© Rockefeller University Press, 0022-1007 $8.00
JEM, Volume 199, Number 5, 607-615
This Article
Abstract
PDF (Full Text)
Supplemental Material Index
Alert me when this article is cited
Citation Map
Services
Alert me to new content in the JEM
Download to citation manager
Cited by other online articles
Google Scholar
Articles by Borowski, C.
Articles by von Boehmer, H.
Articles citing this Article
PubMed
PubMed Citation
Articles by Borowski, C.
Articles by von Boehmer, H.
Pre-TCR{alpha} and TCR{alpha} Are Not Interchangeable Partners of TCRß during T Lymphocyte Development

Christine Borowski, Xiaoyan Li, Iannis Aifantis, Fotini Gounari, and Harald von Boehmer

Department of Pathology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA 02115

Address correspondence to Harald von Boehmer, Dept. of Pathology, Harvard Medical School, Dana-Farber Cancer Institute, Smith Building Room 736, 1 Jimmy Fund Way, Boston, MA 02115. Phone: (617) 632-6880; Fax: (617) 632-6881; email: harald_von_Boehmer{at}dfci.harvard.edu


   Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In contrast with the {alpha}ß T cell receptor (TCR), the pre-TCR spontaneously segregates to membrane rafts from where it signals in a cell-autonomous fashion. The disparate behaviors of these two receptors may stem either from differences inherent to the distinct developmental stages during which they are expressed, or from features intrinsic and unique to the receptor components themselves. Here, we express TCR{alpha} precisely at the pre-TCR checkpoint, at levels resembling those of endogenous pre-TCR{alpha} (pT{alpha}), and in the absence of endogenous pT{alpha}. Both in isolation and more dramatically when in competition with pT{alpha}, TCR{alpha} induced defective proliferation, survival, and differentiation of {alpha}ß T lymphocyte precursors, as well as impaired commitment to the {alpha}ß T lymphocyte lineage. Substitution of TCR{alpha} transmembrane and cytoplasmic domains with those of pT{alpha} generated a hybrid molecule possessing enhanced competitive abilities. We conclude that features intrinsic to the pre-TCR, which are absent in TCR{alpha}, are essential for its unique function.

Key Words: T lymphocyte subsets • cell lineage • receptor-mediated signal transduction • receptor antigen • T-cell {gamma}{delta}


The online version of this article includes supplemental material.

The present address of I. Aifantis is The University of Chicago, Dept. of Medicine, Section of Rheumatology, Chicago, IL 60637.

The present address of F. Gounari is Tufts University, New England Medical Center, Molecular Oncology Research Institute, Boston, MA 02115.

Abbreviations used in this paper: DN, CD4-8- double negative; EGFP, enhanced green fluorescent protein; FTOC, fetal thymic organ culture; HSA, heat-stable antigen; pT{alpha}, pre-TCR{alpha}; WTpT{alpha}, wild-type pT{alpha}.


   Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thymocytes harboring a productive rearrangement at the TCRß locus generate a TCRß molecule that pairs covalently with the invariant pre-TCR{alpha} (pT{alpha}) and noncovalently with CD3 signal–transducing molecules, resulting in formation of a pre-TCR complex at the cell surface. The failure to identify an extracellular ligand for the pre-TCR, as well as the functional capabilities of a mutant pre-TCR complex lacking all extracellular domains (1), could indicate that pre-TCR signaling may be initiated in a manner independent of extracellular ligation. Indeed, pre-TCR complexes on the surface of a SCID murine thymoma cell line localize to membrane rafts, from where they signal in a cell-autonomous fashion (2). The ensuing phosphorylation and activation of p56Lck and ZAP-70 (2), mobilization of intracellular Ca2+ stores (3), and nuclear translocation of nuclear factor {kappa}B and NFAT transcription factors (3, 4) presumably mediate survival, proliferation, differentiation, and TCRß allelic exclusion during {alpha}ß T lymphocyte development (5). The constitutive internalization and ubiquitin-mediated proteasome-dependent degradation of the pre-TCR complex terminates this signal transduction process (6, 7).

In sharp contrast, TCR{alpha}ß complexes localize to membrane rafts, from where they trigger signal transduction cascades only in response to stimulation with costimulatory molecules and agonist peptides presented by MHC molecules (8, 9). Like the pre-TCR, TCR{alpha}ß complexes are constitutively internalized. However, in contrast to the pre-TCR, unstimulated TCR{alpha}ß complexes are recycled back to the cell surface (10, 11). Only in response to stimulation are internalized TCR{alpha}ß complexes degraded (12).

The lack of pre-TCR-induced survival, proliferation, and differentiation is evident in pT{alpha}-/- mice, even though some cells still pass the pre-TCR-controlled checkpoint by virtue of their ability to form TCR{gamma}{delta} and TCR{alpha}ß complexes (13, 14). Whether or not a TCR{alpha} molecule can functionally replace pT{alpha} in the pre-TCR complex is a matter of considerable controversy.

One view suggests that the cell-autonomous nature of pre-TCR signaling is dependent on the identity of the TCRß partner component. Several papers document that, in contrast with pre-TCR expression, premature expression of a TCR{alpha}ß complex promotes a "{gamma}{delta}-like" T cell lineage fate, and results in impaired proliferation and differentiation to the CD4+8+ stage of thymocyte development (1518). More specifically, some evidence has implicated the cytoplasmic domain unique to pT{alpha} as essential for pre-TCR function. Provision of a wild-type pT{alpha} transgene to pT{alpha}-/- mice restored pre-TCR-induced proliferation, survival, and differentiation better than a transgene lacking the pT{alpha} cytoplasmic domain or the proline-rich regions thereof (19). Pulse chase experiments documented cell-autonomous internalization and degradation of TCR and CD3{varepsilon} surface receptor components in cell lines expressing pre-TCR components, but not in those expressing TCR{gamma}{delta} or TCR{alpha}ß components (7). Mutagenesis studies identified the cytoplasmic domain of human pT{alpha} as essential for cell-autonomous receptor internalization and degradation (6). These latter studies further implied that cell-autonomous constitutive internalization might represent one mechanism by which pre-TCR surface expression and signaling is self-regulated. An apparent requirement for strict regulation of surface expression level may also exist in the analogous cell-autonomous pre–B cell receptor signaling cascade (20).

