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Opposite Ability of Pre-TCR and TCR to Induce Apoptosis

Ann-Muriel Steff^*, Sébastien Trop^1,, Mario Maira, Jacques Drouin and Patrice Hugo^2,*,

^* Division of Research and Development, PROCREA BioSciences, Montreal, Quebec, Canada; Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada; and Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada

	Abstract

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In early CD4^-CD8^- pro-thymocytes, signaling through the pre-TCR is crucial for survival and differentiation into CD4⁺CD8⁺ cells. At this more mature stage, interactions between TCR and self-Ag/MHC complexes in turn lead either to cell survival and differentiation (positive selection) or to cell death (negative selection). Intrinsic differences must therefore exist between pre-TCR signals in CD4^-CD8^- thymocytes and TCR signals in CD4⁺CD8⁺ cells, since only the latter can mediate a death signal. In this work, we directly compared the capability of pre-TCR and TCR to induce apoptosis in a CD4^-CD8^- thymoma cell line following receptor cross-linking with mAbs. Cross-linking of TCR triggered high levels of programmed cell death, mimicking the negative selection signal usually induced in CD4⁺CD8⁺ thymocytes. In contrast, pre-TCR was very inefficient at inducing apoptosis upon cross-linking, despite similar levels of surface receptor expression. Importantly, inefficient apoptosis induction by the pre-TCR did not result from its weak association with TCR chain, since TCRs containing -pT chimeric chains, binding weakly to TCR, were still able to induce apoptosis. Although similar tyrosine phosphorylation and calcium influx were induced after either pre-TCR or TCR cross-linking, the two pathways diverged at the level of Fas ligand induction. Among putative transcription factors involved in Fas ligand mRNA induction, Nur77 and NFAT transcriptional activities were readily induced after TCR, but not pre-TCR, stimulation. Together, these results support the view that the structure of the pre-TCR and TCR directly influences their apoptosis-inducing capabilities by activating distinct signaling pathways.

	Introduction

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T cell development is controlled by the sequential expression of, and signaling through, pre-TCR and TCR. In early CD4^-CD8^- double-negative (DN)³ thymocytes, the productive rearrangement of a TCR gene leads to surface expression of a pre-TCR consisting of pT, TCR, and associated CD3 and TCR chains. Signaling through this complex is crucial for the survival and differentiation of these early precursors into CD4⁺CD8⁺ thymocytes, a process termed selection (1). At the CD4⁺CD8⁺ stage, the TCR chain is rearranged and pairs with TCR to form a mature TCR (reviewed in Ref. 2). Following low affinity/avidity interaction with self-Ag/MHC complexes, TCR triggers signals leading to cell survival and differentiation (positive selection). However, the same TCR can also transduce a death signal (negative selection), when its interaction with the self-Ag/MHC complex is of high affinity/avidity (reviewed in Ref. 3). Thus, intrinsic differences exist between pre-TCR signals in CD4^-CD8^- thymocytes and TCR signals in CD4⁺CD8⁺ cells, since only the latter have been shown to be capable of inducing apoptosis.

Various hypotheses might account for the distinct outcomes resulting from pre-TCR and TCR engagement at the surface of CD4^-CD8^- and CD4⁺CD8⁺ thymocytes, respectively. First, pre-TCR expression is extremely low on CD4^-CD8^- thymocytes, as compared with TCR on CD4⁺CD8⁺ cells. Second, specific gene products present at distinct stages of T cell development would cause TCR-mediated signals to be interpreted differently by CD4^-CD8^- and CD4⁺CD8⁺ thymocytes. Finally, structural differences between the pre-TCR and TCR might endow these surface receptors with unique signaling capabilities. Indeed, although both pre-TCR and TCR are multimolecular complexes composed of the TCR chain (associated either with pT or TCR, respectively), CD3, CD3, CD3, and TCR components (2), some structural dissimilarities between these receptors have been noted. First, analysis of mice deficient in various components of the TCR/CD3 complex revealed that, whereas the CD3, CD3, and CD3 chains are all essential for the function of the TCR (4), the CD3 chain, although present in the pre-TCR complex, is not required for its function (5). Second, it has recently been shown that TCR and pre-TCR are differently associated with lipid rafts (6, 7). Third, while TCR chains are functionally linked to both TCRs, physical association of TCR is much stronger with the TCR than with the pre-TCR (8, 9, 10, 11, 12). Notably, TCR has been shown to play a major role in TCR-mediated apoptosis in mature T cells (13, 14, 15, 16).

