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The Journal of Immunology, 1998, 161: 1718-1727.
Copyright © 1998 by The American Association of Immunologists

Receptor-Specific Allelic Exclusion of TCRV{alpha}-Chains During Development

Richard Boyd*, Ivona Kozieradzki{dagger}, Ann Chidgey*, Hans-Willi Mittrücker{dagger}, Dennis Bouchard{dagger}, Emma Timms{dagger}, Kenji Kishihara{dagger}, Christopher J. Ong{ddagger}, Daniel Chui{ddagger}, Jamey D. Marth§, Tak W. Mak{dagger} and Josef M. Penninger1,{dagger}

* Department of Pathology and Immunology, Monash Medical School, Melbourne, Victoria, Australia; {dagger} Amgen Institute, Ontario Cancer Institute, and Departments of Medical Biophysics and Immunology, University of Toronto, Ontario, Canada; {ddagger} The Biomedical Research Centre and Departments of Medical Genetics and Biochemistry, University of British Columbia, Vancouver, Canada; and § Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCR{alpha}ß transgenes that allelic exclusion at the TCR{alpha} locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRV{alpha}-chain. TCRV{alpha} allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRV{alpha}+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRV{alpha}-chain, but lose the nonfunctional receptor. Whereas activation of TCRVß-chains or CD3{epsilon} leads to receptor internalization, TCRV{alpha} ligation promotes retention of the TCR on the cell surface. Although both TCRV{alpha}- and TCRVß-chains trigger phosphotyrosine signaling, only the TCRVß-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRV{alpha}-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Expression of a single Ag receptor on lymphocytes and sensory receptors in neurons is maintained via allelic exclusion, which ensures the generation of cells that express distinct and clonal receptors (1, 2, 3, 4, 5, 6, 7). Expression of a single olfactory receptor and pheromone receptor on the surface of distinct sensory neurons might allow discrimination of diverse stimuli that regulate behavior (1, 2). T and B lymphocytes express unique and specific Ag receptors through genetic recombination to generate a clonal and self-restricted receptor repertoire against myriads of different Ags (3). Although developing lymphocytes have the potential to rearrange two TCR or Ig alleles, most T and B cells express only one Ag receptor on the cell surface (one cell/one receptor rule) (4, 5, 6, 7).

During thymic development, T cells rearrange TCR{alpha} and TCRß genes to generate a functional TCR{alpha}ß heterodimer that can mediate thymic selection (3). TCRVß-chains are allelically excluded in T cells at the genetic level, and almost all mature mouse T cells express only one TCRVß-chain on the cell surface (4, 7, 8, 9, 10). Allelic exclusion at the TCR{alpha} locus is incomplete in TCR{alpha}ß transgenic (Tg)2 mice (11, 12, 13, 14), and as many as 20% of peripheral human T cells (15) and 10% of peripheral mouse T cells (16) can coexpress two different TCRV{alpha}-chains on the cell surface. It also has been shown that thymocytes can undergo multiple TCR{alpha} gene rearrangements (17) and that immature CD3low thymocytes can express two TCRV{alpha}-chains on the cell surface (18). In addition, T cell clones display frequent in-frame rearrangements at both TCR{alpha} alleles and can express two functional mRNA species, but express only one TCR on the cell surface (19, 20, 21). Similarly, B cells can undergo multiple IgH gene rearrangements (22), have the potential to express dual {kappa}/{lambda} B cell receptors on the cell surface (23), and can produce two Igs with dual specificity (24, 25). Although positive selection and TCR/CD3 ligation terminate RAG expression and TCR rearrangement in thymocytes (11, 12, 26, 27), allelic exclusion is intact in Tg mice overexpressing the recombination genes RAG-1 and RAG-2 (28, 29).

We report that a large number of immature thymocytes from normal mice and mice expressing rearranged TCR{alpha}ß transgenes express two or even three distinct TCRV{alpha}-chains on the cell surface, indicating that allelic exclusion of TCRV{alpha}-chains is not functional in immature thymocytes. Mature thymocytes expressed only one TCRV{alpha} on the cell surface and expressed the second TCRV{alpha}-chain in the cytoplasm. Genetic rescue of selection in mice lacking the CD45 protein tyrosine phosphatase restored expression of a single surface TCRV{alpha}-chain in mature, but not in immature, thymocytes. Allelic expression of a single TCRV{alpha}-chain was dependent on specific peptide/MHC recognition events during selection and, in an in vitro assay for positive selection, thymocytes were selected for expression of the peptide-specific TCRV{alpha}-chain and lost surface expression of the nonselectable TCRV{alpha}-chain. Ligation of both TCRV{alpha}- and TCRVß-chains induces tyrosine phosphorylation, but only TCRVß activation mediates recruitment of the GTPase dynamin to the cell membrane, required for receptor endocytosis. These data suggest a novel mechanism of receptor-based selection and allelic exclusion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

CD45 Tg (CD45RO and CD45ABC-splice variants) (30), H-Y-specific TCR{alpha}ß Tg (31), P14 lymphotoxic choriomeningitis virus (LCMV)-specific TCR{alpha}ß Tg (32), cytochrome c-specific TCR{alpha}ß Tg (33), CD45-exon 6 gene-deficient (34), TAP1 gene-deficient (35), CD45Tg/CD45-/- (36), P14 Tg/TAP1-/- (37), P14 Tg/CD45-/- mice (38), and P14 Tg/ß2-microglobulin-/- (ß2m-/-) mice (39) have been described. The genetic background of mouse strains was confirmed by Southern blotting and by immunofluorescence staining using mAbs specific for the Tg TCR. Care of mice was in accordance with guidelines of Canadian Medical Research Council.

