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The Journal of Immunology, 2004, 173: 5591-5600.
Copyright © 2004 by The American Association of Immunologists

Exclusion and Inclusion of TCR{alpha} Proteins during T Cell Development in TCR-Transgenic and Normal Mice1

H. Daniel Lacorazza2,* and Janko Nikolich-Zugich3,*,{dagger}

* Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and {dagger} Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Allelic exclusion of immune receptor genes (and molecules) is incompletely understood. With regard to TCR{alpha}{beta} lineage T cells, exclusion at the tcr-b, but not tcr-a, locus seems to be strictly controlled at the locus rearrangement level. Consequently, while nearly all developing TCR{alpha}{beta} thymocytes express a single TCR{beta} protein, many thymocytes rearrange and express two different TCR{alpha} chains and, thus, display two {alpha}{beta}TCRs on the cell surface. Of interest, the number of such dual TCR-expressing cells is appreciably lower among the mature T cells. To understand the details of TCR chain regulation at various stages of T cell development, we analyzed TCR expression in mice transgenic for two rearranged {alpha}{beta}TCR. We discovered that in such TCR double-transgenic (TCRdTg) mice peripheral T cells were functionally monospecific. Molecularly, this monospecificity was due to TCR{alpha} exclusion: one transgenic TCR{alpha} protein was selectively down-regulated from the thymocyte and T cell surface. In searching for the mechanism(s) governing this selective TCR{alpha} down-regulation, we present evidence for the role of protein tyrosine kinase signaling and coreceptor involvement. This mechanism may be operating in normal thymocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vigorous reactivity against pathogens and the lack of overt reactivity to self are the defining features of a functional adaptive immune system. As postulated by the clonal selection theory (1), both are best achieved when each lymphocyte bears one functional Ag receptor. This clonal distribution of Ag receptors allows lymphocytes to be specifically and maximally activated only by their cognate Ags: those reactive to pathogens will be allowed to divide and grow, and those reactive to self-Ags will be eliminated or silenced. Expression of two or more receptors on a single cell is undesirable–integration of signals from two or more receptors might paralyze an antipathogen response and/or prevent deletion or functional silencing of self-reactive cells.

tcr gene rearrangement and expression occur in the thymus, and are controlled in a manner that generally disallows a single T cell to express more than one functional TCR{alpha}{beta} heterodimer (2, 3). Lymphocytes rearrange Ag receptor genes in an orderly manner (4), and thus thymocytes rearrange the tcr-b locus before the tcr-a locus (5, 6, 7). Allelic exclusion is very strict at the tcr-b locus: few T cells (<1%) show evidence of two complete inframe tcr-b V-DJ rearrangements (8, 2, 3, 9) and this fully explains the clonal distribution of TCR{beta} chains on lymphocytes. Experimental evidence supports the critical role of a functional TCR{beta} chain (8), generated from one tcr-b allele, and of the protein tyrosine kinase (PTK)4 Lck (10) in mediating the recombination arrest at the other allele, but the downstream mechanism remains incompletely understood. The tcr-a locus allelic exclusion, if operating at all, is extremely inefficient. Multiple tcr-a rearrangements are allowed (11, 12, 13), apparently to maximize production of thymocytes bearing potentially selectable TCR{alpha}{beta} heterodimers. Positive selection is then believed to cause an arrest in further tcr-a gene rearrangement (13, 14, 15, 16) by a poorly understood mechanism. Both the availability of the recombination-initiating enzymes (RAG-1 and RAG-2) and the changes in locus accessibility may control TCR{alpha} and {beta} rearrangement (2, 3).