We assessed whether structural dissimilarities between the pre-TCR and TCR could explain their differential ability to induce apoptosis. Thus, we directly tested the ability of wild-type pre-TCR, TCR, as well as -pT/TCR chimeric TCRs retaining or not the ability to bind to the TCR chain, to induce apoptosis in an immature CD4^-CD8^- thymic cell line. We show that in this early thymic lymphoma, mAb cross-linking of TCR triggered programmed cell death, as observed in CD4⁺CD8⁺ thymocytes. In striking contrast, similar engagement of the pre-TCR, expressed at the same levels, was totally unable to induce apoptosis in the same cells. Furthermore, cross-linking of /pT chimeras previously shown to bind very weakly to the TCR chain (10) also induced high levels of apoptosis in this cell line, demonstrating that strong physical TCR association is not an absolute prerequisite for the efficient induction of apoptosis upon TCR engagement. In addition, these results favor the hypothesis that the nature of the receptor, namely pre-TCR or TCR, in a given cellular context, can directly impinge on the outcome of receptor signaling.

	Materials and Methods

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Cell lines and constructs

The SL12 cell line was derived from a SCID mouse spontaneous thymoma, and its phenotype corresponds to the CD25⁺CD44 stage of T cell development (pT expression, CD4^-CD8^-CD25⁺CD44^-) (17, 18). SL12 cells were stably transfected either with the pXS expression vector alone (SL12 Neo), or with the same vector encoding the -chain of the 2B4 TCR (19), allowing pre-TCR surface expression (SL12.12) (17). Murine pT cDNA (20) was cotransfected to generate the SL12 pT.5 cell line (17). This clone, however, expresses almost undetectable levels of pre-TCR at the cell surface, as compared with SL12.12, and was used as recipient for the electroporation of the various TCR/pT constructs used in this study. We have shown that coexpression of TCR and pT chains in this cell line prevents pre-TCR expression at the cell surface (21). TCR/pT chimeric molecules, which have been described previously (10), are constituted of V11.1, J, and C domains of the AD10 TCR chain (22), and portions of pT extracellular, transmembrane, and cytoplasmic domains (20). The V-pT chimera was constructed by fusing the V11-J region (first Ig-like domain) of the AD10 TCR chain to a pT cDNA lacking the leader sequence and the first five amino acids (primer sequences available upon request). All constructs were cloned into the SRPuro mammalian expression vector (provided by François Denis, Institut Armand-Frappier, Quebec, Canada), and transfectants were selected with 1 µg/ml puromycin (Life Technologies, Burlington, Ontario, Canada). All cell lines used in this study were grown in RPMI 1640 medium containing 2 mM glutamine (Mediatech, Herndon, VA) supplemented with 5% FCS (BioMedia, Kirkland, Quebec, Canada), 2.3 mM 2-ME (Sigma-Aldrich, Oakville, Ontario, Canada), 100 IU/ml penicillin (Mediatech), 100 µg/ml streptomycin (Mediatech), and 0.5 mg/ml geneticin (G418; Mediatech).

