3/ modules associated with pre-TCR and TCR surface complexes. Rather, evidence is provided that the pT cytoplasmic tail also causes a reduced surface expression of individual membrane molecules such as CD25 and CD4, which are shown to be retained in the endoplasmic reticulum (ER). Native pT is also observed to be predominantly ER localized. Finally, sequential truncations along the pT cytoplasmic domain revealed that removal of the COOH-terminal 48 residues is sufficient to release a CD4-pT chimera from ER retention, and to restore native CD4 surface expression levels. As such a truncation in pT also correlates with enhanced pre-TCR expression, the observed pT ER retention function may contribute to the regulation of surface pre-TCR expression on pre-T cells.
Key Words: human pre–T cell receptor • pT cytoplasmic tail • surface expression • endoplasmic reticulum retention • CD3 complex
In contrast to TCR-
Although the precise biochemical composition of the pre-TCR has remained elusive until recently, evidence has now been provided that, in the mouse, it has the same subunit composition as does the TCR-
Flow Cytometry.
Generation of cDNA Constructs.
Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Development of mature /β T cells inside the thymus is controlled at two distinct check-points by the sequential expression of the pre-TCR and the mature TCR-/β 123. Early in T cell development, pre-T cells that succeed in productive TCR-β gene rearrangements express a functional TCR-β chain which pairs with the invariant pre–TCR- (pT) chain and associates with CD3 components to form the pre-TCR 45678. Signaling through this pre-TCR complex triggers a process, known as TCR-β selection, which induces the cellular expansion and maturation of CD4– CD8– double negative (DN) pre-T cells into CD4+CD8+ double-positive (DP) thymocytes 2391011, and results in the induction of a high rate of TCR- gene rearrangements 12. On productive TCR- gene rearrangements and substitution of pT by TCR-, the TCR-/β is expressed associated with CD3, and DP thymocytes can undergo a second step of selection, known as TCR-/β selection, during which thymocytes are rescued from programmed cell death and induced to differentiate into conventional single positive (SP) thymocytes, upon binding to self-peptide–MHC complexes expressed on thymic stromal cells 3. /β selection, current data support the view that TCR-β selection is independent of binding to an extracellular (EC) ligand 1314 and no evidence for the existence of such a ligand has so far been provided. However, exit from the endoplasmic reticulum (ER) and expression at the cell surface is mandatory for the pre-TCR complex to exert its function 15, although rules controlling the assembly and intracellular transport of pre-TCR and TCR complexes may differ markedly, as the pre-TCR is expressed only transiently during thymocyte development, and at extremely low levels, 
50–100-fold lower than those of the TCR-/β on mature T cells 11316. The inefficient expression of pre-TCR complexes on pre-T cells is reminiscent of the inefficient expression of the pre–B cell receptor (BCR) on pre-B cells 117, and might be related to the particular signaling function of pre-TCR/BCR complexes during lymphoid development. The question is, therefore, whether such differences in surface expression levels are determined by specific structural properties of the receptors or are intrinsic to the particular developmental stage at which the receptors are expressed. /β complex, differing only in that the TCR- subunit has been replaced by pT and that association has been significantly weakened relative to its association with the mature TCR-/β 47818. Because is required for efficient surface expression of the other subunits of the mature TCR-/β including the CD3 molecules 192021222324, its weak biochemical association with the pre-TCR could well result in a decreased stability of the receptor complex at the cell surface. An alternative possibility is that, as reported for the pre-BCR, surface pre-TCR expression is controlled by a retention mechanism that is not selective for the pre-TCR, but is inherent to the pre-T cell stage 117. In this study, we have examined this issue by performing transfections of human chimeric TCR-–pT molecules comprising distinct domains of TCR- and pT into either pre-T or mature T cell lines, and provide evidence that low surface expression of the human pre-TCR is intrinsic to the pT chain. Particularly, our results show that the cytoplasmic (Cyto) domain of pT is sufficient to promote retention in the ER and to reduce surface expression levels of individual transmembrane (TM) proteins. The possibility that the pT Cyto tail functions as an ER retention signal that contributes to the regulation of pre-TCR expression levels on pre-T cells is discussed.
Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Isolation of Thymocyte Subsets.
Postnatal thymocytes isolated from thymus samples removed during corrective cardiac surgery of patients aged 1 mo to 3 yr were fractionated by centrifugation on Percoll (Amersham Pharmacia Biotech) density gradients as described 25. pre-TCR/CD3low pre-T cells were isolated from the large-sized cell fraction by immunomagnetic sorting (Dynal) as described 16.
PE-labeled anti-CD3 (Leu-4-PE), biotin-conjugated anti-CD25 (7D4), and Cy5-PE-labeled anti-CD4 mAbs were obtained from Becton Dickinson, BD PharMingen, and Caltag Laboratories, respectively. The BMA031 mAb, raised against a monomorphic determinant of TCR-/β 26 was provided by Dr. R. Kurrle (Behringwerke AG, Marburg, Germany), and the 6D6 mAb, against the human V12.1 TCR- domain 27, was the gift of Dr. M. Brenner (Brigham and Women's Hospital, Boston, MA). Surface expression of the human pT chain was determined by staining with a rabbit antiserum (ED-1) raised against a synthetic peptide present in the pT EC domain 16. PE-labeled streptavidin and goat anti–mouse fluorescein-, PE-, or PE-Cy5–coupled F(ab')2 Igs were purchased from Caltag Laboratories. Stained cells were analyzed in a flow cytometer (EPICS XL; Coulter Corp.) as described 25. For flow cytometry, COS cells were removed from culture 48 h after transfection with PBS containing 0.02% EDTA.