Before further recombination at the tcr-a locus is stopped by positive selection, thymocytes will be produced that potentially express two TCRs, consisting of a single TCR{beta} paired with two different TCR{alpha} chains (17, 18, 19, 20, 21, 22). Owing to the paucity of TCR{alpha}-specific Abs, the exact quantification of cells expressing two TCRs has been elusive, with estimates ranging between 2 and 21% of murine peripheral T cells and up to 60% of murine thymocytes (19, 20) to 30% of human T cells (17). It is thus controversial how many dual-TCR thymocytes are produced and how many are selected and exported to the periphery. It is even less clear how many of those would be able to use both TCRs to respond to two different Ags, an issue of obvious importance for autoimmunity. Regardless of the exact numbers, these data strongly suggest that other mechanisms must exist to ensure functional monospecificity of T cells (3).

The probability that two productively rearranged tcr-a genes should yield TCR{alpha} proteins that both pair with a single TCR{beta} chain in a manner allowing positive selection in the thymus is rather low. Experimental evidence suggested that the low probability of these events helps dictate which TCR{alpha} should be expressed–obviously the one that pairs well with TCR{beta} and/or forms a heterodimer that is positively selected (20, 23). However, it is precisely the situation where both receptors can successfully pair and be selected that poses the most rigorous challenge to the system. To investigate this problem, we studied TCR regulation in mice whose T cells were forced by transgenesis to express two rearranged TCR{alpha}{beta} receptors, both of which could be positively selected (TCR{alpha}{beta} doubly transgenic (Tg), TCRdTg in the text). In this setting, we found that one TCR{alpha} chain was selectively and severely down-regulated at the protein level. Results presented herein provide evidence for a mechanism likely to ensure a high degree of phenotypic and functional exclusion of one TCR among immature double-positive (DP) and mature, selected single-positive (SP) thymocytes and peripheral T cells.


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

CD8–/– and MHC class I–/– animals were obtained from The Jackson Laboratory (Bar Harbor, ME). The {alpha}{beta}TCR Tg mice 2C (24), H-Y (25), and OT-1 (26), bred and maintained under the specific pathogen-free conditions at Memorial Sloan-Kettering Cancer Center and Oregon Health and Science University Animal Facilities, and backcrossed to C57BL/6 for >14 generations, were used at 6–12 wk of age and were age- and sex-matched within experiments. TCRdTg mice were obtained by interbreeding single TCRTg strains at B6 or one of the alternative backgrounds (N2 backcrosses); genotypes were confirmed by PCR using the oligonucleotides that span the V{alpha}J{alpha} junction and/or by flow cytofluorometric (FCM) analysis of the peripheral blood lymphocytes.

FCM analysis

Single cell suspensions of lymphoid organs were stained with the indicated mAb and analyzed using FACScan or FACSCalibur instruments (BD Biosciences, Mountain View, CA). FITC-conjugated anti-CD8, CyChrome-conjugated anti-CD4, and FITC- or PE-conjugated anti-V{alpha}2 mAb were purchased from BD Pharmingen (San Diego, CA). mAb 1B2, T3.70, and F23.1 were purified from ascites and conjugated to biotin in our laboratory. PE-labeled streptavidin was purchased from Caltag Laboratories (South San Francisco, CA). Intracellular staining of cells permeabilized with paraformaldehyde was per BD Pharmingen’s protocol booklet.

T cell proliferation assay

Spleen cells (1–5 x 104 for OT-1 or 2C stimulation, 2–5 x 105 for H-Y stimulation) were incubated in 0.2 ml of RP7.5 medium (RPMI 1640, 7.5% FCS, 5 µM HEPES, 20 mM glutamine, antibiotics, 2-ME) with irradiated (30 Gy) stimulator spleen cells (C57BL/6 male for H-Y, BALB/c for 2C, and C57BL/6 + 100 µg of OVA-8 peptide (SIINFEKL) for OT-1) in the absence or the presence of the IL-2-rich Con A supernatant. Cultures were incubated in flat-bottom 96-well plates at 37°C in 5% CO2/air atmosphere for 72 h, and [3H]thymidine (1 µCi/well) was added 6–8 h before harvesting (Filtermate Harvester; Packard Instrument, Meriden, CT). The incorporation of [3H]thymidine was determined using a microplate scintillation counter (TopCount NXT; Packard Instrument). The results are the average of quadruplicates, and are expressed as mean values ± SD. All experiments were repeated at least four times, with identical results.