Immunocytometry

Blood samples (20 µl) were collected in heparinized capillary tubes and washed once in immunofluorescence staining buffer (30 min, 4°C, PBS, 1% FCS, 0.1% NaN3). Single cell suspensions of thymocytes, spleen cells, and lymph node cells were prepared as described (38), resuspended in PBS, and incubated with the appropriate Abs. The following mAbs were used: pan-CD45 (FITC or PE labeled; PharMingen, San Diego, CA); anti-CD3{epsilon} (PE labeled; PharMingen); anti-pan TCRß (PE or FITC labeled; PharMingen); anti-TCRVß8.1/Vß8.2 (clone KJ16; PE or biotin labeled; PharMingen); anti-TCRVß6 (FITC or biotin labeled; PharMingen); anti-TCRVß14 (FITC labeled; PharMingen); T3.70 (a mAb reactive against the H-Y-specific Tg TCRV{alpha}-chain; FITC or biotin labeled); anti-TCRV{alpha}11 (PE labeled; PharMingen); anti-TCRV{alpha}2 (PE or biotin labeled; PharMingen); anti-TCRV{alpha}3.2 (FITC labeled; PharMingen); anti-CD8{alpha} (FITC, PE labeled, or biotinylated; PharMingen); and anti-CD4 (FITC or PE labeled; PharMingen). All staining combinations were as indicated in the figure legends. Biotinylated Abs were visualized with streptavidin-RED670 (Life Sciences, St. Petersburg, FL). Samples were analyzed using a FACScan (Becton Dickinson, Mountain View, CA).

Immunostaining for cytoplasmic TCRV{alpha}-chains

Thymocytes from H-Y Tg mice were purified and surface stained with anti-CD3{epsilon} (FITC labeled) and anti-TCRV{alpha}2 (PE labeled) for 30 min on ice. Cells were then washed, fixed, and permeabilized using a commercial kit (Caltag Laboratories, San Francisco, CA). Fixing destroys the TCRV{alpha} and TCRVß epitopes on the cell surface (not shown). For intracellular staining, permeabilized cells were incubated with anti-T3.70 biotin for 30 min at room temperature. T3.70 biotin was visualized using streptavidin RED670, and CD3{epsilon}highTCRV{alpha}2high thymocytes were analyzed for intracellular staining of the Tg TCRV{alpha}-chain (mAb T3.70) using live acquisition gates on a CellQuest program (Becton Dickinson). Similarly, thymocytes were surface stained with T3.70 (biotin labeled) and anti-TCRV{alpha}2 (PE labeled), fixed to destroy surface epitopes, and subsequently stained for cytoplasmic expression of the H-Y-specific Tg TCRV{alpha}-chain using T3.70 FITC. This protocol allowed to exclude all TCRV{alpha}2/T3.70 surface double-positive cells from the analysis. As a control, cells were surface stained using T3.70 biotin and anti-TCRV{alpha}2 PE, fixed, permeabilized, and stained for cytoplasmic TCRVß expression using anti-TCRVß6 FITC. Both anti-TCRVß6 and T3.70 are rat IgG mAbs. Similar to TCRV{alpha} staining, thymocytes were surface stained with anti-TCRVß8.1/8.2 PE and anti-CD3{epsilon} FITC, permeabilized, and stained for intracytoplasmic expression of TCRVß6 biotin or TCRVß14 biotin.

Northern blot analysis

RNA was isolated from total thymocytes using guanidine isothiocyanate extraction. Five micrograms of total RNA were electrophoresed on a 5.5% formaldehyde/1% agarose gel, transferred to a nitrocellulose membrane, and hybridized with RAG-1, RAG-2, or ß-actin probes. Hybridized membranes were exposed and imaged, and RAG-1 and RAG-2 mRNA levels were compared with ß-actin mRNA. The relative levels of RAG-1 and RAG-2 mRNAs were quantified using a PhosphorImager (ImageQuant software; Molecular Dynamics, Sunnyvale, CA).

Cell sorting

P14 TCR-{alpha}ß Tg mice expressing a V{alpha}2J{alpha}TA31/Vß8.1DJß2.4 TCR specific for a LCMV glycoprotein peptide (p33 peptide; amino acids 33–41) in association with MHC class I (H-2Db) (32) were backcrossed into a TAP1-/- (35) or a ß2m-/- (39) background. Both P14/TAP1-/- and P14/ß2m-/- mice have a block in positive selection of the P14 TCR (37, 39). Thymocytes were isolated from P14/TAP1-/- or P14/ß2m-/- mice and triple stained for TCRV{alpha} expression using anti-TCRV{alpha}2 biotin, anti-TCRV{alpha}11 PE, and anti-CD3{epsilon} FITC. Biotinylated anti-TCRV{alpha}2 was visualized using streptavidin-RED670. Dual TCRV{alpha}2+TCRV{alpha}11+CD3low and dual T3.70+TCRV{alpha}2+CD3low thymocytes were purified using a FACS Power Sorter (Coulter). Similarly, thymocytes from H-Y/CD45-/-/CD45Tg mice (36) were stained using anti-T3.70 biotin (H-Y Tg TCRV{alpha}-chain), anti-TCRV{alpha}2 PE (endogenous TCRV{alpha}-chain), and anti-CD3{epsilon} FITC. In all experiments, postsorting purity of dual TCRV{alpha}+ thymocytes was >98%.

In vitro T cell selection

Stromal cell cocultures for in vitro positive T cell selection have been described (40). Briefly, dual TCRV{alpha}2+TCRV{alpha}11+CD3low thymocytes were isolated from thymi of P14 TCR Tg/TAP1-/- mice (Ly-5.2+) using cell sorting (see above). Sorted TCRV{alpha}2+TCRV{alpha}11+ thymocytes (purity >98%) were prepared at a concentration of 3.4 x 106 cells/ml in RPMI medium (2 mM L-glutamine, 0.05% benzyl penicillin, 0.05% streptomycin (Sigma, St. Louis, MO), 10% heat-inactivated FCS (Life Sciences), and 5 x 10-5 mol/ml 2-ME (Sigma)). Stromal cells from Ly-5.1+ congeneic C57BL6 (H-2b/b) and CBA (H-2k/k) mice were prepared by enzymatic digestion of lymphocyte-depleted thymi using 0.15% collagenase/0.1% DNase (Boehringer Mannheim, Indianapolis, IN). Stromal cells were enriched by elutriation (40) and resuspended at a concentration of 6.7 x 105 cells/ml, then pulsed with 10-9 M LCMV-p33 peptide. Sorted TCRV{alpha}2+TCRV{alpha}11+CD3low thymocytes were mixed with stromal cells at a ratio of 5:1 and cocultured as hanging drops in inverted Terasaki plates at 37°C (5% CO2). Cells were harvested on day 4 and stained with anti-TCRV{alpha}2 (biotin, visualized by RED670), anti-TCRV{alpha}11 (PE), Ly-5.2 (FITC), and anti-CD3{epsilon} (APC). Cells were analyzed by flow cytometry. Transgenic Ly-5.2+ thymocytes were distinguished from Ly-5.1+ congeneic stromal cells by Ly-5.2 surface expression.