Full-length cDNAs encoding the human pT and the AV12S1 TCR chain, respectively, were cloned into the BamHI site of the pcDNA3 plasmid vector (Invitrogen), as described previously 16. TCR-–pT chimeric constructs (see Fig. 2 A) comprising the V12.1 and J domains of the AV12S1 TCR chain fused to pT cDNA lacking the leader sequence (I), or encoding a AV12S1 TCR chain in which either both the TM and Cyto domains (II) or exclusively the Cyto domain (III) of TCR- have been replaced with homologous domains from pT, were generated by PCR using TCR- and pT cDNAs as templates, and specific oligonucleotides. The sense 5'-ATG GAT CCT CTA GAT GAT TTT TGC CAG CCT GTT G-3' primer (with a BamHI site), corresponding to the NH2-terminal region of the AV12S1 TCR chain 16, was used in combination with three distinct antisense primers: 5'-CCC GGA TCC TGG TGC CTG TTC CTG TTC-3' (with a BamHI site), 5'-CGG AAT TCG GTT TTG AAA GTT TAG GTT CG-3' (with an EcoRI site), or 5'-CGG AAT TCC CAG CGT CAT GAG CAG ATT AAA-3' (with an EcoRI site), which correspond to the COOH-terminal region of either the J, the EC, or the TM domains of the AV12S1 TCR chain, respectively. PCR products were either BamHI or BamHI/EcoRI digested, and ligated into pcDNA3 plasmid containing, respectively, a BamHI/XhoI PCR fragment coding for pT lacking the leader sequence, an EcoRI/XhoI PCR fragment encoding both the TM and Cyto pT domains, or an EcoRI/XhoI PCR fragment encoding the Cyto tail of pT. Such pT PCR products were amplified from pT cDNA with the antisense 5'-CCG CTA ACG AGT CAG GCA GCA GCT CCA GCC TGC AG-3' primer, corresponding to the COOH-terminal region of the pT Cyto domain, and containing an XhoI site, in combination with the sense primers 5'-CCC-GGA TCC ATA TGC TAC CCA CAG GTG TGG GC-3', including a BamHI site, or 5'-CGG AAT TCT GTG GCT GGG GGT CCT GCG-3', with an EcoRI site, or 5'-CGG AAT TCT TAC CTG CAG CTG CCT GTG CG-3', including an EcoRI site, respectively.
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and CD4-pT chimeric constructs, comprising the complete EC and TM domains of CD25 or CD4 fused to the pT Cyto tail, were generated by PCR amplification. CD25 PCR products obtained with the sense 5'-ATG GAT CCA AGA TGG AGC CAC GTC TGC TG-3' primer and the antisense 5'-GCG AAT TCC CCA GGT GAG CCC GCT CAG G-3' primer were digested with BamHI/EcoRI, and CD4 PCR products obtained with the sense 5'-AGA GAG AGA GAG AAG CTT TCG GCA AGG CCA CAA TGA AC-3' primer and the antisense 5'-GCG AAT TCC GAA GAA GAT GCC TAG CCC AAT G-3' primer were digested with HindIII/EcoRI. Digested PCR products were independently ligated into pcDNA3 containing the EcoRI/XhoI PCR fragment encoding the pT Cyto tail described above. Truncated CD4-pT chimeras lacking either the COOH-terminal 22 (CD4-pTt22) or 48 (CD4-pTt48) residues of the pT Cyto tail were generated essentially as described for CD4-pT chimeric constructs, except that pT PCR was performed with the sense primer corresponding to the NH2-terminal region of the pT Cyto domain described above, in combination with the antisense 5'-AGA GAG AGA GAG GAT ATC TCA AGC CCT GAG GCG AGA TCT TG-3' or 5'-AGA GAG AGA GAG GAT ATC TCA TAC TGG GCT CCC GGG CTT C-3' primers, for CD4-pTt22 and CD4-pTt48, respectively. EcoRI/EcoRV-digested PCR products were then ligated into pcDNA3 containing the HindIII/EcoRI-digested CD4 product described above. These antisense primers were independently used with a previously described sense primer 16 corresponding to the NH2-terminal pT region to generate truncated pT proteins with identical deletions in the Cyto tail (pTt22 and pTt48). EcoRI/EcoRV-digested products were independently ligated into pcDNA3. Both cDNAs as well as full-length pT and TCR- cDNAs 16 were subsequently subcloned into the bicistronic expression vector pCIGFP. To construct the pCIGFP plasmid, a NotI cassette containing an internal ribosomal entry site (IRES) sequence followed by the enhanced green fluorescent protein (EGFP) cDNA was transferred from the pLZRS retroviral vector 29, provided by Dr. H. Spits (Netherlands Cancer Institute, Amsterdam, The Netherlands), into the NotI site in the pcDNA3 vector. Tailless pT cDNA was generated by amplification with the sense primer described previously 16 in combination with the 5'-GGG GGA TCC TAC AGG AGC AGG TCA AAC AG-3' antisense primer, BamHI digested, and ligated into the pCIGFP bicistronic plasmid. Each construct was sequenced directly in pcDNA3. The pT-EGFP construct has been described previously 16.