RT-PCR

Total RNA was extracted from thymocytes using RNAIsolator (Sigma-Genosys, The Woodlands, TX) according to the manufacturer’s instructions. The RNA was reverse transcribed with a Stratagene kit (La Jolla, CA). PCR was performed with the following primers that span the V{alpha}J{alpha} junction: 5'-AATCTCTGACAGTCTG-3' and 5'-GATCAACTGATAGTTG-3' for OT-1, 5'-TGGACAGCCTGATGCTCATGTCA-3' and 5'-TCACTGTCAGCTTTGTCCCCTCCC-3' for H-Y, 5'GCGACACCTTATCTGTTCTGG-3' and 5'-AACAATGACTTTTGTGCCAGA-3' for 2C, 5'-TGTGATGGTGGGAATGGGTCAG-3' and 5'-TTTGATGTCACGCACGATTTCC-3' for {beta}-actin. Quantification of the band intensity was performed using the Bio-Rad Molecular Imager (Hercules, CA).

Thymocyte treatment with metabolic and signaling inhibitors

Total thymocytes (2 x 106 cells/ml in RPMI 1640/10% FCS) were incubated for 17 h at 37°C/5% CO2 in the absence or the presence of 3 µM herbimycin A (Invitrogen Life Technologies, Gaithersburg, MD), 20 µM PP1 (Calbiochem, San Diego, CA), 10 µM lactacystin (Calbiochem), 20 mM ammonium chloride (Sigma-Aldrich, St. Louis, MO), or 20 µM monensin (Sigma-Aldrich), as described previously (27), washed and analyzed by FCM.

Pulse-chase experiments

Twenty x 106 cells/ml were starved for 1 h at 37°C in a methionine/cysteine-free medium supplemented with 3% dialyzed FBS (Invitrogen Life Technologies) and labeled with 2 mCi of [35S]methionine (Trans-label; Amersham, Arlington Heights, IL) for 30 min. Cells were washed with PBS, incubated ("chased") for indicated times, and lysed with the lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% BSA, 0.5 mM phenyl-methyl-sulfonate, 0.24 U/ml aprotinin, and 5 mM iodoacetamide). Lysates were precleared with normal rabbit serum and zysorbin (Zymed Laboratories, San Francisco, CA), immunoprecipitated with anti-{alpha} or -{beta} TCR Abs for 2 h at 4°C followed by 1 h with protein-A Sepharose CL-4B (Sigma-Aldrich), and analyzed by nonreducing SDS-PAGE (with the 35S enhancer Amplify; Amersham) and autoradiography.

Confocal microscopy

Thymocytes were settled onto slides by cytospin, fixed with methanol, and stained in PBS/0.5% BSA/0.1% Triton X-100. The cells were mounted with Vectashield and analyzed by confocal microscopy. For TCR{alpha} or {beta} chains, biotinylated mAbs were revealed using Streptavidin-Alexa 568 (Molecular Probes); Lamp-2 FITC (BD Pharmingen) was used as a lysosomal marker. Nuclei were stained using 4',6'-diamidino-2-phenylindole (Sigma-Aldrich), but that staining was subtracted from the final image for clarity. Nuclei occupied the entire dark area of the cells as shown. Images were processed using Adobe Photoshop software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dominant expression of one Tg TCR{alpha} among thymocytes of TCRdTg mice

TCRdTg mice were generated by interbreeding of 2C (specific for p2Ca + Ld, Ref. 24), H-Y (H-Y + Db, Ref. 25), and/or OT-1 (OVA257–264 + Kb, Ref. 26) mice. TCRdTg mice were born and survived within the expected Mendelian frequencies. Typically, each F1 litter also contained TCR single Tg (TCRsTg) and non-Tg littermates, that were used as internal controls, but in some cases nonlittermate TCRsTg mice, obtained from breeding of TCRsTg mice to B6, were used as well. No differences between these two types of TCRsTg mice were apparent (not shown).

