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Low Activation Threshold As a Mechanism for Ligand-Independent Signaling in Pre-T Cells ¹

Mariëlle C. Haks^*, Stanley M. Belkowski^*, Maria Ciofani, Michele Rhodes^*, Juliette M. Lefebvre^*, Sebastién Trop, Patrice Hugo, Juan Carlos Zúñiga-Pflücker and David L. Wiest^2,*

^* Division of Basic Sciences, Immunobiology Working Group, Fox Chase Cancer Center, Philadelphia, PA 19111; Department of Immunology, University of Toronto, Sunnybrook & Women’s College Health Sciences Center, Toronto, Ontario, Canada; PROCREA BioSciences Inc., Montréal, Québec, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pre-TCR complexes are thought to signal in a ligand-independent manner because they are constitutively targeted to lipid rafts. We report that ligand-independent signaling is not a unique capability of the pre-TCR complex. Indeed, the TCR subunit restores development of pT-deficient thymocytes to the CD4⁺CD8⁺ stage even in the absence of conventional MHC class I and class II ligands. Moreover, we found that pre-TCR and TCR complexes exhibit no appreciable difference in their association with lipid rafts, suggesting that ligand-independence is a function of the CD4^-CD8^- (DN) thymocytes in which pre-TCR signaling occurs. In agreement, we found that only CD44^-CD25⁺ DN thymocytes (DN3) enabled activation of extracellular signal-regulated kinases by the pre-TCR complex. DN thymocytes also exhibited a lower signaling threshold relative to CD4⁺CD8⁺ thymocytes, which was associated with both the markedly elevated lipid raft content of their plasma membranes and more robust capacitative Ca²⁺ entry. Taken together these data suggest that cell-autonomous, ligand-independent signaling is primarily a property of the thymocytes in which pre-TCR signaling occurs.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Development of immature CD4^-CD8^- double negative (DN)³ thymocytes to the CD4⁺CD8⁺ double positive (DP) stage is linked to productive rearrangement of the TCR locus by signals transduced through a developmental forerunner of the TCR termed the pre-TCR complex. Although it is clear that the transition from the DN to DP stage requires pre-TCR signaling, the way in which those pre-TCR signals are initiated remains unclear.

Whereas signaling through most lymphoid surface receptors, including the mature TCR complex, is triggered by ligand-engagement, this does not appear to be the case for the pre-TCR complex, which is thought to signal in a cell-autonomous, ligand-independent manner. In support of this view, the extracellular Ig domains of both pT and TCR are dispensable for pre-TCR function (1). In the absence of the exodomain Ig loops of pT and TCR, the only potential ligand-binding sites remaining appear to be the exodomains of the CD3 subunits. CD3 subunits are expressed on the surface of thymocytes in the form of clonotype-independent CD3 complexes even in the absence of pT or TCR, yet these CD3 complexes fail to transduce -selection signals unless engaged by mAbs (2). Moreover, mAb engagement of pre-TCR complexes on pre-T cells arrests thymocyte development before their arrival at the DP stage (3). Taken together, these data suggest that engagement by a specific ligand is not responsible for initiation of pre-TCR signaling in vivo.

The ability of the pre-TCR to signal in a ligand-independent manner can be explained in two ways. First, it is possible that the ligand independence of the pre-TCR complex is an intrinsic property of the thymocyte subpopulation. Immature thymocytes in which pre-TCR signaling is initiated may have signaling thresholds that are sufficiently low to obviate the need for ligand engagement. Alternatively, the ability to transduce -selection signals when not engaged by a ligand may be an innate property of the pre-TCR complex conferred upon it by pT because pT is the only receptor subunit unique to the pre-TCR complex (4). A recent report suggested that the ligand independence of the pre-TCR results from its constitutive targeting to specialized membrane domains termed rafts, which are highly enriched in sphingolipid, cholesterol, and signaling protein components and are important in activation of immunoreceptors (5, 6, 7). Because signaling molecules have been shown to be targeted to rafts via posttranslational modification with lipids, it was proposed that pre-TCR complexes are likewise targeted to rafts via palmitoylation of a juxtamembrane cysteine residue of pT (pT-C₁₇₆) (7, 8, 9). However, mutagenesis of pT-C₁₇₆ did not impair the ability of the pre-TCR complex to signal development (10). Nevertheless, it was possible that another pT motif might enable pre-TCR complexes to signal in a ligand-independent manner. pT mutagenesis intended to address this possibility produced discrepant results. Two reports collectively indicated that the ectodomain as well as a large proportion of the intracellular tail of pT were dispensable for proper pre-TCR function (11, 12); however, another report suggested that deletion of the cytoplasmic tail moderately impaired thymocyte development (10). Even in the lone report suggesting that the cytoplasmic tail of pT plays a role in pre-TCR signaling, deletion of the tail only partially blocked thymocyte development suggesting that this is not the entire explanation. Consistent with this view, there is evidence suggesting that the pre-TCR is not unique in its ability to promote traversal of the -selection checkpoint. Indeed, despite the fact that TCR contains neither a juxtamembrane cysteine nor an extensive cytoplasmic tail and requires ligand engagement for raft targeting, the TCR complex was able to restore development of pT-deficient thymocytes beyond the -selection checkpoint (6, 13, 14). However, because the H-Y transgenic (Tg) TCR complexes used in these experiments were expressed in thymi containing selecting ligands, it is possible that MHC-ligand engagement of the TCR was responsible for the ability of TCR to substitute for pT during -selection. Consequently, it remains unclear whether the ability to signal in a ligand-independent manner is a unique property of the pre-TCR conferred upon it by pT or alternatively is an intrinsic property of the DN thymocytes in which -selection occurs.

