Identification of a novel pre-TCR isoform in which the accessibility of the TCRß subunit is determined by occupancy of the `missing' V domain of pre-T{alpha}

Marc A. Berger1,5, Michael Carleton1, Michele Rhodes1, J. Michael Sauder2, Sébastien Trop3, Roland L. Dunbrack2, Patrice Hugo4 and David L. Wiest1

1 Immunobiology Working Group, and
2 Biomolecular Structure and Function Working Group, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
3 Department of Medicine, Division of Experimental Medicine, McGill University, Montréal, Québec H3A 1A3, Canada
4 PROCREA BioSciences, Inc., Montreal, Québec H4P 2R2, Canada

Correspondence to: D. L. Wiest


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have identified a novel pre-TCR isoform that is structurally distinct from conventional pre-TCR complexes and whose TCRß chains are inaccessible to anti-TCRß antibodies. We term this pre-TCR isoform the MB (masked ß)-pre-TCR. Pre-T{alpha} (pT{alpha}) subunits of MB-pre-TCR complexes have a larger apparent mol. wt due to extensive modification with O-linked carbohydrates; however, preventing addition of O-glycans does not restore antibody recognition of the TCRß subunits of MB-pre-TCR complexes. Importantly, accessibility of TCRß chains in MB-pre-TCR complexes is restored by filling in the `missing' variable (V) domain of pT{alpha} with a V domain from TCR{alpha}. Moreover, the proportion of pre-TCR complexes in which the TCRß subunits are accessible to anti-TCRß antibody varies with the cellular context, suggesting that TCRß accessibility is controlled by a trans-acting factor. The way in which this factor might control TCRß accessibility as well as the physiologic relevance of TCRß masking for pre-TCR function are discussed.

Keywords: pre-TCR, pT{alpha}, structural model, thymocyte development, Vpre-T


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Maturation of immature CD4CD8 (double-negative [DN]) thymocytes to the CD4+CD8+ (double-positive [DP]) stage requires traversal of a developmental checkpoint termed ß-selection. ß-Selection stipulates that only those DN precursors that maintain the translational reading frame of TCRß during rearrangement of the TCRß gene locus will survive and mature beyond the CD44CD25+ stage; those which fail to do so will die by apoptosis (15). Expression of a functional TCRß protein enables CD44CD25+ precursors to traverse the ß-selection checkpoint by facilitating assembly and activation of a developmental forerunner of the {alpha}ßTCR complex termed the pre-TCR. The pre-TCR complex is distinguished from the {alpha}ßTCR complex by the presence of an invariant 33 kDa subunit termed pre-T{alpha} (pT{alpha}) (6). Interestingly, because pT{alpha} contains only a single extracellular Ig-like domain equivalent to the constant domain of TCR{alpha}, the existence of an additional pre-TCR component equivalent to the TCR{alpha} variable (V) domain has been proposed (79); nevertheless, no evidence for such a component has been presented to date. Pre-TCR complexes comprise disulfide-linked heterodimers of pT{alpha} and TCRß non-covalently associated with the CD3{gamma}{delta}{varepsilon} and TCR{zeta} signaling components. All of these biochemically defined pre-TCR subunits (except {delta}) are required for pre-TCR function since their elimination by gene targeting inhibits development of DN thymocytes beyond the CD44CD25+ stage (1022). However, despite the clear genetic evidence that pre-TCR signaling is required for precursors to traverse the ß-selection checkpoint, our understanding of how expression of the TCRß protein product triggers those pre-TCR signals remains rudimentary at present (23,24).

Most receptor signals are initiated by ligand engagement, for which antibody usually serves as an effective surrogate; however, this does not appear to be the case for the pre-TCR complex. Indeed, the exodomains of pT{alpha} and TCRß can be eliminated without adversely affecting the pre-TCR's ability to signal thymocyte development (2528). Moreover, antibody engagement of surface pre-TCR complexes actually inhibits development of thymocytes to the DP stage (2931). Two different models of ligand-independent pre-TCR signaling have been proposed: (i) the surface expression model postulates that pre-TCR signaling requires accumulation of completely assembled pre-TCR complexes within the plasma membrane, possibly because of their apposition with glycolipid-enriched microdomains which are important in T cell activation (25,3234), or (ii) the internal signaling model postulates that pre-TCR signals are initiated during assembly of pre-TCR complexes within the endoplasmic reticulum or during their transport to the cell surface (32,35). These models are currently unresolved.

One of the impediments to understanding how thymocyte development is controlled by the pre-TCR complex is the possibility that immature thymocytes express variant pre-TCR isoforms which have different functions. Such isoforms of the pre-B cell receptor (BCR) have been detected in B lymphoid precursors (8). The pre-BCR comprises Ig{alpha} and Igß signaling subunits in association with a dimer of Igµ heavy chains, each of which is disulfide-linked to surrogate light chain (SLC), a dimeric complex containing a constant (C) domain-like {lambda}5 subunit and V domain-like Vpre-B subunit (36,37). An isoform of the pre-BCR which lacks Igµ is expressed on the surface of pro-B cells (38,39). It has not been established whether an equivalent isoform of the pre-TCR complex lacking TCRß is expressed on early thymocytes.

We report here that immature thymocytes (and thymic lymphoma cells) express a pre-TCR isoform whose TCRß chains are unrecognizable by antibody specific for either the Vß or Cß domains. Consequently, we termed this novel pre-TCR isoform the MB (masked ß)-pre-TCR. MB-pre-TCR complexes represent approximately half of the pre-TCR complexes expressed on the surface of both normal thymocytes and also most thymic lymphoma lines. However, one of the lymphoma lines (SL-12ß.12) is virtually devoid of the MB-pre-TCR isoform. The differential representation of MB-pre-TCR complexes in different lymphoma lines suggests that their expression may be controlled by some trans-acting factor(s). The masking of TCRß in MB-pre-TCR complexes depends upon association of TCRß with pT{alpha} and correlates with extensive O-glycosylation of pT{alpha}; nevertheless, preventing O-glycosylation does not restore antibody access to the TCRß subunits of MB-pre-TCR complexes. Antibody access is restored if the vacant V region of pT{alpha} is filled in with a V domain from a TCR{alpha} subunit. Taken together, these data provide indirect evidence that antibody access to TCRß may be governed by the presence of an additional pre-TCR subunit (possibly the `Vpre-T' subunit proposed by others) that occupies the vacant V domain of pT{alpha} (7,23). The mechanistic basis for masking of the TCRß subunits of MB-pre-TCR complexes and its potential impact on pre-TCR function are discussed.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
The C57BL/6 mouse strain as well as mice lacking TCR{alpha} expression (TCR{alpha}-/–) due to gene targeting (20,40) were obtained from Jackson Laboratories (Bar Harbor, ME) and then maintained in the Fox Chase Cancer Center animal facility.

Cell lines and antibodies
Scid.adh, SL-12 and SL-343 are spontaneous thymic lymphomas derived from mice with the scid mutation (Scid mice), and so they lack the clonotypic subunits of the TCR, {alpha} and ß. The SL-4210 lymphoma also arose spontaneously in a Scid mouse; however, SL-4210 does express a TCRß subunit from the Vß16 family (M. Rhodes, unpublished observation). SL-12, SL-343 and SL-4210 were provided by Dr M. Bosma (Fox Chase Cancer Center, Philadelphia, PA) (41,42). SL-12ß.12 cells were generated by electroporating a Vß3 cDNA into the pre-TCR-deficient parental SL-12 cell line, as described (10). Cells were maintained in RPMI supplemented as described (43). The following mAb were used: anti-TCRß (H57-597) (44), anti-CD3{delta}{varepsilon}/{gamma}{varepsilon} (145-2C11) (45), anti-CD3{gamma}{varepsilon} (7D6) (46), anti-TCR{alpha} (H28-710) (47), anti-Vß3 (KJ-25) (48), anti-Vß8.1, 8.2, 8.3 (F23.1; PharMingen, San Diego, CA) (49), anti-2B4 TCR{alpha} (A2B4) (50), anti- influenza virus hemaglutinin (HA) (12CA5; Boehringer Mannheim, Indianapolis, IN), anti-TCR{delta} (GL-3) (51) and anti-V{alpha}11.1, 11.2 (RR8-1) (52). The following polyclonal rabbit antibodies were used: anti-TCR{zeta} (551) (53), anti-CD3{delta} (R9, provided by Dr L. Samelson, NIH, Bethesda, MD) (54) and anti-pT{alpha} (10). Anti-pT{alpha} antibodies were raised against a GST-fusion protein encompassing either the cytoplasmic domain (pT{alpha}-cyt) or the exodomain (pT{alpha}-exo) of pT{alpha} as described (10,53).