Examination of thymic cell numbers and CD8/4 phenotype showed general similarity between TCRdTg and TCRsTg mice (an accumulation of nonprecursor TCR+ CD8–4– mature double-negative (mDN) cells (28, 29), fewer CD8+4+ DP intermediate cells and more CD8+ SP cells, all compared with non-Tg littermates), except that the skewing of thymic CD8/4 profiles toward the CD8+ SP lineage (a consequence of more efficacious positive selection) was less pronounced in TCRdTg mice (our unpublished results), consistent with findings of Dave et al. (30) in a similar system. The overall TCR expression levels did not significantly differ between TCRsTg and TCRdTg thymocytes. The mean TCR{alpha}{beta} fluorescence intensity (MFI) assessed by the pan-TCR{beta} mAb staining, in TCRsTg and TCRdTg thymocytes was within ~35% of each other, with no systematic variation (not shown). Thus, coexpression of two TCR transgenes did not grossly affect the overall TCR expression levels in TCRdTg thymocytes.

We next examined the regulation of the two Tg TCR{alpha}{beta} heterodimers at the surface of TCRdTg thymocytes. If selective gene rearrangement was the main mechanism that mediates allelic exclusion, one would expect most DP and CD8+SP thymocytes in TCRdTg animals to express both Tg receptors. This was the case with both TCR{beta} chains, which were expressed at comparable levels, typically 2- to 3-fold lower by MFI than the levels expressed on the TCR{alpha}{beta}high cells of TCRsTg mice (Fig. 1A for OT-1/2C combination), consistent with the idea that exclusion of TCR{beta} occurs mostly at the level of tcr-b rearrangement. We have observed the same result examining OT-1/H-Y doubly Tg (data not shown). The above slight reduction in expression of each TCRTg{beta} chain is likely due to competition for CD3 and/or other folding intermediate partners. By contrast, expression of TCR{alpha} chains was unequal, as assessed using TCR{alpha}{beta} clonotypic (2C and H-Y TCRs, detected by mAbs 1B2 and T3.70, respectively) or TCRTg{alpha}-family specific (OT-1 TCR, detected by a V{alpha}2-specific mAb B20.1) mAbs–one of them dominated heavily over the other one (Fig. 1A, lower panels, and B). Analysis of >100 TCRdTg mice showed that the TCR{alpha} dominance was not random. In all TCRdTg mice that expressed 2C, the 2C TCR{alpha} chain was dominating over the other two TCR; OT-1 dominated at the surface of the TCRdTg OT-1/H-Y thymocytes (Fig. 1B). Thus, a hierarchy of TCR{alpha} chain dominance existed, in the order 2C > OT-1 > H-Y. These finding were consistent with two scenarios: 1) one of the TCR{alpha} was truly down-regulated and lost from the surface of most (although not all) thymocytes; or 2) one TCR{beta} preferred to pair with the other TCR{alpha}, rather than with its natural partner, leading to the loss of TCR{alpha}{beta} pairing (Id), but not loss of chains, from the cell surface. If 2) was correct, one would expect that the 2C x OT-1 thymocytes should exhibit decreased 1B2 staining compared with TCRsTg 2C thymocytes, because the anti-idiotypic mAb 1B2 can detect only the 2C{alpha}{beta} combination, and not individual 2C{alpha} or {beta} chains paired to other proteins; by contrast, the level of V{alpha}2, detected by mAb B20.1 irrespective of its V{beta} partner should remain comparable, perhaps only slightly lower, than in TCRsTg OT-1 mice. In fact, precisely the opposite was observed–the percentage of 1B2+ cells was greatly increased in 2C x OT-1 dTg thymocytes, while the V{alpha}2 levels were drastically decreased, compared with sTg cells. This ruled out possibility 2) and strongly suggested that other mechanisms operate to ensure differential expression of TCR{alpha} chains.