The alternative view implies that the cell-autonomous nature of pre-TCR signaling depends on the developmental stage at which the receptor is expressed rather than on qualities inherent to the TCRß partner component itself. This view suggests that pT{alpha} represents merely a "surrogate TCR{alpha} molecule," whose function is restricted to stabilizing surface expression of a productively rearranged TCRß molecule. Indeed, a recent paper implicated elevated raft content, stronger capacitative Ca2+ entry, and increased extracellular signal-related kinase activation as factors generating a unique developmental environment in CD4-8- double negative (DN) 3 thymocytes (21).

Thus far, observations concerning the potential interchangeability of pT{alpha} and TCR{alpha} have been inconclusive. Utilization of TCR{alpha} and TCRß transgenes (14, 1618), or a TCR{alpha} transgene expressed at developmental stages different from those of endogenous pT{alpha} (15, 22), has precluded a direct assessment of the performance of a TCR{alpha} molecule expressed at precisely the same developmental stage as that of endogenous pT{alpha}. Although controlled by the p56Lck proximal promoter, the wide variation among founders in the expression of transgenes encoding either pT{alpha} or pT{alpha} substituted with the connecting peptide, transmembrane, and cytoplasmic domains of TCR{alpha} made difficult a comparison between these two transgenes (23). The proliferative potential of pT{alpha}-/- fetal thymocytes retrovirally transduced with either pT{alpha} or TCR{alpha} may have been obscured by analysis of their proliferation in fetal thymic lobes (21), which is much reduced when compared with that of thymocytes developing in situ. Finally, analyses of the reconstitution of empty adult or fetal thymi in which there is no competition for resources or niche space may grossly overestimate the ability of TCR{alpha} molecules to mimic the functions of pT{alpha}.

Here, we analyze the interchangeability of WTpT{alpha} (WTpT{alpha}) with TCR{alpha}, and with a TCR{alpha}/pT{alpha} hybrid molecule consisting of the extracellular domain of TCR{alpha} joined to the transmembrane and cytoplasmic domains of pT{alpha}. By placing each transgene under the control of the p56Lck proximal promoter, we ensured that each potential TCRß partner component is expressed at an equal and relevant stage of thymocyte development. By crossing each transgene onto the pT{alpha}-/- genetic background, we allowed each receptor to perform in the absence of endogenous pT{alpha}. Furthermore, by introducing equal numbers of precursors expressing different receptors into a single thymus, we forced them to compete for available space and resources, revealing differences in receptor function that may be obscured by analyses of the developmental potential of cells that express only a single type of receptor in a noncompetitive environment.

We found that the TCR{alpha} molecule was unable to fully restore the proliferation, survival, differentiation and {alpha}ß T cell lineage commitment induced by the WTpT{alpha} molecule. When placed in direct competition, the superiority of WTpT{alpha} became more dramatically apparent with regard to proliferation and progression to the CD4+8+ stage of thymocyte development. Substitution of the transmembrane and cytoplasmic domains of TCR{alpha} with those of pT{alpha} generated a TCR{alpha}/pT{alpha} hybrid molecule exhibiting enhanced performance in competition with WTpT{alpha} when compared with TCR{alpha}, supporting the view that some of the cell-autonomous nature of pre-TCR signaling is bestowed on the complex by properties unique and intrinsic to the pT{alpha} molecule.


   Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice and Cell Lines.
C57Bl/6 and Rag-/-{gamma}c-/- mice were purchased from Taconic Farms. pT{alpha}-/- mice were generated as described previously (13). All mice were kept in specific pathogen-free animal facilities at the Dana-Farber Cancer Institute. All animal procedures were performed in compliance with the guidelines of the Dana-Farber Cancer Institute Animal Resources Facility, which operates under regulatory requirements of the U.S. Department of Agriculture and Association for Assessment and Accreditation of Laboratory Animal Care. The 58{alpha}-ß+ T cell hybridoma (provided by F. Grassi, Institute for Research in Biomedicine, Bellinzona, Switzerland; reference 28) was maintained in IMDM containing 10% FBS, penicillin-streptomycin, and 0.1% ß-mercaptoethanol.

Generation of Transgenic Mice.
WTpT{alpha} and pT{alpha}-PRO{Delta} transgenic mice were generated as described previously (19, 24). The TCR{alpha}/pT{alpha} hybrid construct was generated by PCR of an N15 TCR{alpha} cDNA template with the primers TCRaBamup, 5'-ACGGATCCTTTCCACCATGAACATGCGTCC-3', and TCRalo, 5'-AGGAATTCTGAAAGTTTAGGTTCATATCTGT-3'; and a pT{alpha} cDNA template with the primers pTaEcoup, 5'-CAGAATTCCTGGCTGAGCCTACTGCGCCTGCT-3', and pTaBamlo, 5'-TGGGATCCAGGGGTGGGTAAGATCTAA-3'. The two amplification products were cut with EcoRI, and the cohesive EcoRI sites were fused. cDNA encoding the TCR{alpha} chain of the N15 TCR (provided by L. Clayton, Dana-Farber Cancer Institute, Boston, MA) was inserted pUC1017 vector, 3' to the p56Lck proximal promoter. p56Lck proximal promoter TCR{alpha} or TCR{alpha}/pT{alpha} hybrid-hGH minigene fragments released by NotI digestion were microinjected into fertilized eggs. Founders were screened for transgene insertion by amplification of tail DNA with the primers lckp, 5'-AACCCAGTCAGGAGCTTGAA-3'; mus3, 5'-CATCGAGCAGAAGCAGTTTGA-3'; and TCRalo. pT{alpha} deficiency was assessed by amplification of tail DNA with the primers pTaF, 5'-TCACAGTGCTGGTAGATGGAAGG-3'; pTaKOR, 5'-GTTTGCTCGACATTGGGTGG-3'; and pTaWTR, 5'-GGCTCAAGAGATAACCTGAACCATG-3'.

Antibodies and Reagents.
Anti-CD8, anti-CD4, anti-TCR{gamma}{delta}, anti-TCRß, anti-CD25, anti-CD44, anti-CD24 (heat-stable antigen [HSA]), anti–Gr-1, anti–Ter-119, anti-Dx5, anti-CD19, anti-CD3{varepsilon}, anti-CD45.1, anti-CD45.2, and Annexin V were purchased from BD Biosciences. Each mAb was either biotinylated or directly conjugated to FITC, PE, cychrome, or allophycocyanin fluorophores. Cychrome- or allophycocyanin-conjugated streptavidin (BD Biosciences) was used to reveal staining with biotinylated mAbs. Surface staining of thymocytes and BM was performed as described previously (14, 19). Cells were analyzed using a FACSCaliburTM flow cytometer (Becton Dickinson) and sorted using a MoFlo cell sorter (DakoCytomation). Propidium iodide was obtained from Sigma-Aldrich.