TCRV{alpha} cross-linking

For TCR cross-linking, goat anti-rat IgG (10 µg/ml; Southern Biotechnology Associates) was bound to round-bottom 96-well plates (Nunc) for 24 h at 4°C. Then plates were washed with PBS and further coated with unconjugated anti-CD3{epsilon} (clone KT3, rat IgG), anti-TCRV{alpha}11 (clone RR8.1, rat IgG), anti-TCRV{alpha}2 (clone B20.1, rat IgG), and anti-TCRVß8.1/8.2 (clone KJ16, rat IgG) for 4 h at 37°C. All cross-linking Abs were used at a concentration of 1 µg/ml. TCRV{alpha}2+TCRV{alpha}11+CD3{epsilon}low thymocytes were sorted from P14 TCR Tg/ß2m-/- mice, as described above, and purified (>99%) dual TCRV{alpha}+ thymocytes (5 x 104/well) were incubated with anti-CD3{epsilon}-, anti-TCRV{alpha}11-, anti-TCRV{alpha}2-, or anti-TCRVß8.1/8.2-coated wells (37°C, 5% CO2). After incubation for 24 h, cells were harvested and stained with anti-CD3{epsilon} (FITC), anti-TCRV{alpha}2 (biotin), and anti-TCRV{alpha}11 (PE), and live cells were analyzed for TCRV{alpha} expression using a FACScan.

Phosphotyrosine signaling and immunoprecipitations

Freshly isolated thymocytes from P14 Tg/ß2m-/- mice (39) or P14 Tg/CD45-/- mice (38) were activated with anti-CD3{epsilon} (clone KT3, rat IgG), anti-TCRV{alpha}2 (clone B20.1, rat IgG), or anti-TCRVß8.1/8.2 (clone KJ16, rat IgG), and a goat anti-rat IgG cross-linking Ab (Jackson ImmunoResearch, West Grove, PA). Cells were lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 100 mM Tris buffer, pH 8, 100 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin) for 2 h on ice. Protein lysates were resuspended in running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) and electrophoresed on a 8% polyacrylamide gel (10% SDS, 10% APS). Proteins were transferred onto nitrocellulose filters using a MilliBlot-Graphite Electroblotter I (Bio-Rad, Hercules, CA), and filters were blocked with TBS, 0.1% Tween-20, and 1% BSA (overnight at 4°C). The filters were then incubated with a horseradish peroxidase-conjugated anti-phosphotyrosine mAb (Upstate Biotechnology, Lake Placid, NY) for 1 h at room temperature, followed by washing in TBS/0.1% Tween-20. Immunoprecipitations using anti-Cbl-2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Vav (Santa Cruz Biotechnology), anti-phospholipase C{gamma}1 (Upstate Biotechnology), anti-ZAP70 (gift of Dr. A. Weiss), anti-Grb2 (Transduction Laboratories, Lexington, KY), and anti-dynamin (Transduction Laboratories) were performed as described (41). Total cell lysates, cell membrane-bound protein fractions, and cytosolic protein fraction were prepared as described (42). Proteins were visualized on Western blots using enhanced chemiluminescence (ECL; Amersham, Little Chalfont, U.K.).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The CD45 protein tyrosine phosphatase controls the signal for allelic exclusion of TCRV{alpha} surface expression

To test whether immature CD3low thymocytes and mature CD3high thymocytes can express two distinct TCRV{alpha}-chains on the cell surface (18), we analyzed dual TCRV{alpha} expression on immature CD3low and mature CD3high thymocytes in normal mice and mice that harbor a mutation in the CD45 protein tyrosine phosphatase gene (34). CD45-/- mice have a block in T cell development from immature CD4+CD8+ to mature CD4+ and CD8+ thymocytes, which correlates with a block in positive selection (Fig. 1GoA). Figure 1GoB shows that CD3low immature, but not CD3high mature, thymocytes or peripheral T cells (not shown) from normal CD45+/- mice expressed two distinct TCRV{alpha}-chains, TCRV{alpha}3.2 and TCRV{alpha}2, on the cell surface. Thymocytes from CD45-/- mice were blocked at the CD3low stage of development and expressed two distinct TCRV{alpha}-chains, TCRV{alpha}3.2 and TCRV{alpha}2, at a high frequency (Fig. 1GoB). It should be noted that TCR/CD3 expression levels (and increased CD4 and CD8 surface expression levels) in immature thymocytes of CD45-/- mice are higher than in normal mice, although these cells are blocked at the first step of positive selection and display a CD4+CD8+CD69-HSA-CD5lowCD44- phenotype (35).



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  		FIGURE 1. Allelic exclusion of TCRV{alpha}-chains is not functional in immature thymocytes. A, Transgenic expression of CD45 (CD45RO isoform) rescues thymocyte development. CD45 expression levels (left), as well as CD4+CD8+ double-positive and mature CD4+ or CD8+ single-positive cell populations (right) were quantified using freshly isolated thymocytes from CD45+/-, CD45-/-, and CD45RO/CD45-/- (CD45Tg) mice. Single thymocyte suspensions from 6-wk-old mice were stained with anti-pan CD45 FITC (histograms on left side) or double-stained using anti-CD4-PE and anti-CD8-FITC (right). Values on histograms indicate the mean value of fluorescence intensity on a log scale. Note up-regulated CD4 and CD8 expression on CD45-/- thymocytes. Numbers on dot blots indicate percentages of thymocyte subsets. B and C, Surface expression of TCRV{alpha}2- and TCRV{alpha}3.2-chains (B) and TCRVß8.1/8.2- and TCRVß6-chains (C) on immature and mature thymocytes from CD45+/- and CD45Tg mice and immature thymocytes of CD45-/- mice. Percentages of immature CD3lowTCRV{alpha}2low (gate R2) and mature CD3highTCRV{alpha}2high (gate R1) thymocytes that express two different TCRV{alpha}-chains are indicated in the histograms. Cells were surface stained with anti-CD3{epsilon} biotin, anti-TCRV{alpha}3.2 FITC, and anti-TCRV{alpha}2 PE (B), or surface stained with anti-CD3{epsilon} biotin, anti-TCRVß6 FITC, and anti-TCRVß8.1/8.2 PE (C). Gates were drawn using staining with isotype-matched control Abs. One of three experiments is shown.