Cell Lines and Transfection.
The TCR-–deficient JR3.11 mutant, derived from the mature human T cell line Jurkat (30; provided by Dr. B. Rubin, Centre National de la Recherche Scientifique, Toulouse, France), the human pre-T cell line SUP-T1 31, and the murine lymphoid cell line BW, were grown in RPMI 1640 (Biowhittaker) supplemented with 10% FCS (GIBCO BRL). COS cells were grown in DMEM (Biowhittaker) supplemented with 5% FCS (GIBCO BRL). Transfections were carried out by electroporation as described 16.
Generation of Polyclonal Anti–Human pT Abs.
A rabbit antiserum (CT-1) was generated against a synthetic peptide corresponding to the human pT sequence 200–212 present in the Cyto domain 25, essentially as described previously for the ED-1 antiserum 16. The specificity of the CT-1 antiserum was assayed by immunofluorescence microscopy of COS cells transfected with a pT cDNA tagged with a c-myc epitope recognized by the specific 9E10 mAb (unpublished results).
Cell Surface Radioiodination, Immunoprecipitation, and N-Glycosidase F Digestion.
Cells (107) were washed with PBS, resuspended in 0.5 ml of PBS containing 0.5 mg/ml of sulfo-succinimidyl-3-(4-hydroxyphenyl) propionate (SHPP) (Boltont-Hunter reagent; Pierce Chemical Co.), and incubated on ice for 30 min. The reaction was stopped by diluting the cells with 10 ml of 10 mM L-lysine (Sigma-Aldrich) in PBS. Cells were centrifuged, resuspended in 150 µl of PBS, and 125I-labeled by the lactoperoxidase method. In brief, 1 mCi Na125I (Amersham Pharmacia Biotech) and 30 µl of 140 IU/ml lactoperoxidase (Sigma-Aldrich) solution were added to the cells, and 10 µl aliquots of 0.06% H2O2 solution were then added three times, at 7-min intervals. The reaction was stopped by adding 20 mM KI (Sigma-Aldrich) and 1 mM L-tyrosine (Sigma-Aldrich) in PBS. Subsequently, the cells were lysed in 1% Brij 96–containing (Sigma-Aldrich) lysis buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 10 mM iodoacetamide, 1 mM PMSF, and 1 µg/ml each of leupeptin, pepstatin, and aprotinin) and centrifuged for 14,000 rpm for 30 min at 4°C. The supernatants were precleared three times with normal mouse serum (NMS) Igs coupled to protein A/G-Sepharose beads (Amersham Pharmacia Biotech) and subjected to immunoprecipitation with the indicated Abs coupled to protein A/G-Sepharose beads. The following Abs were used: the 6D6 anti–human TCR V12.1 mAb 27; the UCHT1 anti–human CD3
mAb 32; the 448 anti–human TCR- rabbit antiserum, generously provided by Dr. B. Alarcón (Centro de Biología Molecular Severo Ochoa, Madrid, Spain); and the CT-1, anti–human pT rabbit antiserum, generated in this study. The immunoprecipitates were washed five times with 1% Brij 96–containing lysis buffer and, when indicated, digested overnight with N-glycosidase F (N-Gly; 0.2 U/sample; Roche Diagnostics). For analysis, the immunoprecipitates were separated by SDS-12% PAGE under nonreducing conditions or, alternatively, by two-dimensional gels using SDS-10% polyacrilamide tube gels in the first dimension (nonreduced), which were subsequently resolved by SDS-12% PAGE in the second dimension (reduced).
Immunofluorescence and Confocal Microscopy.
48 h after transfection, COS cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin in PBS containing 1% BSA, and stained either with the PC61 fluorescein-labeled anti–mouse CD25 mAb (BD PharMingen), or with the HP2.6 anti–human CD4 mAb (provided by Dr. F. Sánchez-Madrid, Hospital de la Princesa, Madrid, Spain), followed by goat anti–mouse fluorescein-coupled F(ab')2 Igs (Caltag Laboratories). The coverslips were viewed using a Radiance 2000 confocal microscope (Bio-Rad Laboratories).
For confocal analysis, pT-GFP SUP-T1 stable transfectants were adhered to Poly L-Lys (Sigma-Aldrich) precoated coverslips (5 x 105 cells/coverslip). Coverslips were then washed in PBS, fixed with 2% paraformaldehyde in PBS for 10 min, permeabilized for 5 min with 0.05% Triton X-100 (Sigma-Aldrich), and blocked with 2% BSA/PBS. Cells were then consecutively stained with the anti–TCR-β βF1 mAb (provided by Dr. M. Brenner) in combination with Cy5-conjugated goat anti–mouse Igs (Jackson ImmunoResearch Laboratories), and with a rabbit antiserum against the ER resident protein PDI (protein disulfide isomerase; StressGen Biotechnologies) plus Cy3-conjugated goat anti–rabbit Igs (Jackson ImmunoResearch Laboratories). Confocal microscopy was performed on a Radiance 2000 (Bio-Rad Laboratories) system coupled to an Axiovert S100TV inverted microscope (ZEISS). Fluorescein and EGFP, Cy3, and Cy5 fluorescence were detected using bandpass filter HQ515/30, longpass filter HQ600/50, and longpass filter HQ660LP, respectively.