RT-PCR.
High pure RNA isolation kit (Roche) was used to isolate thymocyte RNA. SuperScript First-strand Synthesis System for RT-PCR (Invitrogen) was used to generate cDNA. Fivefold serial dilutions of cDNA were used for semi-quantitative PCR analysis of transgene and actin levels with the primers lckp; mus3; A2, 5'GGCACCCCCTTTCCGTCTCT-3'; TCRalo; actinup, 5'-TGGAATCCTGTGGCATCCATGAAAC-3'; and actinlo, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'.

Competition Assay.
BM cells lacking the lineage markers Gr-1, Ter-119, Dx5, CD19, TCRß, and CD3{varepsilon} were sorted from WTpT{alpha}, TCR{alpha}, TCR{alpha}/pT{alpha} hybrid, pT{alpha}-PRO{Delta}, and pT{alpha}-/- mice. 1–2 x 105 cells of each population were mixed and injected intravenously into the tail vein of each irradiated (500 rad) Rag-/-{gamma}c-/- mouse. Thymic reconstitution was analyzed 5 wk after injection.

Retroviral Infection.
WTpT{alpha}, TCR{alpha}, or TCR{alpha}/pT{alpha} hybrid constructs, upstream of an internal ribosomal entry site and enhanced green fluorescent protein (EGFP; CLONTECH Laboratories, Inc.), were cloned into a modified Moloney murine leukemia virus-based retroviral vector (provided by R. Mulligan, Children's Hospital, Boston, MA). Retroviral supernatants were generated as described previously (19). 105 58{alpha}-ß+ T cell hybridoma cells were infected on ice for 3 h in the presence of 8 µg/ml polybrene.

Online Supplemental Material.
Surface expression of TCRß protein on CD4-CD8-CD25+CD44- (DN3) thymocytes from WTpT{alpha} and transgenic mice was analyzed using anti-TCRß from BD Biosciences using a FACSCaliburTM flow cytometer (Becton Dickinson). Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20031973/DC1.


   Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Generation of Transgenic Mice.
To directly compare the performance of WTpT{alpha} with TCR{alpha}, constructs encoding the TCR{alpha} component of the N15 TCR (V{alpha}8), or a TCR{alpha}/pT{alpha} hybrid molecule consisting of the extracellular domain of the N15 TCR{alpha} molecule fused to the transmembrane, and cytoplasmic domains of pT{alpha} were placed under the control of the p56Lck proximal promoter (Fig. 1 A). A previously described founder containing two to three copies of a WTpT{alpha} transgene (F63), which was also controlled by the p56Lck proximal promoter, was selected for comparison (19, 24). This WTpT{alpha} founder was provided by the lab of J. Nikolich-Zugich (Oregon Health and Science University, Beaverton, OR). All founders expressing each transgene were crossed to the pT{alpha}-/- background.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 1. Generation of transgenic mice. (A) Structures of the WTpT{alpha}, TCR{alpha}, and TCR{alpha}/pT{alpha} hybrid molecules. (B) Semi-quantitative RT-PCR analysis of primer efficiency with ß-actin control. Fivefold serial dilutions of the same sample of TCR{alpha}/pT{alpha} hybrid whole thymocyte cDNA were used in all samples. Lckp recognizes transcript p56Lck proximal promoter sequences, mus3 recognizes WTpT{alpha} sequences, and TCRalo recognizes TCR{alpha} sequences. (C) Semi-quantitative RT-PCR analysis of transgene expression (top) in transgenic founders with ß-actin controls (bottom). Fivefold serial dilutions of whole thymocyte cDNA were used. (D) Total thymocyte numbers in transgenic mice. Multiple founders of each transgenic construct were analyzed. Mice were analyzed at 3–5 wk of age.

 
To ensure that TCR{alpha} and TCR{alpha}/pT{alpha} hybrid transgenes were expressed at or near levels of the WTpT{alpha} transgene as well as physiological levels of endogenous pT{alpha} transcripts, RNA prepared from total thymocyte suspensions was analyzed by semi-quantitative RT-PCR (Fig. 1 C). A single pair of primers was used to demonstrate similar levels of transcript expression of the WTpT{alpha} transgene in DN3 thymocytes of this founder with those of endogenous pT{alpha} in DN3 thymocytes of C57Bl/6 mice (19). A common 5' primer (Lckp), which recognizes a portion of the p56Lck proximal promoter, was used to amplify all transgene transcripts. Distinct 3' primers that recognize the cytoplasmic domain of pT{alpha} (mus3) and the extracellular region of TCR{alpha} (TCRalo) were used to quantify WTpT{alpha} and TCR{alpha} transgene transcripts. The inclusion of both of these domains in the TCR{alpha}/pT{alpha} hybrid molecule permitted use of either 3' primer in the amplification of TCR{alpha}/pT{alpha} hybrid transcripts. To ensure the validity of a comparison of different transgenes amplified with distinct 3' primers, we compared the amplification efficiency achieved by each primer pair using the same sample of TCR{alpha}/pT{alpha} hybrid whole thymocyte cDNA as a template (Fig. 1 B). Using these primer pairs of comparable efficiency, four founders of TCR{alpha} transgenic mice and two founders of TCR{alpha}/pT{alpha} hybrid transgenic mice expressing transgene transcripts at levels similar to those of the WTpT{alpha} transgene (F63) were selected for analysis. With regard to surface expression of TCRß, CD4-CD8-CD25+CD44- (DN3) thymocytes from WTpT{alpha} and TCR{alpha} transgenic mice were indistinguishable (Fig. S1 available at http://www.jem.org/cgi/content/full/jem.20031973/DC1).

Neither TCR{alpha} nor TCR{alpha}/pT{alpha} Hybrid Molecules Functionally Replace pT{alpha} in the pre-TCR Complex.
When compared in noncompetitive conditions with WTpT{alpha}, neither TCR{alpha} nor the TCR{alpha}/pT{alpha} hybrid molecule successfully recapitulated pre-TCR function. In contrast with that of the endogenous pT{alpha} promoter, the activity of the p56Lck proximal promoter persists in the CD4+8+ stage of thymocyte development. Expression of a WTpT{alpha} transgene controlled by the p56Lck proximal promoter resulted in a copy number–dependent increase in apoptosis of CD4+8+ thymocytes, and a concomitant decrease in thymic cellularity (24). Although this effect was severe in transgenic founders containing >20 copies of the WTpT{alpha} transgene, little or no reduction in thymic cellularity was apparent in low copy number transgenic founders. Accordingly, the WTpT{alpha} transgenic founder used in the present work contained two to three transgene copies (24). Cellularity of thymi in TCR{alpha} and TCR{alpha}/pT{alpha} hybrid transgenic mice was only one third of that in WTpT{alpha} mice (Fig. 1 D). The fraction of thymocytes remaining at the CD4-8- stage of development was increased three- to fourfold in TCR{alpha} and TCR{alpha}/pT{alpha} hybrid thymi, whereas the proportion of thymocytes in the CD4+8+ compartment was decreased (Fig. 2 A).