Retroviral gene transfer
The {Psi}2 retroviral packaging cell lines expressing the MFG-2B4 (Vß3) and MFG-D011.10 (Vß8) constructs were provided by Dr L. Spain (Holland Laboratory, American Red Cross, Rockville, MD) (55). The murine stem cell virus (MSCVneo, MSCVpuro and MSCVhygro) expression vectors were provided by Dr R. Hawley (American Red Cross, Rockville, MD). The LZRSpBMN-linker-IRES-EGFP (LZRS) retroviral vector was provided by Dr A. Kruisbeek (Netherlands Cancer Institute, Amsterdam, The Netherlands) with permission from Dr Garry Nolan (Stanford University, Stanford, CA) (56). The following cDNAs were obtained as inserts: the 2B4 TCRß–HA fusion protein (provided by Dr Y. Takahama, University of Tsukuba, Tsukuba, Japan) was obtained as an insert from the pCAGGS vector and the 2B4 TCR{alpha} chain (provided by Dr J. Bonifacino, NICHD, NIH, Bethesda, MD) was obtained as an insert from the pCDM8 vector. The V{alpha}11–pT{alpha} chimera was constructed by fusing the V{alpha}11–J{alpha} region (first Ig-like domain) of the AD10 TCR{alpha} chain to a pT{alpha} cDNA lacking the leader sequence and the first five amino acids (primer sequences available upon request) (57). 2B4 TCRß–HA, 2B4 TCR{alpha} and V{alpha}11–pT{alpha} were shuttled through the pCR2.1 cloning vector (Invitrogen, San Diego, CA) into either the MSCV (for 2B4 TCRß–HA and 2B4 TCR{alpha}) or LZRS (for V{alpha}11–pT{alpha}) retroviral expression vectors listed above by PCR using standard methodology (58). Briefly, the 2B4ß–HA and 2B4{alpha} inserts were amplified using oligonucleotide primers flanking the translational start and stop codons that were appended with linkers encoding the HpaI (5) and XhoI (3) restriction sites while the V{alpha}11–pT{alpha} insert was amplified with linkers encoding the XhoI (5) and NotI (3) restriction sites. The amplified inserts were directionally subcloned into MSCVneo (2B4ß–HA), MSCVpuro (2B4{alpha}) or LZRS (V{alpha}11–pT{alpha}) using standard techniques. MSCVneo-2B4ß–HA and MSCVpuro-2B4{alpha} were separately transfected into the {Psi}2 retroviral packaging line using lipofectamine (Gibco/BRL, Grand Island, NY) according to the manufacturer's instructions. Stable viral producing cell lines were selected in 500 µg/ml G418, 1 µg/ml puromycin or 250 µg/ml hygromycin. LZRS-V{alpha}11–pT{alpha} was transfected into the {phi}NX-E retroviral ecotropic viral packaging line (generated by Dr Gary Nolan, Stanford University) using lipofection (56). The Scid.adh or SL-343 thymic lymphoma lines were retrovirally infected by co-culture as follows: 3x105 lymphoma cells were combined with 106 {Psi}2 retroviral producers in a final volume of 3 ml RPMI containing 4 µg/ml polybrene and incubated at 37°C for 48 h. For V{alpha}11–pT{alpha} expression, TCRß-expressing SL-343 cells (SL-343ß.1) were retrovirally infected with virus-containing supernatants generated from transfected {phi}NX-E cells. For the 2B4 constructs, the infected thymic lymphoma cell lines were harvested and grown in medium containing the appropriate antibiotic for selection. For the V{alpha}11–pT{alpha} chimera, cells simultaneously expressing high levels of V{alpha}11 and GFP were isolated by cell sorting, following which surface expression of V{alpha}11-pT{alpha} was verified by flow cytometry.

Surface biotinylation, immunoprecipitation and electrophoresis
Biotin surface labeling was performed as previously described (53,59) after which cell viability was consistently >98%. Labeled cells were lysed in buffer containing 1% digitonin (Wako, Kyoto, Japan) and immunoprecipitated for 2 h with antibody pre-adsorbed to Protein A–Sepharose (Sigma, St Louis, MO). Eluted immune complexes were resolved by one-dimensional non-reducing or two-dimensional non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS–PAGE gels as described followed by transfer onto Immobilon PVDF membranes (Millipore, Bedford, MA). Surface-biotinylated proteins were visualized with horseradish peroxidase-conjugated streptavidin (HRP–Av; Southern Biotechnology Associates, Birmingham, AL) as described (53).

Recapture assay
The recapture assay was performed as previously described (10). Briefly, immune complexes from primary immunoprecipitations were disrupted by boiling in 1% SDS. The SDS eluates were quenched with 10 volumes of 1% NP-40 lysis buffer and secondarily immunoprecipitated using anti-pT{alpha}, anti-TCR{alpha} or anti-epitope tag antibody described above. The recaptured immune complexes were resolved either by one-dimensional or two-dimensional SDS–PAGE gels and visualized as described above.

Glycosidase digestion
Recaptured pT{alpha}ß heterodimers were denatured by boiling in 1% SDS either containing 2-mercaptoethanol for NEPHGE analysis or without 2-mercaptoethanol for one-dimensional non-reducing SDS–PAGE. The eluted glycoproteins, which were neutralized with 10% NP-40 and the appropriate stock digestion buffer (provided by the manufacturers listed below), were treated as follows: (i) mock treatment, digestion buffer only; (ii) for N-glycan removal, 2500 U peptide-N-glycosidase F (PNGaseF; New England Biolabs, Beverly, MA); and (iii) for N- and O-glycan removal, PNGaseF in combination with 20 U/ml sialidase from Vibrio cholerae (Oxford GlycoSciences, Wakefield, MA) and 4 mU of O-glycanase (Genzyme, Cambridge, MA). For all groups, samples were incubated for 18–24 h at 37°C followed by quenching with the appropriate sample buffer.

Inhibition of protein O-glycosylation by treatment with benzyl-2-acetamido-2-deoxy-{alpha}-D-galactopyranoside (benzyl-{alpha}-galnac)
Cells were seeded at an initial concentration of 1x105 cells/ml and cultured for 48 h in the presence of benzyl-{alpha}-GalNac (Toronto Research Chemicals, Toronto, Canada) that was solubilized in DMSO and used at a final concentration of 2 mM (60). Mock-treated control cultures were incubated with an equivalent volume of DMSO (0.1% final). The effectiveness of benzyl-{alpha}-GalNAc treatment was verified by the binding of a fluorochrome conjugate of the lectin derived from Helix pomatia (HPA–FITC; EY Laboratories, San Mateo, CA) as analyzed by flow cytometry (60).

Molecular modeling of the pT{alpha}ß heterodimer complexed with anti-TCRß antibody
The model of the pre-TCR complex was based on the crystal structure of the {alpha}ßTCR heterodimer (PDB: 1NFD) solved at 2.8 Å resolution (61). To build the structure model, we replaced the TCR{alpha} chain with a model of pT{alpha}. This model was based on the pT{alpha} sequence (GenBank accession no. 967187) and a homologous sequence of known structure, which was identified using PSI-BLAST 2.0.8 (62). The pT{alpha} sequence was compared to a non-redundant protein sequence database available from GenBank. Four PSI-BLAST iterations were performed and a position-specific similarity matrix was created which was used to search the PDB sequence database. The E-value cut-off for inclusion of new sequences into the position-specific matrix was 0.0001. The sequence with the best E-value was used as the structure template for the pT{alpha} model. Residues 20–129 of the pT{alpha} sequence were assigned the backbone coordinates of residues 110–228 of the FAB CH chain of the mAb IgG2a (PDB: 1IGT), as defined by the PSI-BLAST alignment. Side-chains were built onto the pT{alpha} model backbone using SCWRL 2.1 (63), which uses a backbone-dependent rotamer library (64) to assign the side-chain {chi} angles, followed by a combinatorial search of allowable rotamers to minimize steric clashes. In order to find the proper orientation between the pT{alpha} model and the TCRß chain, it was first necessary to align the constant domains of the template and the TCR structure, then apply this same rotation/translation to the model. The structure of the CH domain of IgG2a was aligned with the structure of the constant domain of the TCRß chain using MINAREA (65). This rotation and translation was then applied to the pT{alpha} model to dock it to the TCR structure with the {alpha} chains removed. The minimal O-glycan structure was predicted to be a tetrasaccharide having a NeuNAc{alpha}2–3Galß1–3(NeuNAc{alpha}2–6)GalNAc structure based on the known mode of action of O-glycosidase, which cleaves de-sialylated Galß1–3GalNAc residues from serine or threonine residues. The coordinates of the O-linked tetrasaccharides were generated with the computer program Sweet and the sugars were manually docked with MidasPlus in proportion onto the residues predicted as probable O-linked glycosylation sites (T24, S28, T75, S79 and T131), according to the NetOGlyc server (6668). The fifth sugar was placed close to the last residue of the model (S129) since T131 is not part of the model. Figure 7Go(A) was produced with MolScript and Raster3D (69,70).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 7. Molecular modeling of pre-TCR complexes. (A) The model of the pT{alpha}ß heterodimer complexed with H57-597 is based on the reported crystal structure of H57-597 interacting with its epitope, the FG loop of the Cß domain of the N15 {alpha}ß TCR heterodimer. For a detailed description of the construction of this model, refer to Methods. (B) Schematic representation of the theoretical mechanisms of TCRß chain masking.