To distinguish between these possibilities, we asked whether the TCR complex could substitute for the pre-TCR complex in the absence of conventional ligand. We demonstrate that TCR is able to complement pT deficiency even in the absence of both MHC class I and class II. Moreover, we found only limited association of the pre-TCR complex with lipid rafts and this did not differ appreciably from that observed for TCR complexes. Importantly, we did observe that thymocytes exhibited developmental differences in their ability to support pre-TCR signaling. Only CD44^-CD25⁺ DN thymocytes (DN3) were able to support cell-autonomous activation of extracellular signal-regulated kinases (ERK) by the pre-TCR complex. DN thymocytes also appear to have a lower signaling threshold than DP thymocytes, as indicated by their ability to mobilize Ca²⁺ in response to ligand engagement. This may result from the high lipid raft content of their plasma membranes, which is far greater than that of DP thymocytes. Alternatively, it may reflect differences in expression or sensitivity of calcium channels, as DN thymocytes also exhibit more robust capacitative calcium (Ca²⁺) entry than do DP thymocytes. Taken together, these data support the view that the ability to signal in a ligand-independent manner is primarily a property of DN thymocytes, rather than a unique feature of the pre-TCR complex.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

All mice were maintained under specific pathogen-free conditions in the American Association for the Accreditation of Laboratory Animal Care-accredited animal colony of the Fox Chase Cancer Center (Philadelphia, PA). Mice double deficient for MHC class I/class II were purchased from Taconic Farms (Germantown, NY); mice deficient for pT were kindly provided by Dr. H. von Boehmer (Harvard University, Cambridge, MA); TCR-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME); and TCR-Tg SCID mice were generated by backcrossing the 2B4 TCR transgene onto the SCID background in our facility (15). The background strains of mice deficient for pT, TCR, and MHC class I/class II were all a combination of 129 and C57BL/6. The background of the TCR-Tg SCID mice was C.B.-17.

Flow cytometry

Thymic lobes were gently ground with a syringe plunger in PBA (PBS, 1% BSA, 0.02% NaN₃) to produce a single cell suspension that was filtered through mesh, dispensed into a round bottom microtiter plate (1 x 10⁵–1 x 10⁶ cells per well), and stained and analyzed as previously described (16). Biotinylated, FITC-, PE- or APC-conjugated mAb specific for murine CD4, CD8, CD8, CD25, CD44, TCR, TCR, and purified anti-mouse MHC class I H-2K^b/H-2D^b and anti-mouse MHC class II I-A/I-E mAb were obtained from BD PharMingen (San Diego, CA). Where appropriate, avidin-Texas Red or streptavidin (SA)-APC (both from BD PharMingen) were used as second-step reagents.

Recombinant retrovirus production

Murine Flag-tagged pT, Flag-tagged pT-C₁₇₆A, TCR, and TCR/connecting peptide (CP)-pT (17) constructs were generated by standard PCR methodologies and subcloned into the retroviral vector LZRSpBMN-linker-IRES-eGFP (LZRS), which encompasses an internal ribosomal entry site (IRES) that allows cap independent translation of the enhanced green fluorescence protein (eGFP) marker. Retroviral vectors were transiently transfected into Phoenix-E packaging cells (Dr. G. Nolan, Stanford University, Stanford, CA) using the calcium phosphate transfection system according to the manufacturer’s protocol (Life Technologies, Paisley, Scotland).

Retroviral transduction of fetal thymocytes and fetal thymic organ culture (FTOC)

Single cell suspensions were prepared from day 14 fetal thymic lobes and transduced as previously described (18). Subsequently, equal numbers (n = 30,000) of thymocytes were transferred together with deoxyguanosine-treated fetal thymic lobes to a hanging drop in an inverted Terasaki well. After 48 h, thymocytes were examined by FACS analysis (2-day hanging drop), or alternatively, seeded lobes were placed on filter discs on gelfoam in a conventional FTOC system previously described (16) and cultured for another 2 days before FACS analysis (2-day hanging drop plus 2-day FTOC).

Analysis of lipid rafts by subcellular fractionation

A total of 20 x 10⁶ cells were surface-labeled with sulfo-NHS-LC-biotin as described (19) and lysed in 500 µl buffer A (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, and protease inhibitors) containing 0.4% Brij 58 for 1 h at 4°C. Cell lysates were adjusted to 40% sucrose (1 ml) and overlayed with 1 ml each of 30%, 20%, and 10% sucrose in buffer A. Lysates were centrifuged for 17 h at 39,000 rpm in an SW60 rotor at 4°C. Six 400-µl fractions encompassing the low-density lipid rafts were collected from the top of the tube leaving 1600 µl as the high-density detergent soluble fraction. Fractions 2–6 (100 µl) were solubilized in 10 vol of buffer A containing 1% octylglucoside. Biotin-labeled proteins were isolated using SA-Sepharose (Pierce, Rockford, IL). The beads were washed with buffer A containing 0.2% octylglucoside and eluted by boiling in SDS. pT-TCR and TCR dimers were specifically isolated from the fractions with rabbit anti-pT serum (4) and anti-TCR mAb (clone H28-710), respectively, resolved on non-reducing SDS-PAGE gels, and visualized by blotting with HRP-SA as described (19). In addition, 100 µl of each gradient fraction were TCA precipitated, resolved by SDS-PAGE, and transferred to Immobilon-P (Millipore, Bedford, MA) membranes. For protein detection, membranes were immunoblotted with the indicated primary Abs. Bound Abs were detected by incubation with either protein A-HRP or protein G-HRP followed by ECL (Renaissance, NEN, Boston MA). For GM1 ganglioside (GM1) detection, blots were incubated with HRP-labeled cholera toxin B (CTxB) followed by ECL.