Ab treatment

Anti-TCR (H57-597), anti-V11.1 (RR8.1), anti-CD3 (145-2C11), or anti-Fas (Jo2; BD Biosciences, Oakville, Ontario, Canada) mAbs were coated on 96-well Probind plates (Falcon; BD Biosciences) at 2.5 µg/ml (H57-597), 5 µg/ml (RR8.1 and 145-2C11), or 10 µg/ml (Jo2) by 2-h incubation at room temperature. For apoptosis induction assays, SL12 cells were plated in triplicates at 8 x 10⁴ cells/well in a final volume of 200 µl for 20 h at 37°C. Where indicated, soluble anti-Fas ligand (FasL) mAb (MFL3; BD Biosciences) was added at a final concentration of 10 µg/ml. For RNA preparation, cells (3 x 10⁶; 5 x 10⁵ cells/ml) were incubated for 6 h at 37°C, in six-well plates precoated with anti-TCR mAb.

RT-PCR

Cells were harvested and RNA was isolated using the RNeasy purification system (Qiagen, Mississauga, Ontario, Canada). One microgram of RNA was treated with DNase I (Roche Diagnostics, Laval, Quebec, Canada), and first-strand cDNA synthesis was conducted using oligo(dT) primers (Amersham Canada, Oakville, Ontario, Canada) and Moloney murine leukemia virus reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. Titrated amounts of cDNA were amplified by PCR using the following primers and conditions (30 cycles): -actin (5'GTGGGCCGCTCTAGGCACCAA-3'/5'-CTCTTTGATGTCACGCACGATTTC-3'), annealing at 48°C; FasL (5'-CAGCTCTTCCACCTGCAGAAGG-3'/5'-AAGATTGAATACTGCCCCCAGG-3'), annealing at 55°C; Egr-1 (5'-AATCCTCAAGGGGAGCCG-3'/5'-GAGTAGATGGGACTGCTGCTG-3'), annealing at 60°C; Egr-2 (5'-CCCCTTTGACCAGATGAAC-3'/5'-TGGATGGCGGCGATAAG), annealing at 49°C; Egr-3 (5'-CGACTCGGTAGCCCATTAC-3'/5'-GAGATCGCCGCAGTTGG-3'), annealing at 56°C; Nur77 (5'-GCTCTGAGTACTATGGCAGTCCC-3'/5'-CGGAAGTGTCACGGTTCG-3'), annealing at 54°C.

Flow cytometric analysis

Detection of apoptosis was performed using merocyanin-540 (MC540; Molecular Probes, Eugene, OR), a dye-detecting membrane lipid unpacking, as described elsewhere (23). Briefly, cells were harvested, washed once with PBS, and resuspended in 100 µl of PBS containing 0.1% BSA and 5 µg/ml MC540. Samples were incubated in the dark for 3 min at 23°C and resuspended in 400 µl of PBS. Similar results were obtained using annexin V or 3,3'-dihexyloxacarbocyanine iodide staining as readouts for apoptosis. Fas Ag staining was performed by incubating cells with 1 µg/ml Jo2 mAb (BD Biosciences) in PBS, 2% FCS, and 0.1% sodium azide, for 25 min at 4°C, followed by FITC-conjugated anti-hamster Ig mAb (HIG88.2, obtained from J. Kappler, National Jewish Medical and Research Center, Denver, CO). Cells were analyzed on a Coulter Epics flow cytometer (Beckman Coulter, Ville St-Laurent, Quebec, Canada) and the WinMDI 2.7 software (J. Trotter, The Scripps Research Institute, San Diego, CA).

Western blot

SL12 cells, 5 x 10⁵, were incubated with 10 µg/ml anti-TCR mAb for 30 min in cold RPMI 1640. Cells were then washed, resuspended in 100 µl of RPMI 1640 containing 30 µg/ml mouse anti-hamster Ig, and incubated 2.5 min at 37°C. Cells were lysed in 100 µl of ice-cold 2x lysis buffer containing 100 mM Tris (pH 8; Sigma), 2% Nonidet P-40 (ICN Biomedicals, Montréal, Quebec, Canada), 40 mM EDTA (Sigma), 2 mM PMSF (Sigma), 20 µg/ml leupeptin (Sigma), 20 µg/ml aprotinin (ICN Biomedicals), and 2 mM sodium orthovanadate (ICN Biomedicals). Control cells (nonstimulated) were treated similarly, but were lysed before addition of the secondary Ab. Total lysates were resolved by 12% SDS-PAGE under reducing conditions and transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore, Mississauga, Ontario, Canada). Tyrosine phosphorylation was detected using 4G10 mAb, followed by HRP-conjugated anti-mouse Ig mAb (Boehringer Mannheim, Laval, Quebec, Canada). TCR was detected using H146 mAb, followed by HRP-conjugated protein A (Amersham, Arlington Heights, IL). Blots were revealed using chemiluminescence (SuperSignal; Pierce, Rockford, IL).