 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of a novel isoform of the pre-TCR
In a previous report, we established that pre-TCR complexes expressed on the surface of primary TCR{alpha}-deficient (TCR{alpha}-/–) thymocytes comprise pT{alpha}ß heterodimers associated with the CD3{gamma}{delta}{varepsilon} and TCR{zeta} signaling subunits (10). In the current report, we wanted to determine if immature thymocytes also expressed biochemically distinct pre-TCR isoforms as has been demonstrated for the pre-BCR in early B lymphoid precursors. Pro-B cells have been shown to express a `pro-BCR' containing SLC but lacking the Igµ heavy chain (38,39). To determine if thymocytes expressed an equivalent isoform of the pre-TCR (i.e. containing pT{alpha} but lacking TCRß), we exhaustively removed TCRß-containing pre-TCR complexes from detergent extracts of biotin-labeled thymocytes from TCR{alpha}-/– mice and then re-immunoprecipitated the depleted extracts with antibody reactive with other pre-TCR components to determine whether any pT{alpha}-containing pre-TCR isoforms remained (Fig. 1Go; see Figs 2b, 3b and 6GoGoGo for evidence that anti-TCRß preclearing was exhaustive). The primary immunoprecipitations were evaluated by the recapture assay, a sensitive means to identify complexes containing pT{alpha}, the hallmark of the pre-TCR complex (10). The recapture assay entails solubilizing the primary immune complexes in SDS to destroy non-covalent interactions between receptor subunits, following which the solubilized proteins are re-immunoprecipitated with antibody specific for pT{alpha}. Consequently, only pT{alpha} and those proteins with which pT{alpha} is covalently associated are `recaptured'. Surprisingly, the anti-TCRß antibody was able to recognize only ~50% of the pT{alpha}ß-containing complexes, demonstrating the existence of an additional pre-TCR isoform(s) (Fig. 1Go). Indeed, after exhaustive anti-TCRß pre-clearing, anti-CD3{varepsilon} antibody recognized an additional pT{alpha}-containing complex comprising CD3{varepsilon}-associated pT{alpha} multimers with an electrophoretic mobility ~10–20 kDa larger than that of the pT{alpha}ß heterodimers recognized by anti-TCRß antibody (Fig. 1, cfGo. lanes 1 and 3). The pT{alpha} multimers from the novel pre-TCR isoform were not immunoprecipitated by control anti-TCR{delta} antibody (Fig. 1Go). Moreover, their expression was not restricted to TCR{alpha}-/– thymocytes as the isoform was also detected in total thymocytes from wild-type C57BL/6 mice (data not shown).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1. Expression of novel MB-pre-TCR on the surface of primary thymocytes. Digitonin extracts of surface biotin-labeled thymocytes from TCR{alpha}-/– mice were sequentially immunoprecipitated with anti-TCRß antibody (H57-597; left panel) or anti-TCR{delta} (GL-3; right panel) followed by anti-CD3{gamma}{varepsilon} antibody (7D6). Immune complexes were eluted by boiling in SDS. After quenching with NP-40, SDS eluates were immunoprecipitated with either anti-pT{alpha} or control rabbit IgG antibody. Samples were resolved on non-reducing one-dimensional SDS–PAGE. Surface-labeled proteins were visualized by HRP–Av and chemiluminescence. We have previously shown that anti-CD3{gamma}{varepsilon} antibody is able to preclear all pT{alpha}-containing complexes from detergent extracts of these cells (10).

 



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 2. Analysis of pre-TCR complex isoform expression on the surface of Scid.adh thymic lymphomas. (A) Surface expression of both novel and conventional pre-TCR complexes requires TCRß chain expression. Digitonin extracts of surface biotinylated TCRß+ Scid.adh cells were sequentially immunoprecipitated with anti-TCRß followed by anti-pT{alpha} Ab. Those of TCRß- Scid.adh cells were precipitated with either anti-TCRß, anti-pT{alpha} or anti-CD3{gamma}{varepsilon} antibody. After boiling in SDS, the eluted immune complexes were neutralized in NP-40 and recaptured with antibody specific for the cytoplasmic tail (anti-pT{alpha} cyt. tail) or the exodomain (anti-pT{alpha} exo) of pT{alpha}. It should be noted that the novel isoform was recaptured by anti-pT{alpha} antibody recognizing both the cytoplasmic tail (cyt. tail) and exodomain of pT{alpha} (exo). (B) The TCRß chain is a component of the novel MB-pre-TCR. Scid.adh cells were retrovirally transduced either with TCRß (2B4ß) or an epitope-tagged TCRß (2B4ß–HA) cDNA. Digitonin extracts of surface biotin-labeled cells were sequentially immunoprecipitated with anti-TCRß followed by anti-CD3{varepsilon}. SDS eluates were neutralized with NP-40 and recaptured either with anti-pT{alpha} or anti-HA (12CA5) antibody. Samples were resolved by non-reducing SDS–PAGE and surface labeled proteins were visualized as above.

 


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3. Masking of the Cß and Vß domains is dependent on association of TCRß with pT{alpha}. (A) Both the V and C domains of TCRß are masked in MB-pre-TCR. Scid.adh cells were retrovirally transduced with cDNAs encoding either Vß3 or Vß8 cDNAs. Digitonin extracts of biotin-labeled cells were first immunoprecipitated with either anti-TCRß (H57-597) or the appropriate anti-Vß antibody (anti-Vß3, KJ-25 or anti-Vß8, F23.1) then the depleted extracts were further immunoprecipitated with anti-CD3{varepsilon} (145-2C11). SDS eluates were neutralized with NP-40 and the immune complexes were analyzed by recapture assay. (B) Masking of the TCRß chain is dependent upon its association with pT{alpha}. Digitonin extracts of biotin-labeled {alpha}ßTCR-expressing Scid.adh cells were sequentially immunoprecipitated with anti-TCRß or anti-Vß3 mAb followed by anti-CD3{varepsilon}. NP-40-neutralized SDS eluates were recaptured with anti-TCR{alpha} (H28-710) antibody. All samples were resolved by non-reducing SDS–PAGE and surface-labeled proteins were visualized as above.

 



View larger version (72K):
[in this window]
[in a new window]
 
Fig. 6. Antibody access to the TCRß subunit of MB-pre-TCR is controlled by occupancy of the `missing' V domain of pT{alpha}. (A) Antibody access to the ß subunit of MB-pre-TCR complexes is restored by pairing with pT{alpha} molecules appended with a V{alpha} domain. SL-343 cells expressing surface complexes containing either a pT{alpha}ß heterodimer, a V{alpha}11–pT{alpha}ß heterodimer or an {alpha}ß heterodimer were surface labeled with biotin. Digitonin extracts of biotinylated cells were sequentially immunoprecipitated with anti-TCRß-specific antibody (H57-597), anti-V{alpha}11 (RR8-1) antibody and anti-CD3 antibody (145-2C11). SDS-eluted immune complexes neutralized in NP-40 were recaptured with either anti-pT{alpha} (for cells expressing pT{alpha}ß or V{alpha}11–pT{alpha}ß heterodimers) or anti-TCR{alpha} (cells expressing {alpha}ß heterodimers). Samples were resolved by non-reducing SDS–PAGE and surface-labeled proteins were visualized by HRP–Av and chemiluminescence. (B) Expression of MB-pre-TCR complexes is controlled by a trans-acting factor. The indicated pre-TCR expressing thymic lymphoma lines were surface labeled and lysed as above. The detergent extracts were halved following which one half was immunoprecipitated sequentially with anti-TCRß (H57-597) followed by anti-CD3{gamma}{varepsilon} (7D6), while the other was immunoprecipitated only with anti-CD3{gamma}{varepsilon}. The immune complexes were analyzed by recapture assay and resolved on SDS–PAGE gels as above.

 
The novel pre-TCR complex isoform contains pT{alpha}ß heterodimers in which the V and C domains of TCRß are masked
There were two possible explanations for the inability of anti-TCRß antibody to recognize the novel pre-TCR isoform: (i) the novel isoform lacks TCRß or (ii) the novel isoform contains TCRß, but those TCRß subunits are not recognizable by the anti-ß antibody. To distinguish between these possibilities we assessed whether or not TCRß subunits were present in the novel pre-TCR isoform. To do so we first determined if the novel pre-TCR isoform could be expressed in cells lacking TCRß. Since the thymic hypocellularity associated with TCRß deficiency makes such an experiment nearly impossible to perform using primary thymocytes, we utilized a thymic lymphoma, Scid.adh, that arose spontaneously in Scid mice (71). Scid.adh lacks TCRß expression because the scid mutation in DNA-dependent protein kinase prevents rearrangement of the TCRß gene locus (72,73). Neither anti-TCRß, nor anti-CD3 nor anti-pT{alpha} antibody were able to precipitate pT{alpha}-containing multimers from detergent extracts of surface-labeled Scid.adh cells (Fig. 2AGo). Note: The band in lane 6 of Figure 2AGo is a nonspecific band and does not represent a pT{alpha} multimer, since it was not observed reproducibly with the anti-pT{alpha} cyt. tail serum or at any time wth the anti pT{alpha} exo serum. The absence of the novel pre-TCR isoform from Scid.adh cells did not result from the inability of Scid.adh cells to express the novel pre-TCR isoform, as retroviral transduction of Scid.adh with a TCRß cDNA restored expression of both the pre-TCR complexes recognized by anti-TCRß antibody and the novel isoform isolated from the anti-TCRß pre-cleared extract using anti-pT{alpha} antibody (Fig. 2AGo). Therefore, surface expression of the novel pre-TCR isoform does require the TCRß subunit.