Metabolic Labeling, Immunoprecipitation, and Endoglycosidase H Treatment.
48 h after transfection, COS cells were rinsed in PBS twice and incubated for 30 min in DMEM without L-cysteine and L-methionine. Then, 500 µCi [35S]methionine/cysteine (Amersham Pharmacia Biotech) were added, and cells were pulsed for 30 min followed by chase periods, in the presence of DMEM supplemented with 10% FCS. Finally, the cells were washed in PBS, lysed in 1% Triton X-100 (Sigma-Aldrich) lysis buffer, and immunoprecipitated either with the PC61 anti–mouse CD25 mAb (BD PharMingen), with the CT-1 rabbit antiserum against the human pT tail, or with the HP2.6 anti–human CD4 mAb. The immunoprecipitates were digested overnight with endoglycosidase H (endo-H; Roche) or left undigested, and resolved by SDS-12% PAGE under reducing conditions. Signal intensity was quantitated by densitometry (Bio-imaging BAS 1500; Fujifilm).
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Chain Dependent. antiserum and an anti-CD3 mAb 16, the human pre-TCR is expressed on the surface of primary pre-T cells at very low levels, compared with expression levels of the mature TCR-/β on SP thymocytes (Fig. 1 A) or peripheral T cells (not shown). Low pre-TCR expression such as that found on primary pre-T cells was detected also on a pre-T cell line, SUP-T1 (Fig. 1 B), which expresses TCR-β and pT chains but lacks TCR- 31. It is thus possible that, as proposed for the pre-BCR 117, the pre-TCR is expressed at low surface levels due to regulatory mechanisms that operate selectively at early developmental stages. In that case, one would expect that only limited amounts of a conventional mature TCR-/β could reach the plasma membrane of pre-T cells. In contrast, we found that the introduction of a conventional TCR chain (V12.1) into SUP-T1 pre-T cells (wt) resulted in a reproducible increase (15-fold) in surface expression levels of CD3, presumably through the formation of heterodimers composed of the transfected TCR- paired with the endogenous TCR-β and associated with CD3. Accordingly, CD3 was found coexpressed in stoichiometric amounts with the TCR-/β on stable SUP-T1 wt transfectants (Fig. 1 B).
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chain, whereas increased TCR-/β surface density on wt transfectants could be the result of TCR- overexpression. To rule out that possibility, a pT–GFP chimeric construct 16 was introduced into SUP-T1 cells, and surface expression of CD3 was analyzed by flow cytometry on stable pT–GFP transfectants traced by their GFP expression. As shown in Fig. 1 C, GFP+ cells displayed surface CD3 levels that were indistinguishable from those observed on nontransfected SUP-T1 cells, but still 15-fold lower than CD3 levels on wt transfectants. Moreover, surface CD3 was coexpressed with the pT–GFP chimeric protein (Fig. 1 B) and with the endogenous TCR-β chain 16 in stoichiometric amounts, indicating that pT overexpression does not alleviate endogenous pre-TCR components from the regulatory mechanisms that control their limited expression on the surface of pre-T cells. We thus concluded that pT amounts are not rate limiting for the assembly and expression of the pre-TCR on the surface of SUP-T1 pre-T cells.
Reciprocal experiments aimed at analyzing surface pre-TCR expression levels on mature T cells were then performed by introduction of pT or TCR- into a TCR-–deficient mutant (JR3.11) derived from the mature T cell line Jurkat 30. As shown in Table , low pre-TCR and high TCR-/β surface expression levels, identical to those observed on SUP-T1 pre-T cells, were consistently detected on JR3.11 T cells. These data indicate that differences in surface expression levels of the pre-TCR and the TCR are not intrinsic to the particular cell type in which these receptors are expressed, but may be due to the presence of a pT chain in the former and of a TCR chain in the latter.
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Is Sufficient to Reduce Surface Expression of a Conventional TCR Chain to pT Expression Levels. molecule that might account for the low surface expression of the pre-TCR, TCR-–pT chimeric chains were generated as illustrated in Fig. 2 A, by replacing the Cyto, TM, and EC Ig-like constant (C) domains of a TCR chain bearing a V12.1 domain with homologous domains from the pT chain. The chimeric constructs were introduced into SUP-T1 pre-T cells, and the resulting stable transfectants were assayed by flow cytometry for surface expression of the chimeric TCRs using a mAb against their shared V12.1 domain. Cells stably transfected with the wild-type TCR chain (wt) were analyzed in parallel to control surface expression levels of conventional TCR-/βs. After screening of up to 70 stable transfectants of each type, no surface expression of the I chimera consisting of a pT chain lacking the peptide leader fused to the V12.1 and J domains of TCR- was found (Fig. 2 B). However, the I chimeric protein could be detected intracellularly in COS transfectants (data not shown), thus confirming that the TCR- modification had not adversely affected protein expression. Therefore, it remains possible that the I chimera does not associate properly with TCR-β, preventing its surface expression and/or detection. Surprisingly, TCR-–pT chimeras in which both the TM and Cyto domains (II) or exclusively the Cyto domain (III) of TCR- were replaced with the equivalent pT domains were found expressed at the cell surface, although at levels that were reproducibly 10–15-fold lower than those of wt (Fig. 2 C), but comparable to those of endogenous pT or transfected pT–GFP chimeric chains (Fig. 1 C). Importantly, when the wt and the II and III chimeric chains were introduced into the TCR-–deficient JR3.11 mature T cell line, their relative surface expression levels were identical to those expressed on SUP-T1 pre-T cells (Table ). Taken together, these data provide evidence that replacing the Cyto domain of TCR- with the pT Cyto tail is sufficient to reduce TCR chain surface expression (and, hence, expression of the chimeric TCR, see below) to conventional pT chain expression levels, whereas the TM domains of the TCR- and pT chains were equivalent for surface expression of the TCR-–pT chimera.