Immunofluorescence

Cells were incubated for 30 min at 37°C to allow attachment to poly-L-lysine-coated chamber slides, then fixed with 1% paraformaldehyde/PBS pH 7.0 for 10 min at room temperature (RT). Fixed cells were quenched in 50 mM NH₄Cl for 10 min at RT and subsequently blocked for 30 min with 1% BSA/HBSS containing 5% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were stained for 30 min at RT with the following reagents: hamster anti-CD3 (clone 500A2), rat anti-TCR-V11 (clone RR8-1), rat anti-transferrin receptor (Tf-R) (clone R17 217.1.3), or biotinylated CTxB (Sigma-Aldrich, St. Louis, MO). After washing twice in 1% BSA/HBSS, bound mAb or CTxB was visualized by incubation for 30 min at RT with the appropriate fluorescent visualizing agent: goat anti-hamster Rhodamine Red, goat anti-rat-Rhodamine Red, goat anti-rat Cy5, or streptavidin-Cy5 (all from Jackson ImmunoResearch Laboratories). After washing three times with 1% BSA/HBSS, stained samples were examined using a laser scanning confocal microscope (Bio-Rad, Hercules, CA).

Measurement of ERK activity

DN3, DN4, and DP thymocytes were sorted from TCR-deficient mice with a FACSDiVa cell sorter. Sorted populations were 99.8% pure, as determined by post-sort analysis. Before cell sorting for DN3 and DN4 cells, DN cells were enriched by anti-CD8 complement-mediated lysis, as previously described (20). TCR⁺ cells were not specifically excluded and in TCR-deficient thymocytes comprise 0.3–0.7%, 8–13%, and 0.04–0.1% of the DN3, DN4, and DP subsets, respectively. Thymocytes were transfected with PathDetect reporter plasmids (Stratagene, La Jolla, CA) by electroporation using a BTX ElectroCell Manipulator 600 (San Diego, CA), as previously described (21). Briefly, for each sample, 6.5 x 10⁶ cells in 250 µl RPMI 1640 supplemented with 20% FCS were incubated on ice for 10 min with either 20 µg of pFR-luc alone or with 20 µg of pFA-Elk. pCMV-gal (10 µg; Stratagene) was included to determine transfection efficiency. DN3 and DN4 thymocytes were pulsed at 260 V, 1700 µF, 186 with a time constant of 85 ms. DP thymocytes were pulsed at 300 V, 800 µF, 186 with a time constant of 45 ms. After electroporation, cells were incubated on ice for 10 min and cultured in medium for 22 h. At 16 h posttransfection, cells were stimulated with 10 ng/ml PMA and 1 ng/ml ionomycin (Sigma-Aldrich), as indicated, and incubated for a further 6 h before analysis. Transfected thymocytes were assayed for luciferase and -galactosidase activities using the Dual-Light reporter gene assay system (Tropix, Bedford MA), as described (21). Results represent the average luciferase activity of assays conducted in triplicate and indexed for -galactosidase activity.

Cytosolic Ca²⁺ mobilization

Thymocytes (5 x 10⁶/ml) were loaded with the acetoxy-methyl ester of Indo-1 (5 µM) (Sigma-Aldrich) by incubation in the dark for 45 min at 37°C in Iscove’s modified Dulbecco’s medium containing 10 mM HEPES buffer and 2% FCS (Ca-buffer) and supplemented with Indo-1 and 0.01% pluronic acid (Molecular Probes, Eugene, OR). Cells were washed twice and incubated with purified anti-V3 (clone KJ25), anti-CD4, anti-CD8, anti-CD44, and anti-CD25 mAb for 30 min at 4°C. After two more washes, the cells were resuspended at 2 x 10⁶/ml in Ca-buffer and immediately analyzed on a FACSVantage SE flow cytometer at 37°C. After 30 s, cells were stimulated by addition of 30 µg/ml goat anti-hamster IgG (ICN Cappel, Costa Mesa, CA). Successful loading with Indo-1 was confirmed by the addition of 1 µM A23187 at the end of the experiment. For intracellular Ca²⁺ store depletion-activated Ca²⁺ influx, cells were loaded with Indo-1 and subsequently cell surface stained with CD4, CD8, CD44, and CD25 as previously described. Cells were washed twice and resuspended in Ca-buffer containing 2% BSA (instead of FCS) and 2 mM EGTA. Analysis followed immediately on a FACSVantage SE flow cytometer at 37°C. After 30 s, cells were treated with 100 nM thapsigargin (Molecular Probes), and after 6 min, 2 mM Ca²⁺ was added to the cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR can overcome the arrest in pre-T cell development caused by pT deficiency