View larger version (36K):
[in this window]
[in a new window]
 
Figure 2. Defective differentiation, cell cycle entry, and survival of thymocytes expressing TCR{alpha} and hybrid transgenes. (A) Dot plots depict the CD4 versus CD8 profile of a minimum of five 3–5-wk-old mice of each transgenic construct. Multiple founders were analyzed. (B) Percentage of sorted Gr-1-Ter-119-Dx5-CD19-TCR{gamma}{delta}-CD4-CD8-CD25-CD44- (DN4) thymocytes in the S/G2/M phases of the cell cycle. DNA content was analyzed by propidium iodide staining. (C) Percentage of DN4 thymocytes expressing externalized phosphatidyl serine apoptosis marker as revealed by staining with Annexin V. (D) Percentage of CD4+8+ thymocytes expressing intracellular TCRß (shaded histogram). Overlay represents intracellular staining with isotype-matched control antibody.

 
To exclude the possibility that most CD4+8+ thymocytes in TCR{alpha} transgenic mice were generated by transgenic TCR{alpha}–endogenous TCR{gamma}4 complexes (22), rather than by transgenic TCR{alpha}–endogenous TCRß complexes, CD4+8+ thymocytes from WTpT{alpha}, pT{alpha}-/-, and TCR{alpha} mice were stained for intracellular TCRß chains. If the majority of CD4+8+ in TCR{alpha} mice were selected for progression to the CD4+8+ stage by TCR{alpha}{gamma}4 complexes, a large proportion of these CD4+8+ would fail to express intracellular TCRß chains (22). However, >80% of the CD4+8+ thymocytes in TCR{alpha} mice expressed intracellular TCRß (Fig. 2 D), indicating that they were selected for progression to the CD4+8+ stage by TCR{alpha}ß complexes, rather than by TCR{alpha}{gamma} complexes.

Pre-TCR signaling in DN3 thymocytes induces down-regulation of CD25 surface expression, survival, entry into the cell cycle, and progression through the DN4 stage, as well as acquisition of CD4 and CD8 surface expression (5). Propidium iodide staining of DN4 thymocytes sorted from TCR{alpha} and TCR{alpha}/pT{alpha} hybrid mice revealed a threefold reduction in the proportion of this population in the S/G2/M phases of the cell cycle (Fig. 2 B), whereas Annexin V staining identified a three- to fourfold increase in the proportion of cells expressing the apoptotic surface phenotype characterized by externalized phosphatidyl serine (Fig. 2 C). Collectively, these data implicate deficits in cell cycle entry and survival initiated by TCR complexes containing TCR{alpha} or TCR{alpha}/pT{alpha} hybrid molecules as two reasons for the observed thymic hypocellularity.

Defective {alpha}ß T Cell Lineage Commitment Induced by TCR{alpha} and TCR{alpha}/pT{alpha} Hybrid Molecules.
In accordance with the behavior of endogenous pT{alpha} (13, 25), provision of the WTpT{alpha} transgene to pT{alpha}-/- mice reduced the proportion and absolute number of thymocytes expressing surface TCR{gamma}{delta} complexes (19). When compared with the CD4-8- compartment in WTpT{alpha} thymi, both the proportion (Fig. 3 A) and absolute number (Fig. 3 B) of TCR{gamma}{delta}+ thymocytes were elevated in mice expressing TCR{alpha} or TCR{alpha}/pT{alpha} hybrid transgenes. In contrast to the pre-TCR, receptors substituted with TCR{alpha} and TCR{alpha}/pT{alpha} hybrid molecules lack the ability to rescue CD4-8- thymocytes from commitment to the {gamma}{delta} TCR lineage.



View larger version (30K):
[in this window]
[in a new window]
 
Figure 3. Defective {alpha}ß T lineage commitment induced by TCR complexes containing TCR{alpha} and hybrid molecules. Proportion (A) and absolute number (B) of CD4-8- thymocytes expressing surface TCR{gamma}{delta}. (C) Percentage of CD4-8- thymocytes coexpressing TCR{gamma}{delta} and TCR{alpha}ß on the cell surface. (D) Histograms depict the CD4-8-TCRß+ population representative of a minimum of five 3–5-wk-old mice of each transgenic construct. Multiple founders were analyzed. (E) Surface HSA expression on CD4-8-TCRß+ thymocytes in TCR{alpha} (overlay) and WTpT{alpha} (fill) mice. (F) Surface HSA expression of CD4-8-TCRß+ (shaded histogram) and CD4-8-TCR{gamma}{delta}+ (overlay) thymocytes in TCR{alpha} mice.

 
Previous papers investigating the effects of premature TCR{alpha} expression using TCR{alpha}ß (17, 18) or TCR{alpha} (15) transgenic mice identified a population of CD4-8- TCRß+TCR{gamma}{delta}+ cells in some transgenic lines. However, differences in the developmental stage at which expression of the various TCR transgenes began made a conclusion about the {alpha}ß lineage commitment capacity of TCR{alpha}ß complexes difficult. To more definitively resolve this issue, we examined the CD4-8-TCRß+TCR{gamma}{delta}+ populations in mice expressing WTpT{alpha} and TCR{alpha} molecules at precisely the same developmental stage. When expressed at the pre-TCR checkpoint, the TCR{alpha} molecule generated a four- to fivefold larger population of CD4-8-TCRß+ TCR{gamma}{delta}+ thymocytes than does the WTpT{alpha} molecule (Fig. 3 C), further supporting the notion that TCR{alpha}ß complexes lack the robust {alpha}ß lineage commitment capacity possessed by pre-TCR complexes.