 
Rescue of T cell development via a CD45 transgene (CD45Tg) (Fig. 1GoA) (36) restored allelic exclusion in mature, but not immature, thymocytes, i.e., immature thymocytes still expressed two TCRV{alpha}-chains (TCRV{alpha}3.2+TCRV{alpha}2+), but mature thymocytes expressed only one TCRV{alpha}-chain on the cell surface (Fig. 1GoB). Similar results were obtained using double staining on CD3low immature and CD3high mature thymocytes from wild-type, CD45-/-, and CD45Tg mice with different combinations of anti-TCRV{alpha}11, anti-TCRV{alpha}3.2, anti-TCRV{alpha}8, and anti-TCRV{alpha}2 mAbs (not shown). By contrast, only one TCRVß-chain was detectable on the surface of immature or mature CD45-/-, CD45Tg, and CD45+/- thymocytes (Fig. 1GoC). Our genetic data in CD45-/- and CD45Tg thymocytes and previous results by Alam et al. (18) indicate that allelic exclusion of TCRV{alpha}-chains in mature thymocytes occurs at the CD45-dependent progression from the CD3low to the CD3high stage of development.

Allelic exclusion of TCRV{alpha}-chains is independent of surface expression of rearranged TCR{alpha}ß transgenes, but regulated via positive selection

To follow the fate of an endogenous TCRV{alpha}-chain in TCR transgenic thymocytes in a scenario of nonselection, we introduced the H-Y-specific TCR{alpha}ß transgene into CD45-/- mice (31, 34, 36). Thymocytes expressing the male H-Y TCR are positively selected in female H-2b mice and blocked at a CD4+CD8+ immature stage in H-2d/d mice (nonselection). Similar to previously published data (11), allelic exclusion of TCRV{alpha}-chains was incomplete in H-Y Tg mice, and ~1 to 2% of developing H-Y Tg thymocytes coexpressed the Tg TCRV{alpha}3J{alpha}27 (detected by the mAb T3.70)- and endogenous TCRV{alpha}2-chains on the cell surface in a nonselecting H-2d/d background (Fig. 2GoA, upper panel).



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  		FIGURE 2. A, CD4 and CD8 expression (left) and coexpression of Tg TCRV{alpha}3J{alpha}27 (T3.70)- and endogenous TCRV{alpha}2-chains (right) on the cell surface of thymocytes from H-Y Tg CD45+/- (upper panel), H-Y Tg CD45-/- (middle panel), and H-Y Tg CD45RO/CD45-/- (CD45Tg) (lower panel) mice. In all cases, thymocytes were isolated from nonselecting H-2d/d mice in which the H-Y-specific TCR cannot mature. Similar data were obtained using a different CD45Tg encoding the high m.w. CD45 isoform (CD45ABC). The CD45ABC Tg is expressed at lower levels on the cell surface and has a different genomic integration site (30), excluding the possibility that the effect of CD45 on allelic exclusion is due to the transgenic integration site or specific CD45 isoforms. One representative experiment of seven experiments is shown. B, Surface expression of three different TCRV{alpha}-chains on thymocytes from nonselecting H-2d/dH-Y/CD45Tg mice. Histograms show TCRV{alpha}3.2 expression on live gated immature (gate R1) and mature (gate R2) TCRV{alpha}2+ thymocytes. Cells were triple stained using T3.70 biotin, anti-TCRV{alpha}2 PE, and anti-TCRV{alpha}3.2 FITC. B, Lower panel, Cytoplasmic expression of the H-Y-specific TCR (T3.70) in live gated TCRV{alpha}2 surface-positive and T3.70 surface-negative thymocytes (gate R2) in nonselecting H-Y CD45Tg mice. Filled histogram, cytoplasmic staining for the Tg TCRV{alpha} (T3.70)-chain; line histogram, control cytoplasmic staining using a mAb for endogenous TCRVß6-chains. Cells were surface stained using T3.70 biotin and anti-TCRV{alpha}2 PE, and for cytoplasmic expression stained with T3.70 FITC. For control staining, cells were surface stained using T3.70 biotin and anti-TCRV{alpha}2 PE, and for cytoplasmic expression stained with anti-TCRVß6 FITC. Both anti-TCRVß6 and T3.70 are IgG rat mAbs. Similar results were obtained using surface staining for CD3{epsilon} FITC and TCRV{alpha}2 PE, and cytoplasmic staining for T3.70 biotin (not shown). One of three experiments is shown.

 
Approximately 10% of TCRV{alpha}3J{alpha}27+ (T3.70+) thymocytes from nonselecting CD45-/- mice coexpressed TCRV{alpha}2-chains on the cell surface, and all thymocytes expressing the endogenous TCRV{alpha}2-chain coexpressed the Tg TCRV{alpha}-chain (Fig. 2GoA, middle panel). Expression of a CD45Tg in nonselecting (H-2d/d) H-Y Tg CD45-/- mice restored development of T cells expressing the endogenous TCRV{alpha}-chain, and thymocytes expressing high levels of endogenous TCRV{alpha}2-chains appeared that did not coexpress the H-Y Tg TCRV{alpha}-chain (T3.70) on the cell surface (Fig. 2GoA, lower panel, and Fig. 2GoB, gate R2). Almost all immature CD4+CD8+ thymocytes in H-Y CD45Tg mice still coexpressed the Tg TCRV{alpha}-chain and endogenous TCRV{alpha}-chains (Fig. 2Go, A, lower panel, and B). All CD45-/- and CD45Tg thymocytes that expressed two distinct TCRV{alpha}-chains on the surface had a CD3lowCD4+CD8+CD69- phenotype. TCRV{alpha}2high cells in CD45Tg mice were mature thymocytes, as defined by CD3highCD4-CD8+CD69+ expression (not shown). Similar results were obtained using H-Y CD45-/- and H-Y CD45Tg mice in a selecting H-2b/b background, i.e., absence of allelic exclusion in CD4+CD8+ immature thymocytes and presence of single TCRV{alpha}+ cells on the selecting background. Moreover, experiments using two other rearranged TCR{alpha}ß transgenes, the MHC class I-restricted LCMV-specific P14 TCR{alpha}ß (TCRV{alpha}2J{alpha}TA31/Vß8.1DJß2.4) (32) and the MHC class II-restricted V{alpha}11.1J{alpha}8Vß3 TCR specific for cytochrome c (33), showed dual expression of two different TCRV{alpha}-chains in immature, but not mature, thymocytes (not shown). Allelic exclusion of TCRVß-chains was functional in all three TCR Tg mouse models, and immature CD45-/- thymocytes expressed the Tg TCRVß-chain, but not endogenous TCRVß-chains (not shown).