One of the most intriguing aspects of pre-TCR complex function is its ability to transduce signals in an apparently cell-autonomous, ligand-independent manner. To determine whether this capability is unique to the pre-TCR complex, we assessed whether TCR was able to overcome the developmental arrest resulting from pT deficiency (22). It is of interest to assess the ability of TCR to do so because TCR can be rearranged and expressed in the CD44^-CD25⁺ DN3 thymocyte subset in which -selection occurs (23, 24), and because TCR lacks the structural features of pT thought to underlie its constitutive targeting to lipid rafts and ligand-independent signaling (10). d14 pT-deficient fetal thymocytes were retrovirally transduced with TCR or pT before reconstituting T cell-depleted C57BL/6 thymic lobes. In pT-deficient mice, the transition from the CD44^-CD25⁺ DN3 to the CD44^-CD25^- DN4 stage of T cell development is severely impaired, with few cells progressing to the DP or CD4⁺ and CD8⁺ single positive stages (22). Clearly, expression of both pT and TCR effectively restored the transition of CD4^-CD8^- DN thymocytes to the DP stage and the cellular proliferation associated with this transition (Fig. 1). Introduction of TCR into the pT-mutant background increased the percentage of DP thymocytes (from 16 to 49%) as effectively as the pT wild-type control (Fig. 1A, top panel). Furthermore, subdivision of the DN population based on CD44 and CD25 expression revealed that TCR transduction induced the formation of a large proportion of DN4 cells and a concomitant reduction in the population size of the preceding DN3 subset, indicating that expression of TCR released the developmental block at the DN3 stage that normally accompanies pT deficiency (Fig. 1A, bottom panel). In addition to overcoming the arrest in pre-T cell differentiation, introduction of TCR also induced a burst in cell proliferation, another hallmark of -selection (Fig. 1B). Although there is a difference in the initial transduction efficiency (Fig. 1B, top panel), a 2-fold increase in the percentage of eGFP⁺ cells and a 4-fold increase in the absolute number of eGFP⁺ cells can be observed for both pT and TCR transduced thymocytes during 2 days in FTOC. Finally, although pre-TCR signaling promotes development of lineage T cells, it has been reported to antagonize T cell development (22). Consistent with these observations, we found that expression of TCR antagonized the development of T cell lineage as well as did pT (our unpublished observation). In agreement with previous studies, we also found that expression of a pT mutant in which the juxtamembrane cysteine (C₁₇₆) was mutated to alanine (pT-C₁₇₆A) complemented pT deficiency as well as did wild-type pT or TCR (Fig. 1). It is important to note that the ability of TCR to complement pT deficiency is not due to its overexpression as surface expression of all of the transduced receptor subunits was comparable to that observed in DN4 thymocytes from C57BL/6 mice (Fig. 1C). Moreover, the ability of TCR to complement pT deficiency is not due to its assembly into TCR complexes, which result in DP thymocytes lacking surface TCR complexes due to silencing of TCR expression in DP thymocytes, (25) as the majority of DP thymocytes generated in our TCR transduced cultures express TCR on the cell surface (our unpublished observation). Taken together, these data demonstrate that pT, pT-C₁₇₆A, and TCR display equivalent abilities to surmount the defect in development associated with pT-deficiency.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Both the pT-C176A mutant and TCR can promote development of pT-deficient thymocytes beyond the -selection checkpoint. Fetal pT-deficient thymocytes transduced with empty vector control (LZRS), pT, pT-C176A, or TCR were used to reconstitute T cell-depleted C57BL/6 wild-type lobes and analyzed immediately after the 2-day hanging drop culture (2d hanging drop) or after the seeded lobes had been cultured an additional 2 days in FTOC (2d hanging drop + 2d FTOC). A, Thymocytes were monitored for the expression of CD4 vs CD8 (top panel) and for expression of CD44 vs CD25 within the DN thymocyte subset (bottom panel). T cells were excluded by negatively gating on TCR+ cells. Only transduced (eGFP+) thymocytes are shown and the percentage of cells within each quadrant is indicated. B, Total thymocytes were analyzed for the expression of eGFP after 2-day hanging drop (top panel) or 2-day hanging drop plus 2-day FTOC (bottom panel). The absolute number of eGFP+ cells present in eight reconstituted lobes is indicated above the corresponding histogram. C, Fetal C57BL/6 thymocytes were transduced with empty vector control (LZRS), and pT-deficient thymocytes were transduced with empty vector control (LZRS), pT, pT-C176A, or TCR and used to reconstitute T cell depleted C57BL/6 wild-type lobes. DN4 (TCR-) thymocytes were monitored for the expression of TCR. Only the transduced (eGFP+) thymocytes are shown and the percentage of TCR+ cells as well as the mean fluorescence intensity (MFI) of the TCR+ population is indicated. All results are representative of three experiments performed.

TCR does not require conventional ligand to complement pT deficiency

In the experimental setting in Fig. 1, it is possible that the mature TCR complex was able to substitute for the pre-TCR because of interaction with ligands present in the thymic microenvironment. To investigate this possibility, d14 pT-deficient fetal thymocytes were transduced with pT and TCR before reconstituting T cell-depleted MHC class I/class II double-deficient lobes (Fig. 2). Strikingly, expression of the TCR subunit can effectively reverse the phenotype associated with pT deficiency even in the absence of its MHC class I and class II ligands (Fig. 2A). It is important to note that the d14 pT-deficient fetal precursors used to reconstitute MHC class I/class II-deficient lobes contain a small subpopulation of cells (0.1%) with the potential of developing into MHC class I/class II-expressing thymic dendritic cells. To ensure that initiation of signaling by the mature TCR at the pre-T cell stage is indeed ligand-independent, blocking mAb to MHC class I H-2K^b/H-2D^b and MHC class II I-A/I-E were added immediately after transduction. The concentration of blocking anti-MHC class I/class II mAb used was sufficient to abolish positive selection in C57BL/6 lobes dissected at day 15 of gestation and cultured in FTOC for 11 days (our unpublished observation). Interestingly, even in the presence of blocking anti-MHC class I/class II mAb, expression of TCR in pT-deficient thymocytes resulted in the efficient generation of DN4 cells (Fig. 2B), strongly suggesting that the TCR complex does not require conventional ligand to signal.