In addition to the abnormally increased population of CD4-8-TCRß+TCR{gamma}{delta}+ thymocytes, an unusually large population of CD4-8-TCRß+TCR{gamma}{delta}- thymocytes was observed in mice expressing TCR{alpha} and TCR{alpha}/pT{alpha} hybrid transgenes (Fig. 3 D). In contrast to the HSAloCD4-8-TCRß+TCR{gamma}{delta}- population observed in C57Bl/6 (26) and WTpT{alpha} mice, the CD4-8-TCRß+ TCR{gamma}{delta}- thymocytes in TCR{alpha} and TCR{alpha}/pT{alpha} hybrid mice bore an immature HSAhi surface phenotype (Fig. 3 E). In fact, their HSAhi surface phenotype most closely resembled that of thymic HSAhi TCR{gamma}{delta}+ thymocytes (Fig. 3 F). Collectively, these observations raise the possibility that TCR{alpha}ß complexes may substitute more effectively for the TCR{gamma}{delta} complex than for the pre-TCR.

Differences in Pre-TCR and TCR{alpha}ß Functionality Become Much More Apparent under Competitive Conditions.
Analysis of the ability of precursors expressing each transgene merely to fill empty thymi, in an environment of unlimited resources and niche space, might obscure functional differences between each TCR complex. To directly assess the ability of TCR{alpha} and TCR{alpha}/pT{alpha} hybrid molecules to function as the partner of TCRß in the pre-TCR complex, we forced precursors expressing each of these molecules to compete with those expressing WTpT{alpha}. To this end, 1–2 x 105 lineage-negative BM cells from mice expressing TCR{alpha} TCR{alpha}/pT{alpha} hybrid transgenes or pT{alpha}-/- mice were sorted and mixed with equal numbers of lineage-negative BM cells sorted from WTpT{alpha} mice. Crossing TCR{alpha}, TCR{alpha}/pT{alpha} hybrid, and WTpT{alpha} transgenes onto the pT{alpha}-/- Ly5.1+Ly5.2- or Ly5.1+Ly5.2+ background allowed us to identify thymocytes derived from each donor. Each BM cell mixture was injected into the tail vein of irradiated Rag-/-{gamma}c-/- recipients. Reconstituted thymi were analyzed 5 wk after injection.

The differences visible between thymi of mice expressing WTpT{alpha} and TCR{alpha} transgenes became much more apparent in the competition assay. Although in noncompetitive assays, WTpT{alpha} transgenic thymi contained on average only threefold more thymocytes than did TCR{alpha} transgenic thymi, WTpT{alpha}-derived thymocytes dominated TCR{alpha}-derived thymocytes in the competitive reconstitution assay by an average of >60-fold (Fig. 4, A and C). The proportion of WTpT{alpha}-derived donor thymocytes that progressed to the CD4+8+ developmental stage was also far greater than that of the competing TCR{alpha}-derived population (Fig. 4 B). pT{alpha}-/--derived thymocytes were dominated by WTpT{alpha}-derived thymocytes by an average factor of thirty (Fig. 4, A and C). This slight (twofold) difference in performance between thymocytes derived from TCR{alpha} and pT{alpha}-/- BM could be the consequence of transgenesis and was not statistically significant (P > 0.09). Therefore, provision of a TCR{alpha} transgene failed to give any competitive advantage to a pT{alpha}-/- population.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 4. Competitive ability of TCR{alpha}, hybrid, and WTpT{alpha}-derived thymocytes. (A) Dot plots depict apparent domination of WTpT{alpha}-derived thymocytes when in direct competition with TCR{alpha} and TCR{alpha}/pT{alpha} hybrid–derived thymocytes. (B) Dot plots depict the CD4 versus CD8 surface profile of WTpT{alpha}, TCR{alpha}, and TCR{alpha}/pT{alpha} hybrid–derived thymocytes during competitive thymic reconstitution. (C) Average fold excess of WTpT{alpha}-derived thymocytes during competitive thymic reconstitution, normalized to any fold excess CD4-8-25-44+ (DN1) cells in either population. WTpT{alpha} versus pT{alpha}-/- competition is included as a negative control to illustrate background. Dots depict individual mice for each type of competition.

 
Properties Inherent to pT{alpha} Bestow Some Competitive Ability on TCR{alpha}.
In contrast with the noncompetitive analyses, the competitive reconstitution assays revealed clear differences in the abilities of cells expressing TCR{alpha} and TCR{alpha}/pT{alpha} hybrid molecules to compete with cells expressing WTpT{alpha} molecules. In comparison with the 60-fold domination of WTpT{alpha}-derived thymocytes over their TCR{alpha} competitors, TCR{alpha}/pT{alpha} hybrid–derived thymocytes were on average dominated by WTpT{alpha}-derived competitors by only a factor of ten (Fig. 4, A and C). Similarly, when compared with TCR{alpha}-derived thymocytes, a larger proportion of TCR{alpha}/pT{alpha} hybrid–derived thymocytes progressed to the CD4+8+ developmental stage (Fig. 4 B). To confirm the significance of the pT{alpha} cytoplasmic domain specifically at the pre-TCR checkpoint, the performance of a pT{alpha} molecule lacking cytoplasmic proline-rich motifs was placed in direct competition with WTpT{alpha}. When controlled by the p56Lck proximal promoter, expression of this pT{alpha}-PRO{Delta} on a pT{alpha}-/- genetic background resulted in defective ß selection and {alpha}ß T lineage commitment (19). pT{alpha}-PRO{Delta}–derived thymocytes were dominated by WTpT{alpha}-derived competitors by a factor of >100 (Fig. 4 C). The TCR{alpha}/pT{alpha} hybrid molecule mediated passage through the CD4-8-CD25+44- (DN3) pre-TCR checkpoint as efficiently as did the WTpT{alpha} molecule (Fig. 5). In contrast, thymocytes expressing a pT{alpha} molecule lacking the proline-rich region of its cytoplasmic tail (pT{alpha}-PRO{Delta}) accumulated at the DN3 developmental stage during forced competition with thymocytes expressing WTpT{alpha} (Fig. 5). Together, these observations solidify the importance of pT{alpha}, specifically its intact cytoplasmic domain, in securing successful passage through the pre-TCR checkpoint.



View larger version (41K):
[in this window]
[in a new window]
 
Figure 5. Enhanced performance of hybrid at pre-TCR checkpoint requires intact pT{alpha} cytoplasmic domain. Percent of CD4-8- thymocytes expressing CD25+CD44- (DN3) surface phenotype in each competing population. Three individual mice are shown for each competition.