Our results suggested that either the novel isoform actually contains the TCRß subunit or, alternatively, that ß expression induces a distinct protein that is required for formation of the novel pre-TCR isoform. To distinguish between these possibilities, we transduced Scid.adh with an HA-tagged TCRß cDNA, allowing the expressed TCRß subunit to be directly identified in recapture assays using the anti-HA antibody. Using the anti-HA antibody we were able to recapture pT{alpha} multimers from both the anti-TCRß precipitable pre-TCR complex as well as from the novel pre-TCR isoform, demonstrating that TCRß is in fact a component of the novel isoform despite its inaccessibility to antibody reactive with the C domain of TCRß (Fig. 2BGo). Interestingly, the V domain of the TCRß subunits in the novel pre-TCR isoform was similarly inaccessible to antibody (Fig. 3AGo). Indeed, neither the novel pre-TCR isoform containing Vß3 nor that containing Vß8 could be recognized by anti-Vß3 or anti-Vß8 antibody respectively (Fig. 3AGo). Consequently, we term this novel pre-TCR isoform the MB (for masked ß)-pre-TCR, because the ß subunit of this complex is inaccessible to both antibody directed against the C domain and V domain of TCRß.

Since the Scid.adh lymphoma represents an early stage of thymocyte development, we reasoned that the masking of TCRß might be a consequence of the stage of development rather than a unique feature of the pre-TCR complex. More specifically, we wanted to determine if TCRß subunits associated with TCR{alpha} would be masked similarly as when associated with pT{alpha}. This was assessed by retroviral transduction of a TCR{alpha} cDNA into Scid.adh cells already expressing TCRß. Because expression of TCR{alpha} prevents pT{alpha} incorporation into receptor complexes, the pre-TCR complexes expressed by the Scid.adh recipient cells were completely replaced by {alpha}ß TCR complexes (S. Trop et al., manuscript submitted). Immunoprecipitation of detergent lysates of surface-labeled {alpha}ßTCR+ Scid.adh cells with either anti-Cß or anti-Vß domains effectively pre-cleared all {alpha}ßTCR complexes, demonstrating that the masking of TCR Cß and Vß domains in MB-pre-TCR complexes is dependent upon pT{alpha} and therefore is a unique property of pre-TCR complexes (Fig. 3BGo). Consequently, the key to understanding the mechanistic basis for masking of TCRß lay in focusing on those features of pT{alpha} that distinguish it from TCR{alpha}.

Conventional and MB-pre-TCR contain differentially O-glycosylated forms of pT{alpha}
Since masking of the TCRß subunit depends upon its association with pT{alpha} and correlates with the increased Mr of pT{alpha}ß heterodimers from MB-pre-TCR complexes, it was possible that ß chain masking was due to differential post-translational processing of pT{alpha}. The most likely post-translational modification of pT{alpha} would be oligosaccharide addition. Note that we employed the pre-TCR+ SL-343ß.1 thymic lymphoma line (generated by retroviral transduction of a TCRß cDNA into the TCRß-deficient parental line) to assess the role of glycans in masking the ß subunits of MB-pre-TCR complexes because SL-343ß.1 expresses higher surface levels of the pre-TCR, making this analysis more straightforward. pT{alpha} contains two Asp-X-Ser consensus motifs for N-glycan attachment (9); however, while removal of the N-linked glycans using PNGase F treatment did reduce the Mr of pT{alpha}ß heterodimers from both the anti-TCRß precipitable and MB-pre-TCR isoforms, their relative size difference remained unchanged (Fig. 4AGo). Thus, differential processing or addition of N-linked glycans to pT{alpha} is not responsible for the increased Mr of the pT{alpha}ß dimers of the MB-pre-TCR complex. Similar results were obtained with TCR{alpha}-/– thymocytes (data not shown).




View larger version (92K):
[in this window]
[in a new window]
 
Fig. 4. Oligosaccharide analysis of pre-TCR complexes by glycosidase digestion. (A) The difference in Mr between the conventional and novel pT{alpha}ß heterodimers is maintained after N-glycan removal. Digitonin extracts of biotin-labeled pre-TCR+ Scid.adh cells were immunoprecipitated sequentially with anti-TCRß followed by anti-CD3{varepsilon}. Recaptured pT{alpha}ß dimers were resuspended in PNGaseF digestion buffer in the presence (digested) or absence (mock) of PNGase F. Samples were resolved on one-dimensional non-reducing SDS–PAGE gels. (B) Conventional and MB-pre-TCR complexes contain differentially O-glycosylated forms of pT{alpha}. Digitonin extracts of biotin-labeled pre-TCR+ SL-343 cells were immunoprecipitated as in (A). Note: SL-343 cells have increased pre-TCR complex surface expression compared with the Scid.adh line and are therefore better suited for rigorous glycoconjugate analysis. The anti-pT{alpha} immunoprecipitates were either treated with PNGaseF alone for N-glycan removal (top panels) or with a combination of PNGaseF, sialidase and O-glycosidase for the removal of N- and O-glycans (bottom panels). Samples were resolved on two-dimensional NEPHGE/SDS–PAGE gels. Surface-labeled proteins were visualized as above.

 
To determine if we could detect additional biochemical differences between the anti-TCRß precipitable pre-TCR and MB-pre-TCR complexes, the PNGaseF-digested pT{alpha}ß heterodimers were resolved by two-dimensional NEPHGE/SDS–PAGE (Fig. 4BGo). PNGase F digestion of pT{alpha} from the anti-TCRß precipitable pre-TCR complexes yielded four de-glycosylated forms ranging from the expected size of ~21 kDa to progressively larger and more acidic forms up to 28 kDa (Fig. 4B, Gotop left). PNGaseF digestion of the pT{alpha}ß heterodimers from MB-pre-TCR revealed that pT{alpha} migrated exclusively as the larger, more acidic forms ranging from 25 to 28 kDa (Fig. 4B, Gotop right). This difference in pT{alpha} migration was suggestive of differences in the proportion of pT{alpha} molecules modified by O-linked glycans and their terminal sialic acids. Consistent with this possibility, analysis of the pT{alpha} amino acid sequence using NetOGlyc, a motif prediction program which estimates the probability that a particular serine or threonine residue will be O-glycosylated, revealed five consensus sites for O-glycosylation (T24, S28, T75, S79 and T131) (68). To test whether the observed differences in migration of pT{alpha} from conventional and MB-pre-TCR resulted from differential O-glycosylation, we treated the pT{alpha}ß heterodimers with a combination of PNGaseF, sialidase and O-glycosidase to eliminate both N- and O-linked glycans. Importantly, this combined treatment eliminated the differential migration pattern of pT{alpha} observed in Fig. 4Go(B), indicating that the differences in migration of pT{alpha} from conventional and MB-pre-TCR were due to differences in pT{alpha} O-glycosylation. Since TCRß chain masking correlated with the presence of a more heavily O-glycosylated form of pT{alpha}, we hypothesized that O-glycosylation might in fact be responsible for masking the TCRß subunits of MB-pre-TCR complexes.

Inhibition of O-glycosylation does not restore antibody access to TCRß
To test whether the extensive modification of pT{alpha} with O-glycans were actually responsible for inhibiting antibody binding to TCRß, we prevented the addition of O-glycans using a competitive inhibitor, benzyl-{alpha}-GalNAc. If O-linked glycans were responsible for masking the ß chains of MB-pre-TCR complexes, then preventing their addition should restore the recognition of pT{alpha}ß heterodimers by anti-TCRß antibody. Detergent extracts of pre-TCR+ SL-343ß.1 cells that were cultured for 2 days with benzyl-{alpha}-GalNAc and surface-biotinylated were immunoprecipitated first with anti-TCRß, then with anti-CD3{varepsilon} antibody. pT{alpha}ß dimers recaptured from the immune complexes were digested with PNGaseF and resolved on two-dimensional gels (Fig. 5Go). Interestingly, despite the fact that benzyl-{alpha}-GalNAc treatment prevented the addition of O-glycans to pT{alpha} (as evidenced by the change in migration), antibody specific for the TCR Cß domain remained unable to pre-clear all pre-TCR complexes (Fig. 5Go, bottom panels). Moreover, benzyl-{alpha}-GalNAc treatment did not even result in a quantitative increase in the proportion of pT{alpha}ß heterodimers accessible to anti-TCRß antibody (compare mock- and BenzylGalNAc-treated samples; Fig. 5Go). These results suggest that the O-linked glycans are not responsible for TCRß chain masking.



View larger version (67K):
[in this window]
[in a new window]
 
Fig. 5. Inhibition of O-glycan addition does not restore antibody accessibility to TCRß subunits of MB-pre-TCR complexes. SL-343ß.1 cells were cultured for 48 h either in culture medium containing 2 mM benzyl-{alpha}-galNAc (bottom panels) or a comparable amount of the vehicle control, DMSO (top panels; see Methods). Digitonin extracts of surface-biotinylated cells from both treatment groups were immunoprecipitated sequentially with anti-TCRß followed by anti-CD3{varepsilon}. NP-40-neutralized SDS eluates were analyzed by recapture assay. For removal or N-glycans, eluted anti-pT{alpha} immunoprecipitates were resuspended in PNGaseF digestion buffer in the presence of PNGase F. Samples were resolved on two-dimensional NEPHGE/SDS–PAGE gels and surface-labeled proteins were visualized as above.