Triple-staining experiments using Abs against the H-Y-specific TCRV{alpha}3J{alpha}27 (T3.70) and two different endogenous TCRV{alpha}-chains (TCRV{alpha}2 and TCRV{alpha}3.2) further showed that immature thymocytes in H-Y Tg mice can express up to three different TCRV{alpha}-chains on the cell surface (Fig. 2GoB, gate R1). Surface staining for the second endogenous TCRV{alpha}3.2-chain was lost upon rescue of selection in mature CD45Tg thymocytes (Fig. 2GoB, gate R2). Moreover, mature thymocytes of nonselecting H-Y CD45Tg mice with surface expression of only one endogenous TCRV{alpha}-chain (TCRV{alpha}2) expressed the H-Y TCRV{alpha}-chain (T3.70) in the cytoplasm, implying that TCRV{alpha} allelic exclusion in H-Y/CD45Tg mice may be regulated at the posttranslational level (Fig. 2GoB, lower panel). Expression of two or even three different TCRV{alpha}-chains on the cell surface correlated with increased mRNA expression levels of RAG1 and RAG2 (Fig. 3Go).



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  		FIGURE 3. Upper panels, RAG-1 and RAG-2 mRNA expression in total thymocytes isolated from H-Y Tg CD45-/-H-2d/d, H-Y Tg CD45+/- H-2b/b, H-Y Tg CD45-/-H-2b/b, and H-Y Tg CD45+/-H-2d/d mice. Expression of ß-actin mRNA is shown as a control. Lower panels, Expression levels of RAG-1 and RAG-2 mRNA in H-Y-specific TCR Tg mice. RAG-1 and RAG-2 expression levels were normalized to ß-actin mRNA levels using densitometry. Relative levels of RAG mRNA expression are shown as arbitrary units on the y-axis. Positive selecting H-2b/b female mice are included, since the H-Y TCR is selected into the CD8+ lineage in CD45+/- mice (not shown), which precludes rearrangement of the endogenous TCRV{alpha} locus. By contrast, RAG-1 and RAG-2 are highly expressed in nonselecting H-Y CD45+/- mice, since these mice rearrange an endogenous TCRV{alpha}-chain that pairs with the H-Y Tg TCRVß8.1-chain to form a novel selectable TCR{alpha}ß heterodimer (11). Positive selection of the H-Y TCR does not occur in H-2b/bCD45-/- female mice (not shown), and these mice have a phenotype similar to that in nonselecting (H-2d/d) CD45-/-H-Y Tg mice (36).

 
Peptide-specific maturation of single TCRV{alpha}+ thymocytes from double TCRV{alpha}+ thymocytes

Data in normal mice, CD45-/-, CD45Tg, and three different TCR Tg mouse models indicated that immature thymocytes express two distinct TCRV{alpha}-chains, whereas mature thymocytes expressed only one TCRV{alpha}-chain on the cell surface, but lost surface expression of the nonselectable Ag receptor. To test this hypothesis, we purified immature CD3low thymocytes expressing two TCRV{alpha}-chains, endogenous TCRV{alpha}11 and Tg TCRV{alpha}2 from LCMV-specific P14 TCRV{alpha}2Vß8.1 Tg/TAP1-/- mice using cell sorting (Fig. 4Go). P14 Tg/TAP1-/- mice have a block in positive selection of the MHC class I-restricted P14 TCR since TAP1-/- mice lack the transporter for MHC class I peptide loading (35, 37). P14 Tg/TAP1-/- mice (and P14 Tg/ß2m-/- mice (39); see below) were used in these experiments to ensure that thymocytes expressing the Tg TCR chain had not been activated previously by the selecting MHC ligand.



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  		FIGURE 4. Peptide-specific selection of dual TCRV{alpha}+ thymocytes. A, Upper panels, Cell sorting of CD3lowTCRV{alpha}2lowTCRV{alpha}11low thymocytes from Ly-5.2+P14 TCR Tg/TAP1-/- mice. Thymocytes from 6-wk-old P14/TAP1-/- mice were triple stained with anti-CD3{epsilon}, anti-TCRV{alpha}2, and anti-TCRV{alpha}11, and the indicated population was sorted using a Coulter Power Sorter. The sorted population was >98 pure for dual TCRV{alpha}+ cells (right panel) and had a CD3low phenotype (not shown). A, Lower panels, Cell-sorted dual TCRV{alpha}2lowTCRV{alpha}11low thymocytes differentiate into TCRV{alpha}2highTCRV{alpha}11- T cells in the presence of the selecting LCMV-p33 peptide presented by selecting H-2b/b thymic stromal cells, but not in the presence of the same peptide presented by nonselecting H-2k/k stromal cells. CD3lowTCRV{alpha}2+TCRV{alpha}11+ thymocytes were sorted from Ly-5.2+ congeneic P14 TCR Tg+/TAP-/- mice and cultured for 5 days with thymic stromal cells isolated from Ly-5.1 congeneic B6 (H-2b/b) mice (left) or nonselecting stromal cells isolated from CBA (H-2k/k) mice (right). Thymic stromal cells were pulsed with 10-9 M LCMV peptide, as described in Materials and Methods. Numbers on dot blots indicate percentages of thymocyte subsets. One experiment representative of three independent experiments is shown. B, Absolute cell numbers in cocultures Dual TCRV{alpha}2+V{alpha}11+ thymocytes (1 x 106) were cocultured with selecting H-2b/b or nonselecting H-2k/k thymic stromal cells in the presence of 10-9 M p33 peptide. Cocultures were harvested at day 5, and absolute numbers of total transgenic Ly-5.2+ cells, Ly-5.2+TCRV{alpha}2+ cells, and Ly-5.2+TCRV{alpha}11+ cells were determined.