CD3/ Components Are Physically Associated in the Wild-type Pre-TCR as well as in the TCR-–pT Chimeric TCRs.
Three-color flow cytometry showed that the wild-type TCR chain and the II and III (I was not studied further) chimeric proteins were expressed at the cell surface in stoichiometric amounts with TCR-β (Vβ1.1) and CD3 (data not shown), indicating that surface expression of wt, II, and III chains must be achieved by pairing them with the endogenous TCR-β chain and CD3/ components. Therefore, the cyto tail of pT appears sufficient to reduce surface expression of the chimeric TCR complex to pre-TCR expression levels. To assess whether this effect could be attributed to defective TCR–CD3/ interactions, we directly compared the biochemical composition of chimeric TCRs with that of conventional TCR-/βs, and pre-TCR complexes containing a wild-type pT chain. To this end, the wt, II, and III stable transfectants were surface iodinated, treated with 1% Brij 96–containing lysis buffer, and surface TCRs were immunoprecipitated from cell lysates with a mAb specific for their shared V12.1 domain. The untransfected SUP-T1 pre-T cell line was simultaneously labeled and the endogenous pre-TCR complex was immunoprecipitated from cell lysates with a rabbit antisera (CT-1) raised against a synthetic peptide contained in the Cyto region of the human pT molecule. Specificity of the affinity-purified Ab was confirmed previously by immunofluorescence microscopy of COS cells transfected with a c-myc–tagged pT chain (data not shown). Subsequently, the immunoprecipitates were mock treated or treated with N-Gly, in order to distinguish between CD3 and CD3
chains, and resolved by SDS-PAGE under nonreducing conditions. As shown in Fig. 3, a conventional TCR-/β heterodimer of 90 kD was coimmunoprecipitated with CD3
from the surface of wt transfectants, whereas fewer CD3-associated chimeric TCRs of an apparent MW of 115 kD were immunoprecipitated from both II and III transfectants. N-Gly treatment of the immunoprecipitated proteins resulted in an increase in their electrophoretic mobility by elimination of their glycosidic component, and revealed no qualitative biochemical differences in the composition of the CD3 modules associated with conventional and chimeric TCRs, as all , , and CD3 components were reproducibly precipitated with both heterodimers. Similarly, CD3, CD3, and CD3 chains were all associated with a pT-containing complex of 100 kD immunoprecipitated from the surface of untransfected SUP-T1 pre-T cells, corresponding to the endogenous pre-TCR. However, coprecipitation of chain components either with the pre-TCR or with the chimeric or the conventional TCR-/βs was not detected under these experimental conditions, likely reflecting poor chain iodination due to its short EC domain 33.
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chain was physically associated with the pre-TCR and the chimeric TCRs as with the conventional TCR-/βs, cell lysates from surface-iodinated SUP-T1 pre-T cells and wt and III transfectants were immunoprecipitated in parallel either with a rabbit anti–human chain antiserum or with an anti-CD3 mAb, and the immunoprecipitates resolved by two-dimensional nonreducing/reducing SDS-PAGE. As shown in Fig. 4, the anti-CD3 mAb coprecipitated the TCR-/β, as well as the III chimeric TCR, and the pre-TCR with CD3, whereas no chain was detected. However, the chain was detected as an individual protein migrating out of the diagonal with an apparent MW of 16 kD in anti- immunoprecipitates. Interestingly, in all samples the anti- Ab coprecipitated an heterodimeric complex together with CD3, indicating that the chain is physically associated at the cell surface with the pre-TCR and with the III chimeric TCR, as observed with the mature TCR-/β. Furthermore, it is worth noting that the wild-type pre-TCR was precipitated with both the anti-CD3 and anti- Abs not only from the untransfected SUP-T1 cells, but also from the surface of wt and III SUP-T1 transfectants (Fig. 4), indicating that the conventional TCR chain (or the III chimeric chain) does not prevent surface expression of the endogenous pre-TCR but, rather, both the pre-TCR and the TCR-/β can be coexpressed at the cell surface.
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Functions As an ER Retention Signal. components (although quantitative differences can not be ruled out), we next investigated whether the pT Cyto tail by itself could be directly responsible for controlling surface expression levels of the whole receptor complex. To test this possibility, we first analyzed the effect of the pT Cyto domain on the surface expression levels of individual plasma membrane reporter proteins. Murine CD25 (IL-2 receptor chain) is a particularly useful marker in this type of study, because transfection of the wild-type protein results in high surface expression level, as assessed by flow cytometry with an anti-CD25 mAb (Fig. 5). Interestingly, we found that replacement of the CD25 Cyto domain with that of pT results in a >50-fold reduction of surface CD25 expression on COS cells transfected with the CD25–pT chimera, compared with surface expression levels of wild-type CD25. A comparable reduction of CD25 expression was observed in SUP-T1 pre-T cells transfected with the CD25–pT chimera (Fig. 5). In both cell types, the relative levels of CD25 expression were not altered by deletion of the CD25 cyto tail (CD25 tailless), confirming that impaired CD25 surface expression was indeed due to the presence of the pT Cyto tail.