Luciferase assays

The luciferase reporter constructs containing the following response element were: NurRE-luc (24), TRE (12-O-tetradecanoylphorbol-13-acetate-response element)-luc (25), NFAT-RE-luc (26), and myocyte enhancer factor (MEF)-2-RE (27). Cells were incubated in DMEM (Mediatech) with 20 µg of luciferase reporter plasmid on ice for 30 min and then at room temperature for an additional 15 min. After electroporation (240 mV and 960 µF), cells were plated in 35-mm dishes coated or not with anti-TCR mAb (2.5 µg/ml) in 2 ml DMEM containing 10% decomplemented FCS. After 4- to 6-h incubation at 37°C and 5% CO₂, cells were harvested and lysed in 200 µl of lysis buffer (100 mM Tris-HCl, 0.5% Nonidet P-40). Lysates were normalized according to protein content, and luciferase activity was assayed using a LB 953 luminometer (Berthold, Nashua, NH). Induction was calculated as the ratio between relative luciferase units for anti-TCR-stimulated cells over unstimulated cells. Luciferase activity in unstimulated cells was similar for TCR- and pre-TCR-expressing cells, and was comparable from one experiment to another. Data are presented as the means ± SEM for three to five independent experiments.

	Results

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Engagement of TCR, but not pre-TCR, induces cell death in an immature thymic lymphoma

Cross-linking of TCR with mAbs is a well-recognized stimulus mimicking negative selection in vitro, in double-positive thymocytes. On the other hand, molecular interactions leading to pre-TCR signaling on DN thymocytes are not well defined (28), and the ability of the pre-TCR to trigger a death signal has never been reported. To clarify the link between cell death induction and the nature of the receptor expressed by developing T cells, we expressed either TCR or pre-TCR at high or intermediate levels at the surface of an immature thymic lymphoma, SL12 (Fig. 1, A and B). We used mAb cross-linking to punctually induce a signal through either pre-TCR or TCR in SL12 cells and to examine the ability of these cells to undergo apoptosis following receptor engagement. As shown in Fig. 1C, incubation of SL12 cells, expressing either high (mean fluorescence intensity (MFI) = 4) or intermediate (MFI = 1) levels of TCR, with plate-bound anti-TCR mAbs resulted in similar induction of cell death (40% over background). These results indicate that early CD4^-CD8^- thymic lymphoma cells are able to undergo apoptosis when stimulated through a mature TCR, similarly to more mature T cells, and this over a wide range of surface receptor levels. In constrast, cross-linking of the pre-TCR (expressed at an MFI = 4) in this cell line, with the same anti-TCR mAb, was less efficient at inducing apoptosis (25% cell death over background or less), at all doses tested (Fig. 1C). When pre-TCR surface expression was further reduced to more physiological levels by cell sorting (either MFI = 0.6 in Fig. 1B, or even MFI = 1, data not shown), receptor engagement by anti-TCR mAbs became completely inefficient at inducing apoptosis. The same results as in Fig. 1C were obtained using an anti-CD3 Ab for TCR cross-linking (data not shown), demonstrating that the inability of pre-TCR to induce apoptosis is not the result of poor recognition of the pre-TCR by anti-TCR mAb. Rather, our results reveal fundamental differences in the cellular response to either pre-TCR or TCR engagement by anti-TCR mAbs, reflected in this study by divergent apoptosis-inducing properties.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Pre-TCR engagement fails to efficiently induce apoptosis. SL12 cells, expressing either TCR or pre-TCR, were sorted to obtain populations displaying high (A) or intermediate (B) levels of receptors. Cells were stained with FITC-conjugated anti-TCR chain Ab, and surface expression of the receptors is shown. C, SL12 cells, expressing either TCR or pre-TCR, at high or intermediate levels, were plated for 20 h with various concentrations of plate-bound anti-TCR mAb. Apoptotic cells were detected by MC540 staining. At least four independent experiments were conducted, and a representative result is shown.