 
Masking of the TCRß chain is eliminated by appending a TCR V{alpha} domain to pT{alpha}
Having found that O-glycosylation of pT{alpha} was not responsible for modulating accessibility of TCRß to antibody, we focused on the other major structural feature of pT{alpha} that distinguishes it from TCR{alpha}—the number of Ig loops in its exodomain. Whereas TCR{alpha} has both a V and a C domain, pT{alpha} lacks a V domain equivalent. Consequently, it was possible that accessibility of TCRß to antibody might be modulated by the presence of an additional pre-TCR subunit that approximates a V-like domain of pT{alpha} (see the schematic in Fig. 7Go). To test whether the presence of a V domain for pT{alpha} could modulate accessibility of TCRß chains to anti-TCRß antibody, we appended pT{alpha} with the V domain of a V{alpha}11 TCR{alpha} subunit (57). The V{alpha}–pT{alpha} chimera was retrovirally transduced into SL-343ß.1 cells, which were surface biotinylated and sequentially immunoprecipitated with anti-TCRß, anti-V{alpha}11 and anti-CD3{varepsilon}. Recaptured immune complexes were resolved by SDS–PAGE (Fig. 6AGo). Importantly, unlike TCRß subunits associated with pT{alpha}, TCRß subunits associated with the V{alpha}11–pT{alpha} chimera were completely accessible to anti-TCRß antibody, as the anti-TCRß antibody pre-cleared all of the pre-TCR complexes containing V{alpha}11–pT{alpha}ß heterodimers (Fig. 6AGo). It should be noted that retroviral transduction of the V{alpha}–pT{alpha} chimera into SL-343ß.1 increased surface expression of pre-TCR complexes containing the V{alpha}–pT{alpha} chimera while at the same time markedly reducing expression of pre-TCR complexes containing endogenous pT{alpha} (Fig. 6AGo; note that there is less surface pT{alpha} co-precipitated by anti-CD3{varepsilon} in cells transduced with the V{alpha}–pT{alpha} chimera). This presumably occurred because the chimera assembled more effectively with the remaining pre-TCR subunits than did the endogenous pT{alpha} (see description of Fig. 6A and SGo. Trop et al., manuscript submitted). However, increased surface expression alone was not responsible for restoration of antibody access to TCRß, because overexpression of retrovirally transduced pT{alpha} also resulted in a comparable increase in pre-TCR surface expression without restoring antibody access to TCRß (data not shown). Consequently, the ability of the V{alpha} appendage to restore antibody accessibility to associated TCRß subunits suggests that the absence of a V domain from pT{alpha} is responsible for modulating antibody access to TCRß.

The two most likely explanations for the ability of the V{alpha} appendage to restore antibody access to associated TCRß subunits are that: (i) an additional pre-TCR subunit occupies the V region of pT{alpha} in about half of pre-TCR complexes and modulates accessibility of the ß subunit to antibody or (ii) TCRß may stochastically adopt alternative structural conformations—one accessible to anti-ß antibody and one inaccessible—with the accessible conformation preferentially stabilized by the V{alpha} appendage on pT{alpha}. The latter possibility predicts that in different pre-TCR-expressing cell populations, the ratio of conventional (i.e. TCRß is accessible to antibody) to MB (i.e. TCRß is inaccessible)-pre-TCR complexes should be constant; however, regulation of ß accessibility by a trans-acting factor (i.e. an additional pre-TCR subunit) should cause this ratio to vary with the expression level of that additional subunit. To distinguish these possibilities we assessed the relative proportion of anti-ß accessible and inaccessible pre-TCR complexes in a panel of pre-TCR-expressing thymic lymphomas (Fig. 6BGo). Interestingly, the ratio of anti-TCRß accessible (Fig. 6BGo; lane 1) to inaccessible (Fig. 6BGo; lane 3) pre-TCR complexes did vary among the cell lines examined, with the extreme example being SL-12ß.12 cells, which expressed almost exclusively conventional pre-TCR in which TCRß is accessible to anti-TCRß antibody (Fig. 6BGo; bottom panel). Since the ratio of anti-ß accessible:inaccessible pre-TCR complexes varied among the lines examined and all of these lines express all of the known components of the pre-TCR complex, these data suggest that accessibility of TCRß to antibody is controlled by a trans-acting factor(s), possibly an additional pre-TCR subunit.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this report we describe the identification of a pre-TCR isoform, termed MB-pre-TCR for `masked ß', that accounts for about half of the pre-TCR complexes expressed on the surface of primary thymocytes. MB-pre-TCR complexes contain TCRß subunits whose V and C domains are inaccessible to antibody binding. Importantly, the masking of TCRß subunits in the MB-pre-TCR complexes is eliminated if the `missing' V domain of pT{alpha} is supplied in the form of a V domain from TCR{alpha}. Moreover, we have identified a thymic lymphoma cell line which expresses conventional pre-TCR complexes in which TCRß is accessible to antibody, but not MB-pre-TCR complexes in which ß is inaccessible to antibody, suggesting that expression of MB-pre-TCR complexes is under control of a trans-acting factor(s). Our finding that expression of MB-pre-TCR complexes appears to be controlled by a trans-acting factor(s) coupled with our observation that antibody access to TCRß is restored by providing a V domain to pT{alpha}, suggests the existence of an additional pre-TCR subunit that normally occupies pT{alpha}'s V region and is able to modulate recognition of the ß subunit by antibody. This putative subunit may be the Vpre-T subunit proposed by others (79).

Prior to the current report, there was functional evidence suggesting that immature thymocytes might express an isoform(s) of the pre-TCR complex (which we define by the presence of pT{alpha}) (74). Indeed, treatment of TCR{gamma} transgenic (V{gamma}2) RAG-deficient mice with anti-V{gamma}2 antibody slightly increased thymic cellularity and the number of DP thymocytes generated, neither of which was observed in similarly treated mice lacking pT{alpha}. Consequently, the effects of anti-TCR{gamma} antibody treatment were attributed to a pre-TCR isoform comprising pT{alpha}, TCR{gamma} and associated CD3 signaling components (74); however, neither the extent to which this complex is expressed nor its relevance during normal thymocyte development are clear at present. The MB-pre-TCR isoform is clearly distinct from the aforementioned isoform in that MB-pre-TCR complexes lack components of the {gamma}{delta}TCR and are expressed extensively, accounting for about half of the pT{alpha}ß-containing complexes on the surface of TCR{alpha}-/– thymocytes. The expression of pre-TCR isoforms on normal thymocytes has also been predicted by analogy with early B lineage precursors, which express a pro-BCR complex comprising surface SLC but not the Igµ heavy chain (38). The analogous `pro-TCR' complex in T lineage precursors would comprise pT{alpha} in association with CD3, but not with TCRß. While MB-pre-TCR complexes are not recognized by anti-TCRß antibody, they do contain TCRß subunits and so are distinct from the aforementioned `pro-TCR' complex. Moreover, we find no evidence for surface expression of pT{alpha}-CD3 complexes lacking ß in our lymphoma cell lines, even when using our sensitive recapture assay (Fig. 2AGo and data not shown). However, we have found abundant pT{alpha}-CD3 complexes retained intracellularly within the endoplasmic reticulum or Golgi complex of thymic lymphoma cells that lack TCRß (data not shown). Consequently, if these pT{alpha}-CD3 complexes are playing a role in early thymocyte development, they presumably do so from an intracellular location.

Because masking of the V and C domains of TCRß occurred when TCRß was associated with pT{alpha}, but not when TCRß was associated with TCR{alpha}, our investigation of the mechanistic basis for masking focused upon structural features of pT{alpha} that differed from those of TCR{alpha}. These features of pT{alpha} were: (i) the presence of consensus motifs for addition of O-linked glycans and (ii) the absence of a V-like domain (9).

First, we found that pT{alpha} is modified by O-linked glycans and that the pT{alpha} molecules associated with masked ß chains are more extensively glycosylated. Nevertheless, preventing the addition of O-glycans to pT{alpha} using the competitive inhibitor benzyl-{alpha}-GalNAc did not alleviate the masking of TCRß subunits in MB-pre-TCR complexes (60). This suggested that while the extent of O-glycosylation did correlate with the masking of TCRß in the MB-pre-TCR, the O-glycosylation of pT{alpha} was not responsible for the masking. Then why might the extent of O-glycan addition correlate with masking of the TCRß subunit? Since masking of TCRß seems to be controlled by a cellular factor, one simple explanation could be that the cellular factor also modulates accessibility of the glycosylation enzymes to the O-glycan addition sites of pT{alpha} (see below). The inability of O-glycosylation of pT{alpha} to account for masking of TCRß in MB-pre-TCR complexes is supported by the structure and location of the O-glycans in a three-dimensional structural model of a pT{alpha}ß dimer that we generated (Fig. 7AGo). The model was based on the crystal structure of the N15 {alpha}ßTCR complexed with a Fab' fragment of H57-597 (anti-TCR Cß) (61) and was produced by replacing TCR{alpha} with a model of pT{alpha} whose consensus O-glycosylation motifs were appended with O-glycans (see Methods). The resulting three-dimensional model revealed that it was unlikely that O-glycans on pT{alpha} could interfere with antibody recognition of Cß or Vß domains both because of their small size and remote location. Moreover, steric clashes would prevent the O-glycans from rotating into physical proximity with the ß chain epitopes. Although O-glycosylation of pT{alpha} is not responsible for masking the TCRß subunits of MB-pre-TCR complexes, the O-glycans may play other important roles in pre-TCR function. Indeed, O-glycan biosynthesis is developmentally regulated by pre-TCR signals during thymocyte maturation (75). Moreover, recent studies have shown that O-glycans are important mediators of neutrophil migration and inflammation (76), peripheral T cell activation, T cell–antigen-presenting cell interaction (77), and apoptosis during thymocyte development (7880). The involvement of O-glycoproteins in apoptosis of developing thymocytes is mediated by an intrathymic O-glycan-binding lectin termed galectin, which when co-engaged with the TCR induces extensive apoptosis of DP thymocytes, but not DN thymocytes (78,79). It is possible that differential O-glycosylation of pT{alpha} and TCR{alpha} may play some role in susceptibility to galectin-mediated apoptosis (80).