 
Dual TCRV{alpha}2+TCRV{alpha}11+CD3lowLy-5.2+ thymocytes were incubated with Ly-5.1+ congeneic thymic stromal cells and the selecting LCMV-p33 peptide, a system that allows in vitro positive selection of thymocytes expressing the LCMV peptide-specific Tg TCRV{alpha}2-chain (40). Figure 4GoA shows that after 5 days of culture, almost all Ly-5.2+ thymocytes expressed the peptide-specific TCRV{alpha}2-chain and had lost expression of the second endogenous TCRV{alpha}11-chain. Positively selected TCRV{alpha}2+ thymocytes had up-regulated surface expression of the TCR/CD3 complex (Fig. 4GoA). The few TCRV{alpha}11+ cells present after the culture displayed up-regulated TCRV2{alpha} expression, but had not up-regulated the TCRV{alpha}11-chain on the cell surface. No TCRV{alpha}2high cells were present in cocultures containing nonselecting stroma (H-2k), and in these cultures the majority of cells had a TCRV{alpha}2lowTCRV{alpha}11low phenotype (Fig. 4GoA). The absolute numbers of total thymocytes, TCRV{alpha}2+, and TCRV{alpha}11+ thymocytes recovered after coculture with selecting and nonselecting stromal cells (Fig. 4GoB) indicate that the observed effect was not due to preferential survival of a small percentage of TCRV{alpha}11low cells. In similar experiments, immature dual TCRV{alpha}+ thymocytes from nonselecting H-Y Tg CD45Tg mice were incubated with thymic stromal cells. In this system, only endogenous TCRV{alpha}2+ thymocytes are allowed to develop, and in these cultures all mature cells lost the second H-Y Tg TCRV{alpha}3 (T3.70)-chain (not shown). Whereas frequent expression of two different TCRV{alpha}-chains on immature thymocyte has been described previously (18), our data provide the first functional and direct evidence that single TCRV{alpha}+ T cells can develop from immature dual TCRV{alpha}+ thymocytes in a peptide-specific manner.

TCRV{alpha}-chains regulate receptor internalization

To further establish the role of TCRV{alpha}-chains in TCR surface expression, we sorted immature CD3low dual TCRV{alpha}+ (Tg TCRV{alpha}2+ and endogenous TCRV{alpha}11+) thymocytes from P14 Tg/ß2m-/- mice and cross-linked the TCR with anti-TCRV{alpha}2 or anti-TCRV{alpha}11 Abs in vitro. Similar to P14 Tg/TAP1-/- mice, P14 Tg/ß2m-/- mice have a block in positive selection due to impaired MHC class I expression (39). Upon anti-TCRV{alpha}2 cross-linking, TCRV{alpha}2+TCRV{alpha}11+ thymocytes retained the TCRV{alpha}2-chain on the cell surface, but lost surface expression of the TCRV{alpha}11-chain. Cross-linking of TCRV{alpha}11 yielded cells that were positive for TCRV{alpha}11 and had lost expression for TCRV{alpha}2 (Fig. 5Go). Cross-linking of the CD3{epsilon} or the Tg TCRVß8.1-chain, which is the only TCRVß-chain expressed in this transgenic system, led to down-regulation of both TCRV{alpha}2 and TCRV{alpha}11 from the cell surface (Fig. 5Go). It should be noted that TCRVß8-mediated TCR down-regulation did occur after purification with the same anti-TCRVß8 Ab (clone KJ16), suggesting that the purification protocol did not interfere with the differential outcome of TCRV{alpha}- and TCRß-mediated receptor down-modulation (not shown). Our genetic data in mice, functional data from a peptide-specific positive selection system, and in vitro Ab cross-linking experiments suggest that developing thymocytes retain the TCRV{alpha}-chain that has been ligated, but lose the nonutilized TCRV{alpha}-chain.



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  		FIGURE 5. In vitro cross-linking of dual CD3lowTCRV{alpha}2+TCRV{alpha}11+ thymocytes using anti-TCRV{alpha}11, anti-TCRV{alpha}2, anti-TCRVß8.1/8.2, and anti-CD3{epsilon} Abs. Dual TCRV{alpha}2+TCRV{alpha}11+ thymocytes were purified from P14 TCR (TCRV{alpha}2Vß8.1) Tg+/TAP-/- mice and activated with the Abs indicated on top of dot blots. Cells were harvested after 24 h in culture and stained for TCRV{alpha}2 and TCRV{alpha}11 surface expression. Numbers indicate percentages of thymocyte subsets. One result representative of three independent experiments is shown.

 
TCRVß, but not TCRV{alpha}, activation leads to dynamin recruitment

Since TCRVß-, but not TCRV{alpha}-chains triggered TCR internalization, we assumed that TCRV{alpha}- and TCRVß-chains mediate distinct signals. However, the levels and kinetics of tyrosine phosphorylation of intracellular substrates were similar in both anti-TCRV{alpha}2- and anti-TCRVß8-mediated activation (Fig. 6GoA). Moreover, immunoprecipitation of molecules involved in TCR signal transduction, including Cbl-2, Vav, ZAP70, or phospholipase C{gamma}1, did not show any apparent differences in the kinetics and levels of tyrosine phosphorylation induced by anti-TCRV{alpha}2 or anti-TCRVß8 activation (not shown). Although we cannot exclude selective and subtle differences in phosphotyrosine signaling, our data imply that the unique function of TCRV{alpha}-chains is independent of phosphotyrosine signaling pathways.



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  		FIGURE 6. A, Phosphotyrosine signaling of P14 (TCRV{alpha}2Vß8.1) Tg/ß2m-/- thymocytes. Total thymocytes (1 x 106 per sample) were activated for 0, 3, and 10 min with anti-CD3{epsilon}, anti-TCRV{alpha}2, and anti-TCRVß8, as indicated, and phosphoproteins were visualized as described in Materials and Methods. B and C, Grb2-dynamin association in anti-TCRV{alpha}2 (V{alpha}2)-, anti-TCRVß8 (Vß8)-, and anti-TCRV{alpha}2 plus anti-TCRVß8 (Vß8 + V{alpha}2)-activated thymocytes from P14 Tg/ß2m-/- (B) and P14 Tg/CD45-/- mice (C). Thymocytes (2 x 107/lane) were activated for different time points, and Grb2 was immunoprecipitated from total cell lysates. Immunprecipitates were separated on a SDS-PAGE gel, and Grb2 and dynamin were visualized using anti-Grb2 and anti-dynamin Abs. D, Recruitment of dynamin to the cell membrane in anti-TCRV{alpha}2 (V{alpha}2)- and anti-TCRVß8 (Vß8)-activated P14 Tg/ß2m-/- thymocytes. Anti-Grb2 and anti-dynamin immunoprecipitations, preparation of membrane and cytosolic fractions, and immunoblotting were as described in Materials and Methods. Similar results were obtained in three independent experiments.