Induction of apoptosis following TCR cross-linking does not require strong association of TCR

One obvious explanation for the divergent outcomes of pre-TCR vs TCR engagement is their differential association with the TCR chain. Indeed, TCR has been proposed to play specialized functions in the apoptotic responses of T cells following TCR stimulation. For instance, while IL-2 secretion is not affected in activated T cell hybridomas or peripheral T cells expressing "ITAM (immunoreceptor tyrosine-based activation motif)-less" TCR chains, the subsequent induction of apoptosis is abrogated (15, 29). More importantly, a mutation in the TCR chain that abolishes the strong physical association of TCR to the TCR complex in Jurkat cells prevents these cells from undergoing activation-induced cell death, without affecting other responses (13). Since the pre-TCR is known to associate very loosely to the TCR chain (11, 17), one could imagine that this would impair apoptosis induction after receptor engagement. To clarify this point, we used mutant SL12 cell lines we had generated in a previous work, which expressed chimeric molecules between TCR and pT chains, and which were previously shown to retain or not the ability to bind strongly to TCR, due to substitution of the pT-connecting peptide in the TCR chain (10). We examined the ability of these chimeric /pT-containing TCRs to trigger apoptosis following cross-linking. Fig. 2 shows that mAb cross-linking of "Cyto" chimeras, containing pT intracellular domain, induced reasonable levels of apoptosis (27%), which were further increased with higher surface expression (60% for a MFI = 3, data not shown). This result clearly demonstrates that pT intracytoplasmic domain has no protective effect toward apoptosis induction. "Intra" and TCR/pT-connecting peptide (-CPpT) chimeras, which do not bind strongly to TCR, induced 57% and 34% cell death over background, respectively. These responses are much higher than those obtained with SL12 cells expressing high levels of pre-TCR (12%). This indicates that cross-linking of /pT chimeric TCRs having lost their strong association with TCR induces apoptosis at levels similar to wild-type TCR, demonstrating that weak physical association of TCR with the pre-TCR cannot be invoked to explain the inability of pre-TCR to mediate an apoptotic signal in SL12 cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. TCRs composed of chimeric /pT chains are able to induce apoptosis upon cross-linking, despite their weak physical association with TCR. SL12 cells expressing TCR together with various chimeric /pT chains, for which association with TCR was previously determined (10 ), were incubated for 20 h with plate-bound anti-TCR mAb (2.5 µg/ml). Apoptotic cells were detected by MC540 staining. Untreated cells were stained with FITC-conjugated anti-TCR mAb, and MFI was determined. Bars represent mean cell death values over background ± SD, obtained from triplicate cultures, performed at least three times.