Our assessment of the other structural feature of pT{alpha} that differed from TCR{alpha} (i.e. the absence of a V-like domain) did provide insight into the basis for masking of the TCRß subunits in MB-pre-TCR complexes. Indeed, filling in the `missing' V domain of pT{alpha} with a V domain from TCR{alpha} completely eliminated the masking of associated TCRß subunits. There are three potential explanations for this result. The first is that TCRß subunits paired with pT{alpha} can stochastically adopt two conformations—one of which is recognizable and one which is not recognizable by anti-TCRß antibody; and the recognizable conformation is favored when ß pairs with a two-domain partner subunit like TCR{alpha} or the V{alpha}–pT{alpha} chimera. This interpretation predicts that the ratio of recognizable and masked conformations should be an inherent property of the ß subunit and should not vary with the cellular context; however, we find that the SL-12ß.12 thymic lymphoma expresses only surface pre-TCR in which TCRß is accessible to anti-ß antibody (Fig. 6BGo), suggesting that accessibility of antibody to TCRß is regulated by a trans-acting factor(s). The second possibility is that a trans-acting factor such as a molecular chaperone could direct TCRß to assume either its antibody-accessible or masked conformation; however, despite the intensive study of chaperone function in protein folding no precedent exists for a chaperone directing two distinct conformations, both of which are competent to be transported to the cell surface. The final possibility, which best fits our experimental evidence, is that accessibility of the TCRß subunit is modulated by the presence of an additional subunit, possibly a Vpre-T-like molecule, that occupies the V region of pT{alpha}. This configuration, i.e. a surrogate {alpha} complex comprising a C-like pT{alpha} and a V-like Vpre-T, is analogous to the two-subunit SLC of the pre-BCR (7,8). Like the C-domain equivalent of the SLC, {lambda}5, which is disulfide linked to the Igµ chain, pT{alpha} is disulfide linked to TCRß (6,9,36). Moreover, the V domain equivalent of the pre-BCR, Vpre-B, is not covalently associated with either the {lambda}5 or Igµ subunit (36). If the putative V-like subunit of the pre-TCR similarly lacks covalent attachment to pT{alpha} or ß, this would explain its apparent absence from the immune complexes of our recapture assay, which retains only those molecules that are covalently coupled to pT{alpha}. In addition, the V-like subunit of the pre-TCR may not be efficiently visualized by surface biotinylation, requiring a distinct method for detection. This was true of the pT{alpha} subunit, which could not be visualized by iodination but could be visualized by biotinylation (6). A resolution to this issue will require further investigation. When taken together our data are most consistent with the interpretation that access of the TCRß subunit of MB-pre-TCR complexes to anti-TCRß antibody is controlled by an additional V-like subunit of the surrogate {alpha} chain of the pre-TCR complex.

If the V-like subunit is responsible for modulating antibody accessibility to the TCRß subunit, then how might it do so? The two most likely possibilities are: (i) pairing of the V-like molecule with TCRß may stabilize TCRß's conformation, facilitating recognition by antibody, or (ii) pairing of the V-like molecule with TCRß may interfere with antibody binding. The first possibility—that accessible ß subunits are paired with the V-like subunit and inaccessible ß chains are not—is consistent with the differential O-glycosylation of pT{alpha} (Fig. 7BGo, upper right). Indeed, the pT{alpha} subunits of pre-TCR complexes in which TCRß is accessible to antibody tend to have fewer O-glycans, which might result from the obscuring of the consensus O-glycan addition sites by the presence of the V-like subunit (Fig. 7A and BGo, upper right). However, the possibility that pairing of the V-like molecule with the TCRß chain is required to maintain TCRß's conformation in a state compatible with antibody recognition appears to be inconsistent with crystallographic analysis of the TCRß subunit. X-ray crystals of TCRß as a monomer, as a dimer with TCR{alpha} and in association with superantigen reveal no significant differences in conformation, suggesting that the TCRß structure is quite rigid and not significantly influenced by the subunits with which it is paired (8183). Nevertheless, no crystal structure of TCRß in association with pT{alpha} exists, leaving open the possibility that pairing of TCRß with a partner subunit bearing only a C domain may influence TCRß conformation. It should also be noted that crystallographic analysis does not always reliably predict the actual structure of proteins in solution. For example, NMR spectroscopy revealed that the structure of the homodimer interface of the UmuD' protein, which regulates SOS mutagenesis, is not the structure deduced from crystallographic analysis (84,85). The other possible explanation for the effect of a V-like subunit on TCRß accessibility is that this additional subunit could interfere with antibody accessibility to the Vß and Cß epitopes (Fig. 7BGo). This explanation requires no conformational changes in ß and so does agree with the crystallographic analysis; however, it is not readily apparent how this possibility explains the more extensive O-glycosylation of pT{alpha} subunits in the MB-pre-TCR complex. Nor is it clear how such a subunit could interfere with antibody binding to both the V and C domains of TCRß. Experimentation attempting to resolve these models is in progress.

While the physiologic relevance of the MB-pre-TCR and putative VpreT subunit is uncertain at present, it may serve to modulate the intensity of pre-TCR signals. Since supraphysiologic pre-TCR signaling, such as that induced by antibody engagement of surface pre-TCR complexes, arrests thymocyte development (2931), the V-like subunit might act to limit the intensity of pre-TCR signals by preventing lateral interactions between the pre-TCR complex and other structures on the surface of the thymocyte or thymic stroma. Conversely, since there appears to be no ligand for the pre-TCR complex, the V-like subunit might serve to amplify pre-TCR signals by facilitating lateral interactions between the pre-TCR and other surface molecules involved in signaling. Experiments are in progress to identify the developmental stages at which MB-pre-TCR complexes are expressed and to assess the relative efficiency with which conventional and MB-pre-TCR complexes promote thymocyte development.

In this report, we characterized a novel pre-TCR isoform, the MB-pre-TCR complex, in which the pT{alpha} subunits are heavily modified with O-glycans and in which the V and C domains of the TCRß subunit are inaccessible to antibody binding. Moreover, we have presented evidence which suggests that antibody access to the ß subunit of MB-pre-TCR complexes is governed by the presence of a trans-acting factor, possibly an additional pre-TCR subunit that occupies the `missing' V region of pT{alpha}. Future studies will focus on identifying the putative V-like subunit as a first step towards understanding its requirements for assembly into the pre-TCR complex and its role in pre-TCR function. Answers to these questions will represent a substantial increase in our understanding of how thymocyte development is controlled by the pre-TCR complex.



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 7AGo. Legend on facing page.

 

    Acknowledgments
 
We thank Drs S. Belkowski, K. Campbell, L. Eisenlohr, D. Kappes, M. Marks, J. Punt and A. Singer for critically reading this manuscript, and Dr Robert Haltiwanger for helpful discussions on glycan analysis. This research was supported by grants from the National Institutes of Health (R29 CA-73656 and Core Grant CA-06927), from the Human Frontier Science Program (3752-01), and by an appropriation from the Commonwealth of Pennsylvania. M. A. B was supported by National Institutes of Health Training Grant AI-07492 and by a Fellowship from the Arthritis Foundation. M. C. and J. M. S. were supported by National Institutes of Health Training Grants CA-09035-23 and CA-09035-24 respectively. In addition, M. C. was supported by a postdoctoral fellowship from the Cancer Research Institute.


    Abbreviations
 
BCR B cell receptor
benzyl-{alpha}-GalNAc benzyl-2-acetamido-2-deozy-{alpha}-D-galactopyranoside
C constant
DN double negative (CD4CD8)
DP double positive (CD4+CD8+)
HA influenza virus hemaglutinin
HRP–Av horseradish peroxidase-conjugated avidin
LZRS LZRSpBMN-linker-IRES-EGFP
MB masked ß
MSCV murine stem cell virus
NEPHGE non-equilibrium pH gradient electrophoresis
PNGaseF peptide-N-glycosidase F
pT{alpha} pre-T{alpha}
SLC surrogate light chain
V variable