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chimeric protein was retained intracellularly, COS transfectants were permeabilized and examined for the intracellular distribution of CD25 by immunofluorescence microscopy. As shown in Fig. 6 A, transfection of wild-type CD25 resulted in a predominant localization of the protein in the Golgi apparatus, although CD25 expression was also detected at the plasma membrane. However, a markedly different staining pattern showing strong perinuclear fluorescence and a tubular-vesicular peripheral staining was observed in COS cells transfected with the CD25–pT chimera, which strongly suggested an ER distribution. To confirm that the pT tail was in fact conferring ER residence to membrane proteins, we next examined susceptibility to endo-H digestion of wild-type and chimeric CD25–pT molecules, as protein transport out of the ER to the Golgi apparatus is marked by acquisition of endo-H resistance 34. To this end, COS transfectants were labeled for 30 min with [35S]methionine and chased for various periods of time up to 8 h, and Triton X-100–solubilized molecules were then subjected to immunoprecipitation with an anti-CD25 mAb or with an antiserum against the pT Cyto tail (CT-1). Mock-treated or endo-H–treated proteins were then analyzed for their glycosylation state by SDS-PAGE, as endo-H sensitivity causes a marked change in the electrophoretic mobility of CD25. As shown in Fig. 6 B (top), a significant fraction (40%, as quantified by densitometric analysis) of wild-type CD25 acquired endo-H resistance after 1 h of biosynthesis, and was completely converted after 4 h. The situation was markedly different for the CD25–pT chimeras analyzed from either anti-pT (Fig. 6 B, bottom) or anti-CD25 precipitates (data not shown), as none of them acquire endo-H resistance, even after a 4-h chase. In contrast to wild-type CD25, which was partly degraded after 4 h and was hardly detectable after 8 h, up to 20% of pulsed CD25–pT was still recovered essentially in an immature form after 8 h (Fig. 6 B). Therefore, the lack of maturation of the chimeric protein does not result from their increased degradation rate in the ER. Rather, we can conclude that a major proportion of the CD25–pT chimeric proteins primarily reside in the ER and, therefore, cannot be processed in the Golgi apparatus. Taken together, the biosynthetic data in conjunction with the flow cytometry and immunofluorescence microscopy analyses provide evidence that the Cyto tail of the human pT molecule confers ER residency to plasma membrane proteins.
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protein in pre-T cells displaying poor surface expression of the pre-TCR (Fig. 7). When permeabilized SUP-T1 pre-T cells were stained with the rabbit anti-pT Ab CT-1 in combination with fluorescein-conjugated goat anti–rabbit Igs, native pT showed a typical intracellular staining compatible with ER location (data not shown). An identical intracellular pattern was revealed by green fluorescence examination of SUP-T1 pre-T cells transfected with a pT–GFP chimeric protein (Fig. 7). To provide formal proof that the intracellular compartment containing pT was in fact the ER, double-color analyses were performed in pT–GFP transfectants stained with a rabbit Ab recognizing the ER resident PDI protein followed by Cy3-conjugated goat anti–rabbit Igs. As shown in Fig. 7, GFP clearly localized to the same intracellular region as the PDI protein in pT–GFP transfectants. Moreover, costaining with an anti–TCR-β (βF1) mAb followed by Cy5-conjugated goat anti–mouse Igs revealed that endogenous TCR-β colocalized with GFP and PDI. Thus, from these experiments we concluded that intracellular pT and TCR-β are predominantly retained in the ER in SUP-T1 pre-T cells.
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Cyto Tail Releases a CD4–pT Chimeric Protein from ER Retention and Restores Cell Surface Protein Expression. is directly responsible for retaining proteins in the ER. Thus, it was reasonable to predict that deletion of the pT tail (and, therefore, the proposed ER retention determinants) would result in enhanced expression of the pre-TCR complex at the plasma membrane. However, the effect of the pT tail deletion was the opposite, as it prevented surface pre-TCR expression. Indeed, JR3.11 cells transfected with a tailless pT chain failed to react with anti-CD3 mAbs (Fig. 8 A), as well as with an anti–TCR-β (Vβ8) mAb and an anti-pT Ab (data not shown). This unexpected discrepancy could be explained if the cytoplasmic domain of pT contains essential structural information for the proper assembly and/or surface expression of a CD3-associated pre-TCR.