Involvement of Fas-FasL interactions in apoptosis induction

To our knowledge, this is the first report of apoptosis induction in an early DN cell line through TCR cross-linking. Therefore, we further investigated the molecular basis for the differences in the apoptotic responses to TCR and pre-TCR engagement and verified whether Fas-FasL interactions were implicated in cell death, as is the case in more mature T cells (30). When TCR-expressing SL12 cells were exposed to TCR cross-linking in the presence of soluble anti-FasL Ab, apoptosis was completely abrogated, as shown in Fig. 3A. Thus, Fas-FasL interactions appeared to mediate cell death in TCR-expressing SL12 cells, and it seemed likely that absence of apoptosis after pre-TCR cross-linking could be due either to a lack of Fas-FasL interactions or to a defect in the pathway of apoptosis induced by Fas receptors. To test these hypotheses, we first examined Fas expression in TCR- or pre-TCR-expressing SL12 cells, before and after TCR stimulation. As detected by flow cytometry, all cell types expressed low, but detectable levels of Fas Ag at their surface, and expression levels did not change after stimulation (Fig. 3B). Furthermore, cross-linking Fas molecules with immobilized mAbs triggered cell death in all cell lines, showing that the Fas pathway is fully functional in all SL12 cells (Fig. 3C). Hence, impaired apoptosis after pre-TCR cross-linking was most probably due to a defect in FasL expression.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Apoptosis induced through TCR cross-linking in SL12 cells is mediated through a Fas/FasL pathway. A, SL12 cells expressing either TCR or pre-TCR were incubated with soluble anti-FasL mAb (10 µg/ml, ) or plate-bound anti-TCR mAb (2.5 µg/ml, ), alone or in combination (), for 20 h. Cell death was assessed by MC540 staining. B, Fas expression. Unstimulated (dashed lines) or anti-TCR-stimulated (gray histograms) SL12 cells were stained with anti-Fas mAb followed by FITC anti-hamster Ig mAb. Control staining (solid line) was performed with the secondary mAb alone. C, The Fas pathway is functional in SL12 cells. SL12 cells expressing either TCR or pre-TCR were incubated with medium () or with plate-bound Abs, either anti-Fas mAb (10 µg/ml, ) or anti-TCR mAb (2.5 µg/ml, ), for 20 h. Cell death was assessed by MC540 staining and flow cytometry. At least four independent experiments were conducted, and a representative result is shown.

View larger version (46K): [in this window] [in a new window]   FIGURE 4. Inability of pre-TCR stimulation to induce cell death is due to a defect in FasL expression. A, Induction of fasL, egr-1, egr-2, and nur77 genes after stimulation of SL12 cells expressing high or intermediate levels of TCR or pre-TCR. cDNAs were obtained from cells unstimulated (-) or stimulated (+) 6 h with 2.5 µg/ml of plate-bound anti-TCR mAb. PCR was performed using cDNAs diluted and calibrated according to -actin. B, Protein tyrosine phosphorylation is equally induced in TCR- or pre-TCR-expressing cells after stimulation. TCRs were cross-linked with anti-TCR mAb, followed by anti-hamster mAb for 2.5 min. Western blot was performed with the 4G10 antiphosphotyrosine mAb, and revealed by chemiluminescence.

TCR and pre-TCR differently induce Nur77 and NFAT transcriptional activity

Studies on the fasL gene promoter have identified binding sites for many transcription factors. In particular, NFAT (32, 33), Egr (34, 35), or NF-B (36, 37) response elements are all activated after TCR engagement, regardless of whether the signal leads to activation or cell death, and thus are not specific for apoptosis triggering. On the other hand, the Nur77 nuclear orphan receptor is intimately linked to TCR-mediated apotosis in vitro (38, 39, 40, 41, 42) and in vivo (i.e., negative selection) (43, 44). Also, the pro-apoptotic activity of Nur77 has been proposed to act through the Fas/FasL pathway (45, 46). In SL12 cells, cross-linking of TCR clearly induced transcription of egr-1, egr-2, and nur77 genes, as shown by RT-PCR analysis in Fig. 4A. However, pre-TCR engagement on pre-TCR^int cells induced dramatically lower levels of egr-1, egr-2, and nur77 transcripts, as compared with TCR^int-expressing cells. Furthermore, we reproducibly observed a slightly reduced transcriptional induction of these genes after stimulation of pre-TCR^high-expressing cells, as compared with TCR^high-expressing cells (Fig. 4A).