    Notes
 
Transmitting editor: A. Singer

5 Present address: Purdue BioPharma, LP, 201 College Road East, Princeton, NJ 08540, USA Back

Received 2 May 2000, accepted 1 August 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Lewis, S. M. 1994. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv. Immunol. 56:27.[ISI][Medline]
  2. Grawunder, U., West, R. B. and Lieber, M. R. 1998. Antigen receptor gene rearrangement. Curr. Opin. Immunol. 10:172.[ISI][Medline]
  3. Dudley, E. C., Petrie, H. T., Shah, L. M., Owen, M. J. and Hayday, A. C. 1994. T cell receptor beta chain gene rearrangement and selection during thymocyte development in adult mice. Immunity 1:83.[ISI][Medline]
  4. Hoffman, E. S., Passoni, L., Crompton, T., Leu, T. M., Schatz, D. G., Koff, A., Owen, M. J. and Hayday, A. C. 1996. Productive T-cell receptor beta-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo. Genes Dev. 10:948.[Abstract]
  5. Fehling, H. J. and von Boehmer, H. 1997. Early alpha beta T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263.[ISI][Medline]
  6. Groettrup, M., Ungewiss, K., Azogui, O., Palacios, R., Owen, M. J., Hayday, A. C. and von Boehmer, H. 1993. A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein. Cell 75:283.[ISI][Medline]
  7. Borst, J., Jacobs, H. and Brouns, G. 1996. Composition and function of T-cell receptor and B-cell receptor complexes on precursor lymphocytes. Curr. Opin. Immunol. 8:181.[ISI][Medline]
  8. Karasuyama, H., Rolink, A. and Melchers, F. 1996. Surrogate light chain in B cell development. Adv. Immunol. 63:1.[ISI][Medline]
  9. Saint-Ruf, C., Ungewiss, K., Groettrup, M., Bruno, L., Fehling, H. J. and von Boehmer, H. 1994. Analysis and expression of a cloned pre-T cell receptor gene. Science 266:1208.[ISI][Medline]
  10. Berger, M. A., Dave, V., Rhodes, M. R., Bosma, G. C., Bosma, M. J., Kappes, D. J. and Wiest, D. L. 1997. Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required. J. Exp. Med. 186:1461.[Abstract/Free Full Text]
  11. DeJarnette, J. B., Sommers, C. L., Huang, K., Woodside, K. J., Emmons, R., Katz, K., Shores, E. W. and Love, P. E. 1998. Specific requirement for CD3epsilon in T cell development. Proc. Natl Acad. Sci. USA 95:14909.[Abstract/Free Full Text]
  12. Dave, V. P., Cao, Z., Browne, C., Alarcon, B., Fernandez-Miguel, G., Lafaille, J., de la Hera, A., Tonegawa, S. and Kappes, D. J. 1997. CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage. EMBO J. 16:1360.[Abstract/Free Full Text]
  13. Fehling, H. J., Krotkova, A., Saint-Ruf, C. and von Boehmer, H. 1995. Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells [published erratum appears in Nature 1995 Nov 23; 378(6555):419]. Nature 375:795.[ISI][Medline]
  14. Haks, M. C., Krimpenfort, P., Borst, J. and Kruisbeek, A. M. 1998. The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages. EMBO J. 17:1871.[Free Full Text]
  15. Jacobs, H., Ossendorp, F., de Vries, E., Ungewiss, K., von Boehmer, H., Borst, J. and Berns, A. 1996. Oncogenic potential of a pre-T cell receptor lacking the TCR beta variable domain. Oncogene 12:2089.[ISI][Medline]
  16. Liu, C. P., Ueda, R., She, J., Sancho, J., Wang, B., Weddell, G., Loring, J., Kurahara, C., Dudley, E. C., Hayday, A., et al. 1993. Abnormal T cell development in CD3-zeta–/– mutant mice and identification of a novel T cell population in the intestine. EMBO J. 12:4863.[Abstract]
  17. Love, P. E., Shores, E. W., Johnson, M. D., Tremblay, M. L., Lee, E. J., Grinberg, A., Huang, S. P., Singer, A. and Westphal, H. 1993. T cell development in mice that lack the zeta chain of the T cell antigen receptor complex. Science 261:918.[ISI][Medline]
  18. Malissen, M., Gillet, A., Rocha, B., Trucy, J., Vivier, E., Boyer, C., Kontgen, F., Brun, N., Mazza, G., Spanopoulou, E., et al. 1993. T cell development in mice lacking the CD3-zeta/eta gene. EMBO J. 12:4347.[Abstract]
  19. Malissen, M., Gillet, A., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E. and Malissen, B. 1995. Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene. EMBO J. 14:4641.[Abstract]
  20. Mombaerts, P., Clarke, A. R., Rudnicki, M. A., Iacomini, J., Itohara, S., Lafaille, J. J., Wang, L., Ichikawa, Y., Jaenisch, R., Hooper, M. L., et al. 1992. Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages [published erratum appears in Nature 1992 Dec 3; 360(6403):491]. Nature 360:225.[ISI][Medline]
  21. Shinkai, Y., Koyasu, S., Nakayama, K., Murphy, K. M., Loh, D. Y., Reinherz, E. L. and Alt, F. W. 1993. Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes. Science 259:822.[ISI][Medline]
  22. van Oers, N. S., von Boehmer, H. and Weiss, A. 1995. The pre-T cell receptor (TCR) complex is functionally coupled to the TCR-zeta subunit. J. Exp. Med. 182:1585.[Abstract]
  23. von Boehmer, H. and Fehling, H. J. 1997. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol. 15:433.[ISI][Medline]
  24. Wiest, D. L., Berger, M. A. and Carleton, M. O. 1999. Control of thymocyte development by the pre-TCR complex: a receptor without a ligand? Semin. Immunol. 11:252.
  25. Irving, B. A., Alt, F. W. and Killeen, N. 1998. Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains. Science 280:905.[Abstract/Free Full Text]
  26. Jacobs, H., Iacomini, J., van de Ven, M., Tonegawa, S. and Berns, A. 1996. Domains of the TCR beta-chain required for early thymocyte development. J. Exp. Med. 184:1833.[Abstract]
  27. Krimpenfort, P., Ossendorp, F., Borst, J., Melief, C. and Berns, A. 1989. T cell depletion in transgenic mice carrying a mutant gene for TCR-beta. Nature 341:742.[ISI][Medline]
  28. Ossendorp, F., Jacobs, H., van der Horst, G., de Vries, E., Berns, A. and Borst, J. 1992. T cell receptor-alpha beta lacking the beta-chain V domain can be expressed at the cell surface but prohibits T cell maturation. J. Immunol. 148:3714.[Abstract/Free Full Text]
  29. Hunig, T. 1988. Cross-linking of the T cell antigen receptor interferes with the generation of CD4+8+ thymocytes from their immediate CD4-8+ precursors. Eur. J. Immunol. 18:2089.[ISI][Medline]
  30. Takahama, Y., Shores, E. W. and Singer, A. 1992. Negative selection of precursor thymocytes before their differentiation into CD4+CD8+ cells. Science 258:653.[ISI][Medline]
  31. Takahama, Y., Sugaya, K., Tsuda, S., Hasegawa, T. and Hashimoto, Y. 1995. Regulation of early T cell development by the engagement of TCR-beta complex expressed on fetal thymocytes from TCR-beta-transgenic scid mice. J. Immunol. 154:5862.[Abstract/Free Full Text]
  32. O'Shea, C. C., Thornell, A. P., Rosewell, I. R., Hayes, B. and Owen, M. J. 1997. Exit of the pre-TCR from the ER/cis-Golgi is necessary for signaling differentiation, proliferation, and allelic exclusion in immature thymocytes. Immunity 7:591.[ISI][Medline]
  33. Xavier, R., Brennan, T., Li, Q., McCormack, C. and Seed, B. 1998. Membrane compartmentation is required for efficient T cell activation. Immunity 8:723.[ISI][Medline]
  34. Zhang, W., Trible, R. P. and Samelson, L. E. 1998. LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation. Immunity 9:239.[ISI][Medline]
  35. Palmer, D. B., Hayday, A. and Owen, M. J. 1993. Is TCR beta expression an essential event in early thymocyte development? Immunol. Today 14:460.[ISI][Medline]
  36. Karasuyama, H., Kudo, A. and Melchers, F. 1990. The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain. J. Exp. Med. 172:969.[Abstract]
  37. Tsubata, T. and Reth, M. 1990. The products of pre-B cell-specific genes (lambda 5 and VpreB) and the immunoglobulin mu chain form a complex that is transported onto the cell surface. J. Exp. Med. 172:973.[Abstract]
  38. Karasuyama, H., Rolink, A. and Melchers, F. 1993. A complex of glycoproteins is associated with VpreB/lambda 5 surrogate light chain on the surface of mu heavy chain-negative early precursor B cell lines. J. Exp. Med. 178:469.[Abstract]
  39. Karasuyama, H., Rolink, A., Shinkai, Y., Young, F., Alt, F. W. and Melchers, F. 1994. The expression of Vpre-B/lambda 5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice. Cell 77:133.[ISI][Medline]
  40. Philpott, K. L., Viney, J. L., Kay, G., Rastan, S., Gardiner, E. M., Chae, S., Hayday, A. C. and Owen, M. J. 1992. Lymphoid development in mice congenitally lacking T cell receptor alpha beta-expressing cells. Science 256:1448.[ISI][Medline]
  41. Schuler, W., Weiler, I. J., Schuler, A., Phillips, R. A., Rosenberg, N., Mak, T. W., Kearney, J. F., Perry, R. P. and Bosma, M. J. 1986. Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency. Cell 46:963.[ISI][Medline]
  42. Schuler, W., Ruetsch, N. R., Amsler, M. and Bosma, M. J. 1991. Coding joint formation of endogenous T cell receptor genes in lymphoid cells from scid mice: unusual P-nucleotide additions in VJ-coding joints. Eur. J. Immunol. 21:589.[ISI][Medline]