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tail prompted us to define more precisely the location of the potential ER retention signal in the pT Cyto domain. To this end, we examined the effect of successive truncations along the pT tail on the intracellular location and surface expression levels of individual CD4–pT chimeric proteins which could be readily detected by staining with a mAb against CD4. As shown in Fig. 8 B, immunofluorescence microscopy analyses of COS cells transfected with CD4–pT chimeric constructs where the Cyto domain of human CD4 was replaced with the full pT tail (CD4–pT) revealed a clear tubular-vesicular staining of the ER that was virtually identical to that recorded for the CD25–pT chimera (Fig. 6 A), and correlated with low surface CD4 expression on both COS (data not shown) and BW cells (Fig. 8 B). No loss of the CD4 ER staining pattern was noted with the removal of the 22 COOH-terminal pT residues by a truncation at position 243 (CD4-pTt22), although this truncation resulted in a partial increase of surface CD4 expression on the corresponding BW transfectants (Fig. 8 B). In contrast, when an additional 26 amino acids (aa) were removed, the resulting CD4–pTt48 truncated chimera was expressed on the surface of BW at levels that were as high as those expressed on BW cells transfected with a CD4-tailless (Fig. 8 B) or a wild-type CD4 (data not shown) construct. As this enhanced expression of surface CD4 correlated with loss of the ER staining pattern and appearance of fluorescence staining at the plasma membrane, we concluded that a region in the pT Cyto domain between aa 217 and 243 is responsible for the observed ER retention function of the pT tail. Accordingly, the CD4–pTt48 chimera as well as the CD4-tailless protein were efficiently transported from the ER to the Golgi apparatus, and displayed similar kinetics, as assessed by pulse-chase metabolic labeling of COS transfectants, anti-CD4 immunoprecipitation, and endo-H treatment. As shown in Fig. 9 A, both proteins acquired endo-H resistance after a 90-min chase, and were mostly converted after 4 h. In contrast, the CD4–pTt22 chimera remained essentially endo-H sensitive after 90 min and up to 50% of pulsed chimeric protein failed to acquire endo-H resistance, even after an 8-h chase.
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protein on pre-TCR surface expression. Bicistronic vectors containing such pT constructs allowed tracing of transiently transfected JR3.11 cells by EGFP expression as a separate but not as a chimeric protein. As shown in Fig. 9 B, although the pT deletions were not sufficient to reach TCR- expression levels, JR3.11 cells transfected with a pT chain lacking the terminal 48 residues (pTt48) consistently showed a modest increase of surface CD3 levels, compared with CD3 expression on wild-type pT transfectants. Thus, as observed with the CD4–pTt48 chimera, this particular truncation may release pT from ER retention, resulting in enhanced surface pre-TCR expression. In contrast, surface expression of a pre-TCR composed of the pTt22 truncated protein was indistinguishable from expression levels of a wild-type pre-TCR. Collectively, our data suggest that the observed pT ER retention function contributes to the regulation of surface pre-TCR expression on pre-T cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain in the former and of a TCR chain in the latter. We have first shown that introduction of a conventional TCR chain into pre-T cells increases surface receptor expression to levels similar to those of TCR-/β on transfected mature T cells. Interestingly, although it has been reported that expression of TCR- precludes the formation of a TCR-β–pT dimer in a murine pre-T cell line 35, TCR- transfection does not impair endogenous pre-TCR surface expression on human pre-T cells, indicating that TCR-β chains are in large excess and that TCR- and pT are not competing each other for pairing with TCR-β. Conversely, transfection of a TCR-–deficient mature T cell line with a pT chain results in low surface expression of the pre-TCR, relative to TCR-/β surface levels induced upon TCR- transfection. Because low levels of pre-TCR expression is a common feature of both cell types, we concluded that this is an intrinsic property of the pre-TCR complex, and therefore, the pT chain itself might play a direct role in controlling pre-TCR expression levels.
In an effort to identify the structural domain/s of pT responsible for such a role, we took advantage of the similarities and differences between the EC, TM, and Cyto domains of pT and TCR- and produced TCR-–pT chimeric proteins that were analyzed for their ability to bring a CD3-associated chimeric TCR complex to the cell surface. We initially focused on the EC pT region, which lacks a covalently associated Ig-like V domain 25, and asked the question of whether replacement of the pT EC domain with the EC region of TCR- could provide a symmetrical shape to the TCR-β–pT heterodimer that could result in an increased expression at the cell surface. Unexpectedly, however, such chimeric TCR-–pT chains (II) did not affect the levels of expression of the pre-TCR, indicating that low surface pre-TCR expression is not dependent on the pT EC domain. From these data one could infer that the lack in pT of a partner domain capable of pairing with the hydrophobic surface of the Vβ domain 36 does not impair expression of the complex at the cell surface. Alternatively, the pre-TCR, by analogy with the pre-BCR, may contain an additional not yet identified VpreT component that fulfils that function, and that might thus be rate limiting for proper assembly and transport of the TCR-β–pT heterodimer to the cell surface 26. Expression of such a putative VpreT is expected to be developmentally regulated, such that its absence would account for the impaired surface pre-TCR expression described in murine mature T cells 6. However, human mature T cells proved to be as efficient as pre-T cells in expressing a functional TCR-β–pT heterodimer on their surface (this study and unpublished results), thus supporting the alternative hypothesis that the pT chain is sufficient to promote surface expression of the pre-TCR complex in the absence of an additional component.