It is known that Nur77 transcriptional activity relies not only on the level and persistence of its expression, but also on many posttranslational modifications, such as phosphorylation, nuclear translocation, or interaction with other partners (47, 48, 49). The fact that nur77 transcripts were induced at almost similar levels in pre-TCR^high and TCR^high cells after receptor cross-linking, despite the fact that these cells displayed very different apoptotic responses, prompted us to directly examine the actual transcriptional activity of Nur77. We transfected SL12 cells, expressing high levels of receptors, with a luciferase-reporter gene under the control of the Nur77 response element, NurRE (24). In a similar manner, we also measured the transcriptional activitiy of NFAT, reported to be induced upon pre-TCR signaling in Jurkat cells (50), and of MEF-2, a factor involved in the calcium-dependent transcriptional activation of nur77 during TCR-mediated apoptosis (41, 51). Fig. 5 shows that NFAT and Nur77 activities were clearly induced after TCR stimulation of SL12 cells. In constrast, pre-TCR engagement only led to a comparatively small/weak induction of NFAT and Nur77 response elements. AP-1 and MEF-2 response elements were very poorly induced after either - or pre-TCR cross-linking, indicating that these factors are not implicated in TCR signaling in immature SL12 cells or that they are activated during a different time frame. Together, these results indicate impaired Nur77 activity in cells expressing and signaling through pre-TCR.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Pre-TCR stimulation fails to induce Nur77 and NFAT transcription. SL12 cells expressing high levels of either TCR or pre-TCR were transiently transfected with luciferase reporter constructs containing binding sites for either Nur77 (NurRE), AP-1 (TRE (12-O-tetradecanoylphorbol-13-acetate-response element)), NFAT (NFAT-RE), or MEF-2 (MEF-2-RE). After 4- to 6-h incubation either in medium or in the presence of plate-bound anti-TCR mAb, cells were lysed and luciferase activity was determined. Induction was calculated as the ratio between relative luciferase units for anti-TCR-stimulated cells over unstimulated cells. Luciferase activity in unstimulated cells was similar for TCR- and pre-TCR-expressing cells and was comparable from one experiment to another. Data are presented as the means ± SEM for three to five independent experiments, each performed in triplicate.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Impaired apoptosis following TCR cross-linking on mature T cells has previously been linked to defects in the association of TCR with TCR (13, 14, 15, 29). From these studies, it could be inferred that the failure of pre-TCR, as opposed to TCR, to induce apoptosis in SL12 cells could be related to the constitutive weak physical association between pre-TCR and TCR (10, 11, 12). We have shown that engagement of an /pT chimera (referred to as "Intra"), which associates loosely with TCR (10), is nonetheless fully competent at inducing apoptosis in SL12 cells (Fig. 2). This demonstration therefore rules out the possibility that loose physical association between pre-TCR and TCR can be invoked to explain the inability of this receptor to induce apoptosis in DN cells.

In the search for other structures conferring inability to the pre-TCR to trigger apoptosis, we reasoned that the missing N-terminal Ig-like domain in pT could be, at least partly, responsible for the distinct apoptosis-inducing capabilities of TCR compared with the pre-TCR. It was conceivable that the presence of this Ig-like domain in TCR induces a particular conformation of the TCR or yet modifies the interaction of the TCR with other surface proteins, necessary for transmitting pro-apoptotic signals. Our experiments with VpT chimeras clearly ruled out this hypothesis. Nonetheless, the common feature of the receptors inducing low levels of apoptosis (namely, pre-TCR and VpT chimeras), compared with those highly competent to trigger cell death (TCR, Intra, Cyto, CPpT chimeras), was the presence of the specific pT Ig-like domain. It is thus possible that a molecule, dampening the apoptotic response, could be interacting with this domain. To clarify this point, we constructed a TCR/pT chimera replacing the C-terminal Ig-like domain of TCR by the one of pT. Unfortunately, despite multiple attempts, we were unable to express this molecule at the surface of SL12 cells. Finally, it has been recently shown that pre-TCR and TCR are differently distributed in the plasma membrane, since pre-TCR is stably associated with lipid rafts, while TCR is not (6, 7). Whether different localization of the receptors before cross-linking impinges on their signaling properties after engagement is not known. Stimulation of our chimeric receptors shows, however, that apoptotic responses are unrelated to the presence of pT cytoplasmic domain (putatively containing the "raft-localization" signal).