View larger version (62K): [in this window] [in a new window]   FIGURE 2. Expression of TCR can restore development of pT-deficient thymocytes in the absence of MHC class I and class II. Fetal pT-deficient thymocytes were transduced with empty vector control (LZRS), pT, TCR, or TCR/CP-pT and used to reconstitute T cell-depleted MHC class I/class II double-deficient lobes (as in Fig. 1) in the absence (A) or presence (B) of 100 µg/ml blocking anti-MHC class I H-2Kb/H-2Db and anti-MHC class II I-A/I-E mAb. Results are representative of three experiments performed. Notably, as was true in Fig. 1B, TCR transduction of thymocytes seeded into MHC-deficient host lobes also resulted in an 4-fold increase in the number of eGFP+ cells following 2 days in FTOC.

Only a small fraction of surface pre-TCR complexes localize to lipid rafts

Previous analysis suggested that pre-TCR complexes signal in a cell-autonomous, ligand-independent manner because most pre-TCR complexes (77%) are constitutively targeted to lipid rafts (5). TCR complexes, in contrast, are thought to require ligand engagement to be targeted to lipid rafts (6, 13). Because our analysis indicated that the pre-TCR was not unique in its ability to signal in a ligand-independent manner, we assessed whether pre-TCR and TCR complexes differed in their targeting to rafts using both immunofluorescence microscopy and subcellular fractionation (Fig. 3). SCID thymic lymphoma cell lines expressing either pre-TCR complexes (SL-343.1) or TCR complexes (SL-343.1) (19) were stained with GM1-binding CTxB subunit as a marker for lipid rafts and either anti-CD3 mAb to identify pre-TCR complexes (SL-343.1) or anti-TCR-V11 mAb to identify TCR complexes (SL-343.1) (Fig. 3, A and B). Evaluation of the extent of overlap of the resultant staining patterns revealed that only a minority (an estimated 10%) of pre-TCR complexes colocalized with rafts, and this was not appreciably different for TCR complexes. Consistent with this observation, the staining pattern for the pre-TCR complex overlapped more extensively with that of the Tf-R, which is excluded from rafts (13, 26) (Fig. 3C). Confocal microscopy on primary thymocytes from TCR-Tg SCID mice also suggested that only a minor fraction of the pre-TCR complexes colocalize with lipid rafts (Fig. 3D). Finally, sucrose gradient fractionation revealed that >80% of surface pre-TCR complexes were excluded from rafts and this fraction was equivalent to that of the TCR complex (Fig. 3E). As previously described, the raft components, LAT and GM1, were detected in low-density raft-containing fractions while the cytosolic protein tubulin and membrane receptor Tf-R were primarily present in the soluble high-density fraction. Taken together, our immunofluorescence and biochemical analyses suggest that pre-TCR complexes associate with rafts in approximately the same low stoichiometry exhibited by TCR complexes.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Only a small proportion of pre-TCR complexes localize in lipid rafts. Subcellular localization of pre-TCR and TCR complexes was assessed by laser scanning confocal microscopy (A–D) and sucrose gradient fractionation (E). Colocalization of pre-TCR complexes with GM1-containing lipid rafts was analyzed using a pre-TCR-expressing (SL-343.1) SCID thymic lymphoma cell line (A) and primary thymocytes from 2B4-TCR-Tg SCID mice (D). Cells were stained with anti-CD3 mAb to identify the pre-TCR complex and with CTxB to identify lipid rafts. We have verified that the anti-CD3 staining detects pre-TCR complexes (not clonotype-independent CD3 complexes) in SL-343.1 cells because the staining is dependent upon transduction with TCR (data not shown). Colocalization of TCR complexes with lipid rafts was assessed by staining TCR-expressing SCID thymic lymphoma cells (SL-343.1) with anti-TCR-V11 mAb and CTxB as previously described (B). It should be noted that because SL-343 lymphoma cells express pT, cells transduced with TCR and TCR (SL-343.1) express both pre-TCR and TCR complexes. This required the use of an anti-TCR Ab to assess the raft association of TCR complexes. Nevertheless, when SL-343.1 cells were co-stained with anti-V11 and anti-CD3 Abs, we could not detect any significant difference in the staining patterns (data not shown). Colocalization of the pre-TCR with Tf-R was assessed by staining for the pre-TCR as in A and with anti-Tf-R mAb (C). Confocal slices of 0.5-µM thickness are depicted and the extent of overlap is revealed by yellow fluorescence in the electronically merged slices (A–D, right panels). E, Association of pre-TCR and TCR complexes with lipid rafts was assessed by sucrose gradient fractionation of surface biotin-labeled SL-343.1 and SL-343.1 cells. Surface-labeled pT-TCR and TCR heterodimers were isolated from the gradient fractions, resolved by non-reducing SDS-PAGE, and visualized by blotting with HRP-SA and chemiluminescence. Western blot analysis was performed on TCA-precipitated sucrose gradient fractions with Ab reactive to the indicated marker proteins. H, high-density detergent soluble fraction.