The finding that the chimeric chain in which the pT EC domain was replaced with the EC TCR- domain behaves as a wild-type pT chain in terms of surface receptor expression was not obvious, because the TCR- C domain displays little homology with the equivalent pT domain 925. However, as the TCR- C domain residues involved in polar interactions with the TCR-β C domain are all conserved in the pT C domain, it has been proposed that association between pT and TCR-β C domains may occur in a way similar to that observed in the TCR CCβ module 337. These data are against an essential function of TCR-β–pT assembly in controlling surface expression levels of the pre-TCR, and suggest that the CD3/ subunits associated with the TCR-β–pT heterodimer probably account for such a regulatory function. Of them, the chain would merely be a dispensable amplification module for the pre-TCR, which increases its assembly rate and stability 1920212224, and CD3 seems dispensable for pre-TCR assembly and function 81338. In contrast, the CD3 pair, which may associate with the pre-TCR EC domain module in a manner similar to that suggested for its association with the TCR-/β 39, has proved mandatory for proper assembly of a functional pre-TCR 8404142. Therefore, it is not surprising that the wild-type pT chain and the chimeric chains containing both the EC and TM domains, or exclusively the EC domain of TCR-, are equally capable of mediating association with CD3, CD3, and CD3 subunits. What is more surprising is that all the wild-type pre-TCR and the chimeric TCR complexes are equally found physically associated with the chain, given that the weak association of the chain with the pre-TCR has recently been shown to map to the connecting peptide (CP) of pT lying between its TM and Ig-like EC domains 43. Therefore, one would expect that the presence of the TCR- CP domain in both chimeras would confer an increased stability to their corresponding receptors that might result in higher levels of surface expression. In contrast, their expression levels were as low as those of the wild-type pre-TCR. Taken together, our results allowed us to conclude that qualitative differences in the contribution of the CD3/ components to pre-TCR or TCR assembly do not influence surface receptor expression levels.
A striking finding was that replacing the Cyto domain of TCR- with the equivalent pT domain is sufficient for inducing low surface expression of the chimeric TCR. It is thus obvious that the pT Cyto tail is directly responsible for low surface pre-TCR expression. A strong argument in favor of the attractive possibility that the pT Cyto tail functions as an ER retention signal was the observation that individual plasma membrane proteins are mostly retained in the ER and fail to reach the Golgi compartment when appended to the pT tail. Accordingly, pT chains are primarily found in internal structures which colocalize with the ER. The demonstration that truncation of the terminal 48 aa of pT (and particularly deletion of the region between residues 217 and 243) releases CD4–pT chimeric proteins from ER retention lends further support to that notion, and suggests that pT ER retention determinants localize primarily in the deleted region. However, the pT tail sequence from position 217 to 243 does not harbor the consensus dilysine cytoplasmic motif for ER retention of TM proteins 34 or the reported CD3 ER retention sequence 44, suggesting the existence of alternative ER retention signals. An extensive series of point mutations will have to be introduced into that particular pT sequence to unravel the precise requirements for its retention in the ER.
As proposed for the mature TCR-/β 45, ER retention of individual subunits or partial complexes might be an essential mechanism for regulating assembly and levels of expression of the pre-TCR. However, additional structural information may be contained in the pT Cyto tail that is essential for surface expression of the pre-TCR, as pT tailless molecules fail to bring a CD3-associated pre-TCR complex to the cell surface. Therefore, efficient surface expression of the pre-TCR complex may be the result of a balance between complex mechanisms controlling retention and assembly of its individual components. This may explain the finding that release of individual chimeras bearing a truncated pT tail from ER retention correlates with recovery of wild-type protein expression levels, whereas pT molecules with identical truncations were unable to recover TCR-/β surface levels, although they were more efficient than wild-type pT in bringing the pre-TCR to the cell surface. Interestingly, an ER retention mechanism has also been proposed to account for the low plasma membrane expression of the pre-BCR 17, although in that case ER retention seems to be inherent to pre-B cells. Despite the striking functional similarities between the pre-TCR and the pre-BCR, the particular structural features of their individual components (i.e., pT and 
5) may explain such a discrepancy.
Finally, although one would expect that a retention mechanism similar to that proposed here for the human pre-TCR would be functional also in mice, the poor conservation of the Cyto tail in both species 625 would make this possibility unlikely. However, conserved residues in the cytoplasmic domain have been identified 25. In addition, two independent findings strengthen our view: first, DN thymocytes from pT–/– mice made transgenic for a pT tailless chain display increased TCR-β surface levels, compared with DN thymocytes from wild-type or pT–/– nontransgenic mice 46, and second, low CD3 expression is common to DP thymocytes from recombination activating gene (Rag)1–/– mice expressing a transgenic functional pre-TCR consisting of truncated TCR-β and pT forms that keep their TM and Cyto regions 14. In conclusion, our data provide evidence that the pT Cyto tail displays a novel ER retention function which may participate in the regulation of surface pre-TCR expression levels on pre-T cells.
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Acknowledgments |
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This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (SAF 97-0161), Dirección General de Enseñanza Superior (PB97-1194), Comunidad de Madrid (08.3/0015.1/99), Fondo de Investigación Sanitaria (FIS 00/1044), and Fundación Eugenio Rodríguez Pascual. We thank the Fundación Ramón Areces for an institutional grant to the Centro de Biología Molecular Severo Ochoa.
Submitted: 18 September 2000
Revised: 27 March 2001
Accepted: 29 March 2001
Abbreviations used in this paper: aa, amino acid(s); BCR, B cell receptor; Cyto, cytoplasmic; DN, double negative; DP, double positive; EC, extracellular; EGFP, enhanced green fluorescent protein; endo-H, endoglycosidase H; ER, endoplasmic reticulum; PDI, protein disulfide isomerase; pT
, pre–TCR-; SP, single positive; TM, transmembrane.
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