 
Receptor-mediated interactions between the molecular adapter Grb2 and the GTPase dynamin and recruitment of dynamin to the cell membrane is an important step for receptor internalization and endocytosis (42, 43). Moreover, it has been reported that Grb2, which is not phosphorylated upon TCR activation, associates with distinct immunoreceptor tyrosine-based activation motifs present in CD3 chains (44). Interestingly, TCRVß8, but not TCRV{alpha}2, activation induced association of Grb2 with dynamin (Fig. 6GoB) and recruitment of dynamin to the plasma membrane (Fig. 6GoD). Coordinate activation of both TCRVß8- and TCRV{alpha}2-chains inhibited dynamin-membrane recruitment (Fig. 6GoD), implying that TCRV{alpha}-chain activation can prevail over the TCRVß signal.

Since CD45-/- mice display higher levels of TCR/CD3 expression on immature CD4+CD8+ thymocytes (Figs. 1Go and 2Go) (34, 36), we tested whether dynamin/Grb2 interactions were dependent on the presence of CD45. In contrast to CD45+ thymocytes (Fig. 6GoB), activation of TCRVß8 in P14 Tg/CD45-/- thymocytes did not induce interactions between Grb2 and dynamin (Fig. 6GoC) or plasma membrane recruitment of dynamin (not shown). These results indicate that anti-TCRVß8-mediated Grb2/dynamin associations and dynamin recruitment require expression of the tyrosine phosphatase CD45. These data also provide a rationale for the increased TCR/CD3 and CD4/CD8 expression in CD45-/- thymocytes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
At the turn of the century, Paul Ehrlich proposed that cells of the immune system display different receptors on the cell surface, each of which has specificity for Ag (side-chain theory) (25, 45). Subsequently, clonal selection theory postulated that every lymphocyte expresses only a single Ag receptor with exquisite and unique specificity (46, 47). Our genetic data and Ab staining data from Alam et al. (18) indicate that a high frequency of immature thymocytes expresses two (or, in the case of TCR Tg mice, even three) distinct TCRV{alpha}-chains on the cell surface, whereas less than 10% of mature thymocytes and peripheral T cells express two TCRV{alpha}-chains. Importantly, our results provide the first experimental evidence that immature dual TCR{alpha}+ thymocytes can be selected for expression of a functional TCR.

Assuming random rearrangement at the TCR{alpha} locus (3) and random V{alpha}-Vß pairing (18), our data in normal mice, CD45 gene-deficient mice, and transgenic mouse models expressing rearranged TCR{alpha}ß heterodimers indicate that a large number of CD4+CD8+ immature thymocytes expresses two different TCRV{alpha}-chains on the cell surface. These findings are in contrast to the lower frequency of dual TCRV{alpha}+ thymocytes in nonselecting TCR{alpha}ß transgenic wild-type mice (11, 12, 13, 14), implying that, although not complete, expression of a rearranged TCR{alpha}ß Tg on the cell surface can partially inhibit rearrangement of endogenous TCR{alpha} genes. Interestingly, in H-Y TCR Tg mice, it has been reported that thymocyte hybridomas expressing an endogenous TCRV{alpha}-chain on the cell surface express mRNA of the Tg TCRV{alpha}-chain (11), indicating that mature T cells in these mice had once expressed the Tg TCRV{alpha}-chain. Since the H-Y-specific TCR is expressed very early in T cell development (31, 48), it is possible that rearrangement of the endogenous TCR{alpha} locus occurs earlier than in wild-type mice. By contrast, Tg TCRV{alpha}-chains accumulate on the cell surface of H-Y CD45-/-, H-Y CD45Tg, P14 CD45-/-, P14 Tg/TAP1-/-, and P14 Tg/ß2m-/- thymocytes, since these cells either never receive the signal required for positive selection or have an increased threshold for Ag receptor signaling. Most importantly, expression of two distinct TCRV{alpha}-chains can be found on immature thymocytes of wild-type mice at a high frequency (this study and 18 , indicating that expression of two different TCRV{alpha}-chains in immature thymocytes is a normal physiologic process.

The question thus arises as to how a T cell can distinguish between a selectable and nonselectable TCR, since both TCRs on the surface should be structurally similar and should be regulated similarly at the molecular level. Loss of the nonselected TCR and clonal surface expression of the activated TCR could be stochastic, i.e., down-regulation of either one TCR and rescue of the useful receptor by positive selection, or instructive, i.e., the signal for positive selection specifically down-regulates the second TCRV{alpha}-chain. Both of these mechanisms appear to be functional in lineage commitment and positive selection of CD4+ or CD8+ thymocytes (48, 49, 50, 51). However, expression of different TCRV{alpha}-chains must be limited due to constraints of possible TCR{alpha} rearrangements in one cell (3).

In vitro ligation of TCRV{alpha}2-chains on immature dual TCRV{alpha}2+V{alpha}11+ thymocytes triggers surface retention of the TCRV{alpha}2-chain and down-regulation of the nonligated TCRV{alpha}11-chain, whereas activation of the TCRV{alpha}11-chain leads to retention of TCRV{alpha}11- and loss of TCRV{alpha}2-chains. Cross-linking of the Tg TCRVß8.1-chain (and CD3{epsilon}) leads to down-regulation of both TCRV{alpha}2- and TCRV{alpha}11-chains from the cell surface, implying that TCR{alpha}- and TCRß-chains mediate distinct signals. Whereas the levels and kinetics of tyrosine phosphorylation of intracellular substrates were similar in response to TCRV{alpha}2 and TCRVß8 activation, stimulation of TCRVß, but not TCRV{alpha}, induced association of Grb2 with dynamin and recruitment of dynamin to the plasma membrane. TCRVß-mediated Grb2/dynamin interactions and dynamin recruitment did not occur in CD45-/- thymocytes, indicating that Grb2/dynamin associations and dynamin recruitment require expression of the tyrosine phosphatase CD45. Receptor-mediated interactions between the molecular adapter Grb2 and the GTPase dynamin and recruitment of dynamin to the cell membrane is an important step for receptor internalization and receptor-mediated endocytosis (42, 43). Moreover, it has been reported that Grb2, which is not phosphorylated upon TCR activation, associates with distinct CD3 chains (44). Besides Grb2/dynamin associations and recruitment of dynamin to the plasma membrane, other signaling pathways might contribute to the observed differences between TCRV{alpha} and TCRVß activation. Our data imply that TCRV{alpha}- and TCRVß-chains associate with distinct CD3 signaling modules (52, 53) and selectively activate (TCRß-chains) or block activation (TCRV{alpha}-chains) of the cellular machinery for receptor internalization. These results provide the first evidence that distinct chains in heterodimeric receptors can selectively inhibit or trigger receptor internalization.