 
"pT{alpha}-like" Performance of TCR{alpha}/pT{alpha} Hybrid Molecule Is Altered by the Presence of the Extracellular Domain of TCR{alpha}.
Although the TCR{alpha}/pT{alpha} hybrid performance was superior to that of the TCR{alpha} and pT{alpha}-PRO{Delta} molecules, it remained inferior to that of WTpT{alpha}. As the only difference between the TCR{alpha}/pT{alpha} hybrid and WTpT{alpha} molecules is the presence of the extracellular domain of TCR{alpha} in the former, perhaps properties inherent to this domain are detrimental to the function of the pT{alpha}-derived portion of the TCR{alpha}/pT{alpha} hybrid molecule.

Specifically, the more efficient pairing of TCRß with TCR{alpha} than with pT{alpha} (27) is attributed to the presence of a second extracellular V{alpha} domain in TCR{alpha}, which is absent in pT{alpha}. In addition, pairing with TCRß is influenced by differences in the position of the interchain cystine residue within the connecting peptide regions of TCR{alpha} and pT{alpha}. The addition of a V{alpha} domain to the extracellular portion of pT{alpha} generated a molecule capable of outcompeting wild-type pT{alpha} for pairing with TCRß molecules in the ER (27). The distance between the transmembrane domain and the cystine residue involved in covalent linkage with TCRß is longer in TCR{alpha} than in pT{alpha}. Relocation of this TCR{alpha} cystine to the position of the pT{alpha} cystine was accompanied by loss of efficiency in pairing with TCRß, to the degree that this mutant TCR{alpha} molecule, when expressed in the same cell as a wild-type TCR{alpha} molecule, could not compete for pairing with limited TCRß molecules (27). By virtue of its TCR{alpha} extracellular domain and connecting peptide sequence, the hybrid may outcompete pT{alpha} for pairing to TCRß in the ER. This higher efficiency of pairing may better protect TCRß monomers from degradation in the ER, resulting in higher levels of hybrid TCR complexes at the thymocyte surface. The apparent strict regulation of pre-TCR surface expression, mediated by its constitutive internalization and ubiquitin-mediated proteasome-dependent degradation (6, 7), highlights the potential link between receptor surface level and function. Thus, deviation from the exquisitely low level of surface receptor expression characteristic of the pre-TCR checkpoint could alter the intensity of signals received by DN3 thymocytes.

Indeed, symmetrical TCR complexes containing TCR{alpha} and TCR{alpha}/pT{alpha} hybrid molecules, both of which contain a second extracellular V{alpha} domain as well as TCR{alpha} connecting peptide sequences, were expressed at levels higher than those of normal pre-TCR complexes on the surface of the 58{alpha}-ß+ T cell hybridoma (28), which is a variant of a DO-11.10.7 mouse T cell hybridoma that does not express functional TCR{alpha} or TCRß chains (Fig. 6). Thus, the possibility exists that higher surface levels might impair the pT{alpha}-like function of TCR{alpha}/pT{alpha} hybrid TCR complexes.



View larger version (23K):
[in this window]
[in a new window]
 
Figure 6. Influence of the extracellular region of TCR{alpha} on the behavior of the hybrid molecule. TCRß levels on the surface of the 58{alpha}-ß+ T cell hybridoma are influenced by the second extracellular domain TCR{alpha}. 58{alpha}-ß+ hybridoma cells were infected with retroviruses encoding WTpT{alpha}, TCR{alpha}, or TCR{alpha}/pT{alpha} hybrid molecules, followed by an internal ribosomal entry site and enhanced green fluorescent protein (EGFP). Infected cells were identified by EGFP expression. Shaded histograms represent TCRß surface staining on cells infected with retroviruses expressing WTpT{alpha}, TCR{alpha}, or TCR{alpha}/pT{alpha} hybrid molecules. The overlay represents TCRß staining on cells infected with a control retrovirus expressing EGFP alone. The 58{alpha}-ß+ T cell hybridoma is a variant of the DO-11.10.7 mouse T cell hybridoma that does not express functional TCRß or TCR{alpha} chains (28).

 

   Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Here, we show that replacement of pT{alpha} with TCR{alpha} precisely at the pre-TCR developmental checkpoint generated a TCR{alpha}ß complex incapable of inducing normal ß selection processes. TCR{alpha} substitution resulted in an increase in apoptosis and decrease in proliferation of DN4 thymocytes, a defect in progression to the CD4+8+ developmental stage, and a failure to rescue thymocytes from the {gamma}{delta} T cell lineage. The similarities between these defects and those observed in pT{alpha}-/- mice emphasize the inability of TCR{alpha} to functionally replace pT{alpha} as the partner of TCRß in the pre-TCR complex. Although visible in noncompetitive conditions, the differences in functional capacity between pre-TCR and TCR{alpha}ß complexes became much more dramatically apparent when placed in direct competition with each other. Addition of the pT{alpha} transmembrane and cytoplasmic domains to the extracellular domain of TCR{alpha} generated a TCR{alpha}/pT{alpha} hybrid molecule possessing enhanced abilities that became evident only when placed in direct competition with WTpT{alpha}. The competitive performance of a pT{alpha} molecule lacking the proline-rich regions of the cytoplasmic domain (pT{alpha}-PRO{Delta}) was inferior to that of both the TCR{alpha}/pT{alpha} hybrid and TCR{alpha} molecules, suggesting that possession of an intact pT{alpha} cytoplasmic domain was required for the improved competitive ability displayed by the TCR{alpha}/pT{alpha} hybrid molecule.

Although the TCR{alpha}/pT{alpha} hybrid performance was superior to that of the TCR{alpha} and pT{alpha}-PRO{Delta} molecules, it remained inferior to that of WTpT{alpha}. One possible explanation for this discrepancy is that the higher surface expression levels of TCR{alpha}/pT{alpha} hybrid TCR complexes may impair their pT{alpha}-like function (Fig. 6). The strict regulation of pre-TCR surface expression, mediated by its constitutive internalization and ubiquitin-mediated proteasome-dependent degradation (6, 7), highlights the significance of the potential link between receptor surface level and function. Alternatively, the pT{alpha}-like function of TCR{alpha}/pT{alpha} hybrid TCR complexes may be affected by the strength of association between TCR{alpha}/pT{alpha} hybrid molecules and TCR{zeta} chains. Sequences unique to the connecting peptide of TCR{alpha}, which are present in the TCR{alpha}/pT{alpha} hybrid but absent in WTpT{alpha}, are required for strong association with TCR{zeta} chains (29). Their potentially stronger association with TCR{zeta} chains might allow TCR{alpha}/pT{alpha} hybrid TCR complexes to activate or augment signaling pathways that are silent or attenuated during normal pre-TCR signaling. A final possibility is that the second extracellular V{alpha} domain in the TCR{alpha}/pT{alpha} hybrid molecule may allow, in addition to higher levels of surface expression, recognition of MHC molecules in the thymic microenvironment. In contrast, the asymmetrical nature of the pre-TCR ectodomain precludes recognition of MHC molecules. The observed spontaneous segregation of pre-TCR complexes to glycolipid-enriched microdomains, which contain a high concentration of signaling molecules (2), may be involved in the cell-autonomous nature of pre-TCR signal initiation. Recognition of thymic MHC molecules might interfere with spontaneous membrane segregation processes, resulting in altered receptor signaling capacity.