The observation that pre-TCR complexes are found in rafts in approximately the same low stoichiometry as TCR complexes are found supports the viewpoint that ligand-independent signaling is not unique to the pre-TCR complex but rather is an intrinsic property of the DN thymocytes in which pre-TCR signaling is initiated. To address this possibility, we asked whether DN and DP thymocytes differed in their ability to support ERK activation by the pre-TCR complex because several reports indicate that the Ras/Raf/MEK/ERK signaling cascade is important for development of thymocytes beyond the -selection checkpoint (27, 28, 29). DN3, DN4, and DP thymocytes were isolated from TCR-deficient mice and the endogenous levels of cellular ERK activity were measured using a reporter plasmid-based system (29). TCR-deficient thymocytes were used because their inability to express the TCR complex enables us to evaluate pre-TCR function in both DN and DP thymocytes. Importantly, ERK activity is not detected in pre-TCR-deficient DN3 thymocytes using this assay (21). Fig. 4A shows that TCR-deficient DN3 thymocytes display a significant level of ERK activity as compared with DN4 or DP thymocytes. It should be noted that the level of ERK activity detected in the DN3 subset is likely to be an underestimate because only 15% of DN3 in TCR-deficient mice is thought to express surface pre-TCR complexes (30). The lack of any detectable ERK activity within DN4 and DP cells does not reflect an inability to induce this signaling cascade, as stimulation with PMA and ionomycin results in a strong induction of ERK activity (Fig. 4A). These data suggest that ligand-independent pre-TCR signaling is able to activate ERK in DN3 cells, but not in later developmental stages.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. DN thymocytes display a lower signaling threshold compared with DP thymocytes. A, DN3, DN4, and DP thymocytes were isolated from TCR-deficient mice and transfected with reporter plasmids (pFR-Luc + pFA-ELK) for the detection of ERK activity. ERK function was measured as induced luciferase activity in the transfected thymocyte subsets. Following transfection, thymocytes were cultured for 22 h; for stimulated DP thymocytes, PMA and ionomycin were added 6 h before analysis. The broken line shown represents the background luciferase activity from thymocytes transfected with the luciferase reporter construct (pFR-Luc) alone. Transfection efficiency was monitored by cotransfection with a control -galactosidase expression plasmid. B, Mobilization of cytosolic Ca2+ was analyzed in electronically gated DN4 thymocytes (thick black line), total DN thymocytes (thick gray line), or DP thymocytes (thin black line) derived from adult 2B4-TCR-Tg SCID mice. Cells were treated with indicated concentrations of anti-V3 mAb (clone KJ25) followed 30 s later by cross-linking with goat anti-hamster (GH) Ab. Successful loading with Indo-1 was confirmed by the addition of 1 µM A23187 at the end of the experiment. Data are shown as the ratio of fluorescence of Ca2+-bound (violet)-Ca2+-unbound (blue) Indo-1. C, Cell surface expression of TCR was assessed by flow cytometry on electronically gated DN (open areas) and DP (shaded areas) thymocytes derived from 2B4-TCR-Tg SCID mice. D, Intracellular Ca2+ store depletion-activated Ca2+ influx was analyzed in electronically gated DN4 thymocytes (thick black line), DN3 thymocytes (thin gray line), total DN thymocytes (thick gray line), or DP thymocytes (thin black line) derived from 2B4-TCR-Tg SCID mice (top panel) and from TCR-deficient mice (bottom panel). Cells were treated with 100 nM thapsigargin (TG) 30 s after the analysis was initiated, and after 6 min 2 mM Ca2+ was added. Data are shown as the ratio of fluorescence of Ca2+-bound (violet)-Ca2+-unbound (blue) Indo-1.

We hypothesized that one explanation for the ability of DN thymocytes to support ligand-independent signaling is that their signaling thresholds might be sufficiently low to obviate the need for ligand engagement. To investigate this possibility, we assessed the relative abilities of pre-TCR complexes expressed on DN and DP thymocytes to mobilize cytosolic Ca²⁺ in response to stimulation by a defined ligand. We used thymocytes from TCR-Tg SCID mice, which like the TCR-deficient mice express the pre-TCR complex (but not TCR complexes) on both DN and DP thymocytes (31, 32). TCR-Tg SCID thymocytes were used instead of TCR-deficient thymocytes, because we were only able to evoke Ca²⁺ responses after stimulation with the potent anti-V3 mAb specific for the V3 Tg (data not shown). Strikingly, V3 cross-linking of pre-TCRs expressed at the surface of DN thymocytes, and in particular the DN4 subset, evoked a marked increase in cytosolic Ca²⁺ (Fig. 4B). In this particular assay, DN4 thymocytes represent the most appropriate population to compare with DP thymocytes because DN4 cells are the earliest population in which most cells express the pre-TCR complex. The increase in intracellular free Ca²⁺ was observed over a wide range of anti-V3 mAb concentrations used and displayed a biphasic pattern with an initial rapid rise followed by decline to a sustained Ca²⁺ plateau. In sharp contrast, stimulation of pre-TCR complexes expressed at the DP stage resulted in only a slight increase in intracellular Ca²⁺. Anti-V3 stimulation of DN3 thymocytes did not evoke a detectable increase in intracellular free Ca²⁺, presumably because only few cells in this subpopulation express the pre-TCR complex. These data, together with the finding that pre-TCR expression may be marginally higher in DP than in DN thymocytes from TCR-Tg SCID mice (Fig. 4C), suggest that DN thymocytes have a lower signaling threshold compared with DP thymocytes.