Distinct capabilities of pre-TCR vs TCR to induce apoptosis do not seem to depend on inefficient Ab recognition or defective proximal signaling of the pre-TCR. This is supported by several observations. We have previously shown that cross-linking either pre-TCRs or TCRs in SL12 cells induced comparable calcium flux and a global increase in protein tyrosine phosphorylation (including the high molecular mass form of CD3) (this study and Ref. 10). Since these two receptors apparently triggered similar proximal signaling events, we looked at a more downstream target specifically involved in cell death, i.e., Fas-FasL interactions. Pre-TCR- and TCR-bearing cells expressed similar levels of Fas, and triggered equivalent cell death upon cross-linking of Fas. This observation, coupled with a complete inhibition of TCR-induced apoptosis with addition of blocking anti-FasL mAbs, suggests that the lack of apoptosis after pre-TCR stimulation was most likely due to defective FasL induction after receptor engagement. This was confirmed by showing very low/nonexistent FasL transcription following pre-TCR stimulation as opposed to that triggered by TCR ligation. This discrepancy at the level of FasL induction clearly shows that, in SL12 cells, TCR and pre-TCR signals diverge at a point other than, or downstream of, protein tyrosine phosphorylation (10) or ERK activation (31). It has been shown that, upon TCR cross-linking in T cell hybridomas (57), the ERK pathway positively regulates the transcription of the nur77 gene. Consistent with this observation, we observed little difference in the transcription of nur77 after stimulation of either TCR^high- or pre-TCR^high-expressing cells. However, it appeared that Nur77 transcriptional activity differed dramatically between these two cell lines after receptor engagement. Whether impaired Nur77 function in pre-TCR^high cells results from defective posttranslational modifications, translocation, or interaction with specific protein partners, is not clear at present. On the other hand, regulation of the fasL promoter appears to be very complex (32, 34, 35, 37, 57, 58, 59, 60, 61, 62), and whether impaired Nur77 and NFAT activities after pre-TCR, as opposed to TCR, engagement in DN cells are the sole reasons for inefficient induction of the fasL gene, remains to be investigated. In addition, such elevated Nur77 activity triggered by TCR ligation is known to mediate negative selection of TCR-bearing double-positive thymocytes, although Fas-FasL interactions do not appear to be essential for this process.

Together, our observations support the view that the structure of the receptors dictates, at least in part, the outcome of pre-TCR vs TCR signaling in thymocytes. We have shown that inefficient induction of apoptosis by the pre-TCR in an immature thymoma cell line is neither attributable to the intrinsic loose physical interaction between the pre-TCR and TCR, nor to the lack of a second Ig-like domain in pT. Rather, TCR and pre-TCR signaling seems to diverge in their ability to activate the transcription factors Nur77 and NFAT, ultimately leading to effective or impaired FasL induction according to the receptor engaged. The model described herein will allow a better definition of the structural motifs and signaling cascades conferring distinct differentiation and proliferation capabilities upon the pre-TCR in DN thymocytes.

	Acknowledgments

 
We thank Dr. S. Latour, Dr. D. L. Wiest, M. Fortin, and F. Philippoussis for critical reading of this manuscript and helpful advice. We acknowledge the Flow Cytometry Unit at the Institut de Recherches Cliniques de Montréal for cell sorting.

	Footnotes

 
¹ S.T. is the recipient of a Doctoral Research Award from the Medical Research Council of Canada.

² Address correspondence and reprint requests to Dr. Patrice Hugo, Division of Research and Development, PROCREA BioSciences, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2 Canada.

³ Abbreviations used in this paper: DN, double negative; ERK, extracellular signal-regulated kinase; FasL, Fas ligand; MC540, merocyanin-540; MEF, myocyte enhancer factor; MFI, mean fluorescence intensity.

Received for publication November 9, 2000. Accepted for publication February 14, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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