Whether or not TCR{alpha} can substitute for pT{alpha} in the pre-TCR complex has been a matter of some controversy and discussion. Conflicting views placing significance either on the receptor components themselves or on the developmental stage at which they are expressed have been the subject of numerous investigations. However, three factors have precluded a complete assessment of the interchangeability of pT{alpha} and TCR{alpha} at this point of development. These factors are as follows: (a) expression of TCRß and/or TCR{alpha} transgenes at developmental stages different than that of endogenous pT{alpha}, either in the presence (1518, 22) or absence (14) of endogenous pT{alpha}; (b) analyses of the ability of precursors to fill empty thymi with no competition for niche space or resources; and (c) use of fetal thymic organ culture (FTOC) systems that may not reveal the full proliferative potential of precursors (21). Here, we observe the performance of a TCR{alpha} molecule expressed in the absence of pT{alpha}, at the same relevant developmental stage and RNA levels as endogenous pT{alpha}, in adult thymi, both by itself and in direct competition for thymic space and resources.

Utilization of p56Lck proximal promoter elements ensured a uniform temporal regulation of WTpT{alpha}, TCR{alpha}, TCR{alpha}/pT{alpha} hybrid, and pT{alpha}-PRO{Delta} transgene expression. Thus, our results fail to support the notion that the developmental stage at which the pre-TCR is expressed, rather than the identity of the receptor components themselves, is the factor on which pre-TCR function is most dependent. A recent work (21), reaching the opposite conclusion, compared the development of pT{alpha}-/- fetal thymocytes retrovirally transduced with either WTpT{alpha} or TCR{alpha} in FTOC. The short time of incubation in FTOC (2 d), which may have obscured differences in the full proliferative potential of precursors retrovirally transduced with each receptor component, raises a question regarding the validity of the conclusion that these two molecules are functionally equivalent at the pre-TCR checkpoint.

Its failure to rescue CD4-8- thymocytes from commitment to the {gamma}{delta} T cell lineage may be perhaps the most significant deficiency in the repertoire of "pre-TCR-like" skills possessed by a TCR{alpha}ß complex. Specifically, this defect highlights a potential reason for the precise temporal segregation of pT{alpha} and TCR{alpha} expression during thymocyte development. By virtue of its failure to direct immature CD4-8- thymocytes through the normal ß selection processes vital to the development of mature CD4+ and CD8+ T lymphocytes, a TCR{alpha} molecule expressed at the pre-TCR checkpoint could instead direct young CD4-8- thymocytes into a pathway leading to an immature {gamma}{delta}-like fate.

Our data in TCR{alpha} transgenic mice and previous data in pT{alpha}-/-TCR{delta}-/- mice (14) indicate that a prematurely expressed TCR{alpha} chain can generate some limited numbers of TCRß-selected CD4+8+ thymocytes and argues that the {alpha}ß TCR, rather than a putative TCR{gamma}4/TCR{alpha} heterodimer (22), is involved in the selection of CD4+8+ thymocytes.

The requirement for the cytoplasmic domain of pT{alpha} at the pre-TCR checkpoint might reside in its ability to recruit the pre-TCR complex to glycolipid-enriched membrane microdomains rich in signaling molecules. An interaction of the proline-rich region of the pT{alpha} cytoplasmic domain with Src homology 3 domains of raft-localized Src kinases could result in both raft localization and, via disruption of the inhibitory Src kinase intramolecular loop, in cell-autonomous activation of Src kinase activity. Alternatively, the pT{alpha} cytoplasmic domain might be required for constitutive internalization and degradation of pre-TCR complexes (6, 7), thereby regulating and maintaining the exquisitely low levels of pre-TCR complexes normally observed on the thymocyte surface.

Future studies analyzing differences in genetic and biochemical profiles induced by expression of WTpT{alpha} and TCR{alpha} at the pre-TCR checkpoint may provide insight as to why TCR{alpha} is unable to substitute for pT{alpha} at this crucial stage of thymocyte development.


   Acknowledgments
 
We thank M. Handley, H. Levine, and J. LaVecchio for excellent cell sorting, and L. Holcomb and E. Smith for superb assistance in preparation of the manuscript. The authors thank Dr. O. Kanagawa for drawing their attention to competitive reconstitution experiments.

This work was supported by National Institutes of Health grants AI47281 and AI45846 (to H. von Boehmer), and a National Science Foundation Fellowship (to C. Borowski). I. Aifantis is supported by the Cancer Research Institute and the Howard Hughes Medical Institute Research Resource Program at The University of Chicago. The authors declare that they have no competing financial interests.