We reasoned that the greater increase in cytosolic Ca²⁺ observed following mAb stimulation of DN4 thymocytes might result in part from a greater capacity to facilitate Ca²⁺ influx through Ca²⁺ release-activated Ca²⁺ (CRAC) channels, which are characterized by receptor-independent opening in response to depletion of intracellular Ca²⁺ stores (33). Consequently we asked whether DN and DP thymocytes (from TCR-Tg SCID and TCR-deficient mice) differed in their capacity to facilitate influx ofextracellular Ca²⁺ following depletion of intracellular Ca²⁺ stores with the SERCA Ca²⁺-pump inhibitor, thapsigargin. Indeed, thapsigargin treatment resulted in a greater influx of extracellular Ca²⁺ in DN4 thymocytes than in DP thymocytes (Fig. 4D). Interestingly, thapsigargin treatment of DN3 thymocytes also resulted in greater Ca²⁺ influx, presumably because thapsigargin-mediated depletion of intracellular Ca²⁺ stores bypasses the requirement for a receptor signal. The increased Ca²⁺ influx in DN3 and DN4 thymocyte populations may result from expression of more CRAC channels or theoretically through their more efficient opening in response to store depletion.

Another parameter that might influence the signaling threshold of thymocytes is the raft content of their plasma membranes. In support, CD4⁺ memory T cells that are thought to have a lower signaling threshold than their naive counterparts also reportedly have a higher density of lipid rafts in their plasma membranes (34). Moreover, rafts have been shown to be required for effective Ca²⁺ mobilization induced by ligand engagement of the pre-TCR (35). Consequently, we assessed raft content of the plasma membranes of thymocyte subpopulations (Fig. 5). Staining of wild-type fetal thymocytes with CTxB suggests that the plasma membranes of DN thymocytes, and in particular DN3 and DN4 cells, exhibit far higher lipid raft content than DP thymocytes (Fig. 5).

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Thymocytes at the DN stage have higher lipid raft content compared with DP thymocytes. Cell surface levels of GM1 were measured on day 17 of fetal thymocytes cultured for 3 days in FTOC (A) or day 15 (B) fetal thymocytes derived from wild-type C57BL/6 mice by staining with biotinylated CTxB followed by avidin-Texas Red. Shown are electronically gated, DN, DP, and SP thymocytes (A) or DN subpopulations (DN1–4) characterized by differential expression of CD44 vs CD25 (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous reports suggested that pre-TCR complexes signal in a cell-autonomous, ligand-independent manner because an undefined structural motif unique to pT constitutively targets them to lipid rafts (5, 10). In this study we present data indicating that the pre-TCR is not unique in its ability to signal in a ligand-independent manner. Indeed, the TCR subunit restores development of pT-deficient thymocytes even in the absence of its MHC class I and class II ligands (Fig. 2), suggesting that when expressed at the DN stage, the TCR complex does not require conventional ligand to initiate signaling. This is significant because TCR lacks motifs equivalent to those of pT thought to underlie ligand independence of the pre-TCR complex. Moreover using both microscopic and biochemical approaches, we found only limited association of the pre-TCR complex with lipid rafts, and the fraction of surface pre-TCRs associated with lipid rafts did not differ appreciably from that observed for TCR complexes (Fig. 3), suggesting that ligand-independent signaling is primarily a function of the DN thymocytes in which -selection signaling occurs.

Lipid microdomains act as repositories of signaling molecules that serve as essential platforms for signaling reactions in many cells (36). Specifically in T cells, the TCR complex is coupled to downstream signaling pathways through its ligand-induced association with rafts (6, 13). Although there has been no definitive demonstration that rafts are required for ligand-independent pre-TCR signaling, there is evidence consistent with that possibility (5), which raises the question of how the pre-TCR interaction with rafts is initiated in the absence of ligand. Previous analyses suggested that a motif particular to pT mediates constitutive targeting of a large fraction of pre-TCR complexes (77%) to lipid rafts (5, 10). However, we find a much lower fraction of pre-TCR complexes associated with lipid rafts, which is estimated to be 10% (Fig. 3). It is unclear why we find a substantially lower fractional association of pre-TCR complexes with rafts, but this may result from differences in experimental procedures. For example, limited fixation conditions are required to maintain plasma membrane integrity of thymocytes and thymic lymphoma lines (data not shown). Consequently, the extensive colocalization of the pre-TCR with lipid rafts reported previously may result in part from staining of internal structures because lipid rafts form in the Golgi complex (36). Our finding that the pre-TCR and TCR do not differ significantly regarding the extent of raft localization has two important implications for ligand-independent signaling. First, because the TCR complex can promote traversal of the -selection checkpoint in the absence of conventional ligand, the structural motif underlying this capability need not be particular to pT. Second, if constitutive raft association is important to signal without ligand engagement, association in relatively low stoichiometry is sufficient to initiate signaling. How then might raft localization be accomplished in the absence of ligand? Lipid modification of cysteine residues has been shown to be required for raft localization of a number of signaling molecules such as LAT, Ras, and Src kinases (7, 8, 37, 38). Nevertheless, the single juxtamembrane cysteine (C₁₇₆) residue of pT, which can be palmitoylated in vitro, appears to be dispensable for pre-TCR function (Fig. 1) (5, 10). Recent analysis suggests that the cytoplasmic tail of pT might be important because its deletion or mutagenesis partially impairs pre-TCR function (10); however, TCR is able to signal without conventional ligand and lacks an extended cytoplasmic tail. Consequently, while the cytoplasmic tail of pT may be important for some aspects of surface pre-TCR expression or function, it remains to be determined whether it contributes specifically to the purported ligand-independence of the pre-TCR complex or its modest association with rafts. The ability of the TCR to signal in the absence of conventional ligand at the DN stage, but not at the DP stage, suggests that ligand-independence is a function of the cells in which -selection signals are transduced. In support, we found that the pre-TCR complex was able to activate ERK signaling in a ligand-independent manner only in DN3 thymocytes. Although the mechanistic basis for the ability of DN thymocytes to support ligand-independent signaling by TCR and pre-TCR complexes remains unclear, we provide evidence that early DN thymocytes exhibit a lower signaling threshold than shown by DP thymocytes. Among the many adaptations (including differential expression of signaling molecules) that might contribute to setting cellular signaling thresholds, we have found that DN thymocytes exhibit both markedly elevated lipid raft content in their plasma membranes and a greater capacity to support capacitative Ca²⁺ entry (Figs. 4B and 5). The elevated lipid raft content could facilitate ligand-independent signaling through increasing the frequency or duration of undirected interactions of the pre-TCR complex with lipid rafts, such that the activation threshold can be exceeded even in the absence of ligand engagement. Alternatively, the frequency/duration of pre-TCR/raft association may not differ appreciably, and the increased raft content may instead serve to amplify comparatively weak signals. It is also possible that relatively weak signals may by amplified due to Ca²⁺ oscillations resulting from increased expression of CRAC channels and/or (theoretically) more efficient coupling of their activation to modest depletion of intracellular Ca²⁺ stores (33). The ability to signal in a ligand-independent manner (as indicated by ERK activity) appears to be lost at later stages of development perhaps due to induction of negative regulators of signaling such as CD5 or the Src-like adaptor protein, SLAP, which are both thought to be induced in response to pre-TCR signaling (39, 40). Interestingly, the signaling threshold can still be overcome in DN4 thymocytes by increasing pre-TCR signal strength through ligand engagement, although this ability is also lost upon arrival at the DP stage (Fig. 4B). Ligand engagement of the TCR complex is able to overcome the signaling threshold of DP thymocytes, presumably because its expression level is far higher than that of the pre-TCR complex.