  43. Wiest, D. L., Kearse, K. P., Shores, E. W. and Singer, A. 1994. Developmentally regulated expression of CD3 components independent of clonotypic T cell antigen receptor complexes on immature thymocytes. J. Exp. Med. 180:1375.[Abstract]
  44. Kubo, R. T., Born, W., Kappler, J. W., Marrack, P. and Pigeon, M. 1989. Characterization of a monoclonal antibody which detects all murine alpha beta T cell receptors. J. Immunol. 142:2736.[Abstract/Free Full Text]
  45. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. and Bluestone, J. A. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl Acad. Sci. USA 84:1374.[Abstract]
  46. Coulie, P. G., Uyttenhove, C., Wauters, P., Manolios, N., Klausner, R. D., Samelson, L. E. and Van Snick, J. 1991. Identification of a murine monoclonal antibody specific for an allotypic determinant on mouse CD3. Eur. J. Immunol. 21:1703.[ISI][Medline]
  47. Becker, M. L., Near, R., Mudgett-Hunter, M., Margolies, M. N., Kubo, R. T., Kaye, J. and Hedrick, S. M. 1989. Expression of a hybrid immunoglobulin-T cell receptor protein in transgenic mice. Cell 58:911.[ISI][Medline]
  48. Pullen, A. M., Marrack, P. and Kappler, J. W. 1988. The T-cell repertoire is heavily influenced by tolerance to polymorphic self-antigens. Nature 335:796.[ISI][Medline]
  49. Staerz, U. D., Rammensee, H. G., Benedetto, J. D. and Bevan, M. J. 1985. Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor. J. Immunol. 134:3994.[Abstract/Free Full Text]
  50. Samelson, L. E., Germain, R. N. and Schwartz, R. H. 1983. Monoclonal antibodies against the antigen receptor on a cloned T-cell hybrid. Proc. Natl Acad. Sci. USA 80:6972.[Abstract]
  51. Goodman, T. and Lefrancois, L. 1989. Intraepithelial lymphocytes. Anatomical site, not T cell receptor form, dictates phenotype and function. J. Exp. Med. 170:1569.[Abstract]
  52. Jameson, S. C., Nakajima, P. B., Brooks, J. L., Heath, W., Kanagawa, O. and Gascoigne, N. R. 1991. The T cell receptor V alpha 11 gene family. Analysis of allelic sequence polymorphism and demonstration of J alpha region-dependent recognition by allele-specific antibodies. J. Immunol. 147:3185.[Abstract/Free Full Text]
  53. Wiest, D. L., Burgess, W. H., McKean, D., Kearse, K. P. and Singer, A. 1995. The molecular chaperone calnexin is expressed on the surface of immature thymocytes in association with clonotype-independent CD3 complexes. EMBO J. 14:3425.[Abstract]
  54. Samelson, L. E., Weissman, A. M., Robey, F. A., Berkower, I. and Klausner, R. D. 1986. Characterization of an anti-peptide antibody that recognizes the murine analogue of the human T cell antigen receptor-T3 delta-chain. J. Immunol. 137:3254.[Abstract/Free Full Text]
  55. Fuller-Espie, S., Hoffman Towler, P., Wiest, D. L., Tietjen, I. and Spain, L. M. 1998. Transmembrane polar residues of TCR ß chain are required for signal transduction. Int. Immunol. 10:923.[Abstract]
  56. Kinsella, T. M. and Nolan, G. P. 1996. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7:1405.[ISI][Medline]
  57. Trop, S., Steff, A.-M., Denis, F., Wiest, D. L. and Hugo, P. 1999. The connecting peptide domain of pTalpha dictates weak association of the pre-T cell receptor with the TCR-zeta subunit. Eur. J. Immunol. 29:2187.[ISI][Medline]
  58. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  59. Wiest, D. L., Bhandoola, A., Punt, J., Kreibich, G., McKean, D. and Singer, A. 1997. Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of `ER-resident' molecular chaperones. Proc. Natl Acad. Sci. USA 94:1884.[Abstract/Free Full Text]
  60. Burchell, J. and Taylor-Papadimitriou, J. 1993. Effect of modification of carbohydrate side chains on the reactivity of antibodies with core-protein epitopes of the MUC1 gene product. Epithelial Cell Biol. 2:155.[ISI][Medline]
  61. Wang, J., Lim, K., Smolyar, A., Teng, M., Liu, J., Tse, A. G., Hussey, R. E., Chishti, Y., Thomson, C. T., Sweet, R. M., Nathenson, S. G., Chang, H. C., Sacchettini, J. C. and Reinherz, E. L. 1998. Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody. EMBO J. 17:10.[Abstract/Free Full Text]
  62. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389.[Abstract/Free Full Text]
  63. Bower, M. J., Cohen, F. E. and Dunbrack, R. L., Jr. 1997. Prediction of protein side-chain rotamers from a backbone-dependent rotamer library: a new homology modeling tool. J. Mol. Biol. 267:1268.[ISI][Medline]
  64. Dunbrack, R. L., Jr. and Cohen, F. E. 1997. Bayesian statistical analysis of protein side-chain rotamer pReferences. Prot. Sci. 6:1661.[Abstract/Free Full Text]
  65. Falicov, A. and Cohen, F. E. 1996. A surface of minimum area metric for the structural comparison of proteins. J. Mol. Biol. 258:871.[ISI][Medline]
  66. Bohne, A., Lang, E. and von der Lieth, C. W. 1998. W3-SWEET: carbohydrate modeling by internet. J. Mol. Model. 4:33.[ISI]
  67. Ferrin, T. E., Huang, C.C., Jarvis. L. E. and Langridge, R. 1988. The MIDAS display system. J. Mol. Graphics 6:13.[ISI]
  68. Hansen, J. E., Lund, O., Tolstrup, N., Gooley, A. A., Williams, K. L. and Brunak, S. 1998. NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconjugate J. 15:115.[ISI][Medline]
  69. Kraulis, P. J. 1991. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24:946.[ISI]
  70. Merritt, E. A. a. M., M.E.P. 1994. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr. 50:869.
  71. Carleton, M. O., Ruetsch, N. R., Berger, M. A., Rhodes, M. R., Kaptik, S. and Wiest, D. L. 1999. Signals transduced by CD3 epsilon, but not by surface pre-TCR complexes, are able to induce maturation of an early thymic lymphoma in vitro. J. Immunol. 163:2576.[Abstract/Free Full Text]
  72. Blunt, T., Finnie, N. J., Taccioli, G. E., Smith, G. C., Demengeot, J., Gottlieb, T. M., Mizuta, R., Varghese, A. J., Alt, F. W., Jeggo, P. A., et al. 1995. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 80:813.[ISI][Medline]
  73. Kirchgessner, C. U., Patil, C. K., Evans, J. W., Cuomo, C. A., Fried, L. M., Carter, T., Oettinger, M. A. and Brown, J. M. 1995. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science 267:1178.[ISI][Medline]
  74. Kang, J., Fehling, H. J., Laplace, C., Malissen, M., Cado, D. and Raulet, D. H. 1998. T cell receptor gamma gene regulatory sequences prevent the function of a novel TCRgamma/pTalpha pre-T cell receptor. Immunity 8:713.[ISI][Medline]
  75. Reed, D. S., Olson, S. and Lefrancois, L. 1998. Glycosyltransferase regulation mediated by pre-TCR signaling in early thymocyte development. Int. Immunol. 10:445.[Abstract]
  76. Ellies, L. G., Tsuboi, S., Petryniak, B., Lowe, J. B., Fukuda, M. and Marth, J. D. 1998. Core 2 oligosaccharide biosynthesis distinguishes between selectin ligands essential for leukocyte homing and inflammation. Immunity 9:881.[ISI][Medline]
  77. Tsuboi, S. and Fukuda, M. 1997. Branched O-linked oligosaccharides ectopically expressed in transgenic mice reduce primary T-cell immune responses. EMBO J. 16:6364.[Abstract/Free Full Text]
  78. Vespa, G. N., Lewis, L. A., Kozak, K. R., Moran, M., Nguyen, J. T., Baum, L. G. and Miceli, M. C. 1999. Galectin-1 specifically modulates TCR signals to enhance TCR apoptosis but inhibit IL-2 production and proliferation. J. Immunol. 162:799.[Abstract/Free Full Text]
  79. Baum, L. G., Pang, M., Perillo, N. L., Wu, T., Delegeane, A., Uittenbogaart, C. H., Fukuda, M. and Seilhamer, J. J. 1995. Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells. J. Exp. Med. 181:877.[Abstract]
  80. Perillo, N. L., Pace, K. E., Seilhamer, J. J. and Baum, L. G. 1995. Apoptosis of T cells mediated by galectin-1. Nature 378:736.[ISI][Medline]
  81. Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K. and Mariuzza, R. A. 1996. Crystal structure of a T-cell receptor beta-chain complexed with a superantigen [see Comments]. Nature 384:188.[ISI][Medline]
  82. Bentley, G. A., Boulot, G., Karjalainen, K. and Mariuzza, R. A. 1995. Crystal structure of the beta chain of a T cell antigen receptor [see Comments]. Science 267:1984.[ISI][Medline]
  83. Fields, B. A. and Mariuzza, R. A. 1996. Structure and function of the T-cell receptor: insights from X-ray crystallography [see Comments]. Immunol. Today 17:330.[ISI][Medline]
  84. Ohta, T., Sutton, M. D., Guzzo, A., Cole, S., Ferentz, A. E. and Walker, G. C. 1999. Mutations affecting the ability of the Escherichia coli UmuD' protein to participate in SOS mutagenesis. J. Bacteriol. 181:177.[Abstract/Free Full Text]
  85. Ferentz, A. E., Opperman, T., Walker, G. C. and Wagner, G. 1997. Dimerization of the UmuD' protein in solution and its implications for regulation of SOS mutagenesis [Letter]. Nat. Struct. Biol. 4:979.[ISI][Medline]