TCRV{alpha}-based inhibition of TCR internalization might resolve the paradox that in vitro activation of the TCR/CD3 complex on CD4+CD8+ thymocytes induces TCR down-regulation (54), whereas in vivo positive selection induces up-regulation of the selected TCR (55). A selective role of distinct TCRV{alpha}-chains has also been suggested for MHC-directed specific T cell selection (56). Moreover, in T cell lines, it has been demonstrated that a conserved motif present in the connecting peptide domain of the TCR{alpha}-chain controls Ag responsiveness and IL-2 production, implying that TCRV{alpha}-chains have a unique role in TCR{alpha}ß heterodimer-induced signal transduction (57).

What is the physiologic role of dual TCRV{alpha}+ thymocytes? The major barrier for thymocyte development is expression of a selectable TCR, and it has been estimated that 90 to 95% of thymocytes undergo programmed cell death due to nonselection (58). Expression of different TCRV{alpha}-chains on the surface of a single immature T cell would increase the probability that thymocytes are positively selected on self MHC molecules. Our findings also suggest that TCR clonality is not an intrinsic quality of murine T cells imposed at the level of V(D)J rearrangement, but is directed by positive selection signals from the thymic microenvironment. Consistent with this hypothesis, overexpression of RAG-1 and RAG-2 in mice does not impair allelic exclusion (28). Accordingly, RAG expression is down-regulated upon TCR ligation in immature thymocytes (12, 17, 27), but highly up-regulated in CD45-/- mice (Fig. 3Go).

In contrast to our results, several laboratories have shown that TCRV{alpha} cross-linking in peripheral T cells and TCR-transfected cell lines leads to down-regulation of the activated TCR (59, 60). The most likely explanation for these differences is that we used freshly isolated immature thymocytes, whereas these studies were done with mature T cells, i.e., immature thymocytes and mature T cells might have different TCR internalization behaviors. Additional experiments are required to determine whether engagement of TCRVß-chains in peripheral T cells also leads to Grb2/dynamin interactions and recruitment of the GTPase dynamin to the plasma membrane, and whether this process can be blocked by activation of the TCRV{alpha}-chain.

Since ~10 to 20% of peripheral human and murine T cells do express two different TCRV{alpha}-chains on the cell surface, it is likely that selection for clonality is leaky and, in some instances, generates T cells with dual TCR specificity (15, 16). How this occurs is unknown and might result from inefficient signaling through the engaged TCRV{alpha}-chain. A critical question is how TCRV{alpha}-regulated allelic exclusion that occurs during positive thymocyte selection can be maintained in peripheral T cells. One explanation is that maturation-triggered down-modulation of the nonengaged TCR might become permanent through a yet unknown mechanism. Alternatively, allelic exclusion in peripheral T cells might be maintained via continuous engagement of the TCR via the selecting MHC molecule. For example, recent reports indicate that the survival of naive peripheral CD4 and CD8 T cells is dependent on the presence of selecting MHC class I and class II molecules and continuous MHC recognition (61, 62).

Based on our genetic and functional results in normal, gene-deficient, and TCR transgenic mice, we propose the following model of TCR selection and allelic exclusion (Fig. 7Go): Immature CD4+CD8+ thymocytes rearrange multiple TCRV{alpha}-chains and can simultaneously express two TCR molecules with distinct specificities on the cell surface. During positive T cell selection, peptide-specific activation induces up-regulation and surface retention of the activated TCR and loss of the second, nonselected Ag receptor. Surface retention of the TCR appears to be a unique function of the TCRV{alpha}-chain, most likely through inhibition of dynamin-regulated receptor endocytosis. This model of receptor-based allelic exclusion might explain allelic inactivation of TCR or Ig genes in cartilaginous fish, despite the fact that these receptors are organized as gene clusters and apparently do not undergo gene rearrangement (63). Our model might also explain allelic expression of single olfactory and pheromone receptors on the surface of distinct sensory neurons that might allow discrimination of diverse stimuli that regulate behavior (1, 2). Similarly to our model for thymocyte selection, it has been demonstrated in vitro that synaptic strength regulates axon withdrawal and innervation by a single motoneuron in skeletal muscle cells (64), suggesting that activation-dependent receptor retention might be a general physiologic mechanism. Our data in T cells suggest a novel mechanism of TCRV{alpha}-mediated T cell selection and allelic exclusion, and provide the first evidence that activation-based allelic receptor expression can occur in vivo.



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  		FIGURE 7. Proposed model for T cell selection. Immature thymocytes have the intrinsic ability to rearrange and express two different TCRV{alpha}-chains on the cell surface. A CD45-dependent positive selection signal mediates allelic exclusion, expression of the activated TCRV{alpha}-chain, and down-regulation of the second, nonselectable TCRV{alpha}-chain. Most mature thymocytes and peripheral T cells only express one TCRV{alpha}-chain on the cell surface.

 


   Acknowledgments
 
We thank L. Harrington, H. Nishina, P. Waterhouse, L. Zhang, M. Saunders, and D. Siderovski for critical comments; and P. Kisielow, P. Poussier, L. Zang, H. P. Pircher, S. Hedrick, S. Tonegawa, P. Ohashi, and C. Paige for reagents and mice.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. J. M. Penninger, Amgen Institute, Ontario Cancer Institute, and Departments of Medical Biophysics and Immunology, University of Toronto, 620 University Avenue, M5G 2C1 Toronto, Ontario, Canada. E-mail address: Back

2 Abbreviations used in this paper: Tg, transgenic; ß2m, ß2-microglobulin; LCMV, lymphotoxic choriomeningitis virus; PE, phycoerythrin. Back

Received for publication March 23, 1998. Accepted for publication April 15, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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