Although we have found that the TCR complex can promote traversal of the -selection checkpoint even in the absence of MHC class I/class II ligands, previous attempts to address this question have produced contradictory results. In one TCR-Tg model, pre-T cell development appeared normal both on wild type and pT-deficient backgrounds (14). However, in other Tg models, premature expression of TCR complexes impaired progression beyond the -selection checkpoint, reduced thymic cellularity, and caused accumulation of TCR-expressing DN thymocytes (41, 42). This may have resulted from ligand engagement of a Tg TCR complex that was overexpressed at an extremely early stage of development, and which induced either negative selection at the DP stage or developmental arrest of DN thymocytes. Therefore, one possible explanation for the contradictory results is that in model systems in which the TCR is expressed at relatively low physiologic levels, such as in our retrovirally transduced thymocytes (Fig. 1C), pre-TCR function is closely mimicked and differentiation toward the T cell lineage is promoted. In contrast, premature overexpression of the TCR may result in an excessively intense signal that retards development toward the T cell lineage. Therefore, it may be the strength of the signal transduced, rather than any intrinsic difference in pre-TCR and TCR structure, that is the primary determiner of a receptor’s ability to promote T cell development. This view is corroborated by studies showing that increasing pre-TCR signal strength, either by ligation with anti-TCR mAb or by overexpressing signaling molecules (p38 mitogen-activated protein kinase and Lck), impairs development of thymocytes (3, 43, 44). Taken together, our findings support the view that the ability to signal in a ligand-independent manner is primarily a property of DN thymocytes rather than a unique feature of the pre-TCR complex. The modulation of cellular signaling thresholds during lymphoid development may be an important general mechanism for controlling receptor function.

	Acknowledgments

 
We thank Drs. D. Kappes and A. Singer for critically reviewing the manuscript, Drs. S. Shinton and J. Boyd for excellent technical assistance, Dr. H. von Boehmer for providing pT-deficient mice, and Dr. B. Freedman for helpful discussions. We are also grateful for the assistance of the following core facilities at Fox Chase Cancer Center: Cell Culture, Cell Imaging, DNA Sequencing, DNA Synthesis, Flow Cytometry, Laboratory Animal, and Special Services.

	Footnotes

 
¹ This work was supported by American Cancer Society Grant RSG-01-084-01-LIB, National Institutes of Health Grants CA73656 and CA087047, National Institutes of Health Core Grant P01CA06927, and an appropriation from the Commonwealth of Pennsylvania. M.C.H. was supported by TALENT-Stipendium S 92-210 from the Netherlands Organization for Scientific Research (NWO-MW) and by the Fox Chase Cancer Center Board of Directors’ Postdoctoral Fellowship.

² Address correspondence and reprint requests to Dr. David L. Wiest, Division of Basic Sciences, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. E-mail address: DL_Wiest{at}fccc.edu

³ Abbreviations used in this paper: DN, CD4^-CD8^-; DP, CD4⁺CD8⁺; CP, connecting peptide; CRAC, Ca²⁺ release-activated Ca²⁺; CTxB, cholera toxin B subunit; Tg, transgenic; DN3, CD44^-CD25⁺CD4^-CD8^-; DN4, CD44^-CD25^-CD4^-CD8^-; eGFP, enhanced green fluorescent protein; ERK, extracellular signal-regulated kinase; FTOC, fetal thymic organ culture; GM1, GM1 ganglioside; LZRS, LZRSpBMN-linker-IRES-eGFP; pT, pre-T; pTC₁₇₆, cysteine 176 of pT; SA, streptavidin; Tf-R, transferrin receptor.

Received for publication October 29, 2002. Accepted for publication January 8, 2003.

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