Submitted: 14 November 2003
Accepted: 31 December 2003


   References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Irving, B.A., F.W. Alt, and N. Killeen. 1998. Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains. Science. 280:905–908.[Abstract/Free Full Text]
  2. Saint-Ruf, C., M. Panigada, O. Azogui, P. Debey, H. von Boehmer, and F. Grassi. 2000. Different initiation of pre-TCR and gammadeltaTCR signalling. Nature. 406:524–527.[CrossRef][Medline]
  3. Aifantis, I., F. Gounari, L. Scorrano, C. Borowski, and H. von Boehmer. 2001. Constitutive pre-TCR signaling promotes differentiation through Ca2+ mobilization and activation of NF-kappaB and NFAT. Nat. Immunol. 2:403–409.[Medline]
  4. Voll, R.E., E. Jimi, R.J. Phillips, D.F. Barber, M. Rincon, A.C. Hayday, R.A. Flavell, and S. Ghosh. 2000. NF-kappa B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development. Immunity. 13:677–689.[Medline]
  5. von Boehmer, H., I. Aifantis, J. Feinberg, O. Lechner, C. Saint-Ruf, U. Walter, J. Buer, and O. Azogui. 1999. Pleiotropic changes controlled by the pre-T-cell receptor. Curr. Opin. Immunol. 11:135–142.[CrossRef][Medline]
  6. Carrasco, Y.R., M.N. Navarro, and M.L. Toribio. 2003. A role for the cytoplasmic tail of the pre-T cell receptor (TCR) alpha chain in promoting constitutive internalization and degradation of the pre-TCR. J. Biol. Chem. 278:14507–14513.[Abstract/Free Full Text]
  7. Panigada, M., S. Porcellini, E. Barbier, S. Hoeflinger, P.A. Cazenave, H. Gu, H. Band, H. von Boehmer, and F. Grassi. 2002. Constitutive endocytosis and degradation of the pre–T cell receptor. J. Exp. Med. 195:1585–1597.[Abstract/Free Full Text]
  8. Montixi, C., C. Langlet, A.M. Bernard, J. Thimonier, C. Dubois, M.A. Wurbel, J.P. Chauvin, M. Pierres, and H.T. He. 1998. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17:5334–5348.[Abstract/Free Full Text]
  9. Viola, A., S. Schroeder, Y. Sakakibara, and A. Lanzavecchia. 1999. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science. 283:680–682.[Abstract/Free Full Text]
  10. Minami, Y., L.E. Samelson, and R.D. Klausner. 1987. Internalization and cycling of the T cell antigen receptor. Role of protein kinase C. J. Biol. Chem. 262:13342–13347.[Abstract/Free Full Text]
  11. Krangel, M.S. 1987. Endocytosis and recycling of the T3–T cell receptor complex. The role of T3 phosphorylation. J. Exp. Med. 165:1141–1159.[Abstract]
  12. Liu, H., M. Rhodes, D.L. Wiest, and D.A. Vignali. 2000. On the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity. 13:665–675.[Medline]
  13. Fehling, H.J., A. Krotkova, C. Saint-Ruf, and H. von Boehmer. 1995. Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells. Nature. 375:795–798.[CrossRef][Medline]
  14. Buer, J., I. Aifantis, J.P. DiSanto, H.J. Fehling, and H. von Boehmer. 1997. Role of different T cell receptors in the development of pre–T cells. J. Exp. Med. 185:1541–1547.[Abstract/Free Full Text]
  15. Fritsch, M., A. Andersson, K. Petersson, and F. Ivars. 1998. A TCR alpha chain transgene induces maturation of CD4- CD8- alpha beta+ T cells from gamma delta T cell precursors. Eur. J. Immunol. 28:828–837.[CrossRef][Medline]
  16. Lacorazza, H.D., C. Tucek-Szabo, L.V. Vasovic, K. Remus, and J. Nikolich-Zugich. 2001. Premature TCR alpha beta expression and signaling in early thymocytes impair thymocyte expansion and partially block their development. J. Immunol. 166:3184–3193.[Abstract/Free Full Text]
  17. Bruno, L., H.J. Fehling, and H. von Boehmer. 1996. The alpha beta T cell receptor can replace the gamma delta receptor in the development of gamma delta lineage cells. Immunity. 5:343–352.[Medline]
  18. Terrence, K., C.P. Pavlovich, E.O. Matechak, and B.J. Fowlkes. 2000. Premature expression of T cell receptor (TCR){alpha}ß suppresses TCR{gamma}{delta} gene rearrangement but permits development of {gamma}{delta} lineage T cells. J. Exp. Med. 192:537–548.[Abstract/Free Full Text]
  19. Aifantis, I., C. Borowski, F. Gounari, H.D. Lacorazza, J. Nikolich-Zugich, and H. von Boehmer. 2002. A critical role for the cytoplasmic tail of pTalpha in T lymphocyte development. Nat. Immunol. 3:483–488.[Medline]
  20. Pogue, S.L., and C.C. Goodnow. 2000. Gene dose-dependent maturation and receptor editing of B cells expressing immunoglobulin (Ig)G1 or IgM/IgG1 tail antigen receptors. J. Exp. Med. 191:1031–1044.[Abstract/Free Full Text]
  21. Haks, M.C., S.M. Belkowski, M. Ciofani, M. Rhodes, J.M. Lefebvre, S. Trop, P. Hugo, J.C. Zuniga-Pflucker, and D.L. Wiest. 2003. Low activation threshold as a mechanism for ligand-independent signaling in pre-T cells. J. Immunol. 170:2853–2861.[Abstract/Free Full Text]
  22. Erman, B., L. Feigenbaum, J.E. Coligan, and A. Singer. 2002. Early TCRalpha expression generates TCRalphagamma complexes that signal the DN-to-DP transition and impair development. Nat. Immunol. 3:564–569.[CrossRef][Medline]
  23. Gibbons, D., N.C. Douglas, D.F. Barber, Q. Liu, R. Sullo, L. Geng, H.J. Fehling, H. von Boehmer, and A.C. Hayday. 2001. The biological activity of natural and mutant pT{alpha} alleles. J. Exp. Med. 194:695–703.[Abstract/Free Full Text]
  24. Lacorazza, H.D., H.E. Porritt, and J. Nikolich-Zugich. 2001. Dysregulated expression of pre-Talpha reveals the opposite effects of pre-TCR at successive stages of T cell development. J. Immunol. 167:5689–5696.[Abstract/Free Full Text]
  25. Aifantis, I., O. Azogui, J. Feinberg, C. Saint-Ruf, J. Buer, and H. von Boehmer. 1998. On the role of the pre-T cell receptor in alphabeta versus gammadelta T lineage commitment. Immunity. 9:649–655.[Medline]
  26. Fowlkes, B.J., A.M. Kruisbeek, H. Ton-That, M.A. Weston, J.E. Coligan, R.H. Schwartz, and D.M. Pardoll. 1987. A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family. Nature. 329:251–254.[CrossRef][Medline]
  27. Trop, S., M. Rhodes, D.L. Wiest, P. Hugo, and J.C. Zuniga-Pflucker. 2000. Competitive displacement of pT alpha by TCR-alpha during TCR assembly prevents surface coexpression of pre-TCR and alpha beta TCR. J. Immunol. 165:5566–5572.[Abstract/Free Full Text]
  28. Letourneur, F., and B. Malissen. 1989. Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin. Eur. J. Immunol. 19:2269–2274.[Medline]
  29. Trop, S., A.M. Steff, F. Denis, D.L. Wiest, and P. Hugo. 1999. The connecting peptide domain of pT alpha dictates weak association of the pre-T cell receptor with the TCR zeta subunit. Eur. J. Immunol. 29:2187–2196.[CrossRef][Medline]