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  		FIGURE 1. Exclusion of TCR{alpha} chains in double TCRTg thymocytes. A, TCR{alpha} and {beta} expression on thymocytes of TCRsTg and TCRdTg mice. OT-1/2C TCRdTg and the control TCRsTg thymocytes were stained with the corresponding anti-{alpha} or anti-{beta} Tg chain-specific mAb and with anti-CD4 and -CD8 mAbs. Dot plot profiles for V{beta}8 (2C) vs V{beta}5 (OT-1) and 1B2 (2C) vs V{alpha}2 (OT-1) are shown for total thymocytes. B, A summary of cell surface expression of OT-1 (B20.1+, {square}), H-Y (T3.70+, {blacksquare}), and 2C (1B2+, {cjs2112}) {alpha} chains among thymocytes of TCRdTg mice. Results are expressed as mean ± SD (n = 10). C, TCR{alpha} expression in DP and CD8 SP thymocytes. TCRsTg and TCRdTg thymocytes were stained as in A with V{alpha} expression on gated DP and CD8 SP populations. Results are representative of seven independently analyzed animals. Similar results were obtained in the other two TCRdTg strains (not shown). D, Inclusion of both TCR{alpha}{beta} heterodimers in mDN thymocytes. Cells were stained and analyzed as in C, except that expression of V{alpha}/TCR{alpha} idiotypic determinants on gated DN thymocytes is shown. Results are shown as x ± SD (n = 8). Same result was obtained in the analysis of H-Y/2C and OT-1/H-Y doubly Tg mice (not shown).

 
The observed down-regulation of one TCR{alpha} chain on most thymocytes was somewhat different from the report by Sant’Angelo et al. (31) where in some cases both TCR{alpha} and TCR{beta} were subject to exclusion at a protein level, but similar to findings of three other groups that all saw selective TCR{alpha} down-regulation (20, 22, 30). We further investigated the developmental stage at which TCR{alpha} chain is down-regulated. As CD8+4+ DP thymocytes become positively selected, they immediately up-regulate TCR expression and selectively commit to one of the two SP (CD8+4– or CD8–4+ SP) lineages. Previously, it was shown that normal thymocytes very frequently express two TCR{alpha} chains at low levels at the DP stage, but that one of them is down-regulated following positive selection and that double expressors are very rare among TCRhigh DP and SP thymocytes (20). We therefore examined the extent of TCR{alpha} dominance in TCRdTg thymocyte subsets. We found that TCR{alpha} down-regulation was operating in both the DP cells and in their positively selected CD8 SP progeny in TCRdTg mice (Fig. 1C). (The results for CD4 SP cells are not shown, because these cells are not positively selected via the TCRTg receptors). This difference between normal and TCRdTg in the stage at which exclusion becomes manifest is likely due to much higher TCR levels on Tg DP than on wt DP cells–indeed, in both normal and Tg thymocytes, exclusion affected TCRhigh cells.

Importantly, TCR{alpha} dominance was not seen in DN cells, where both TCR{alpha} chains (and {alpha}{beta} complexes) coexisted at the cell surface at comparable levels (Fig. 1D). In fact, direct analysis of dual TCR-expressing cells revealed that 46–93% of DN cells are positive for both TCRs (69% in Fig. 6A; and data not shown). These results definitively show that the allelic exclusion at the protein level does not operate in the majority of TCRTg mDN cells. As mentioned, preferential pairing between the TCRTg chains, whereby one TCR{alpha} chain would "steal" TCR{beta} away from its natural partner, was initially invoked to explain TCR{alpha} dominance in normal and TCRTg mice (20, 22, 23, 30). However, our data are not consistent with this explanation, because both TCR{alpha}{beta} pairs are expressed at high levels on mDN cells. This conclusion is further supported by the recent data of Gascoigne and colleagues (32). Therefore, different mechanisms are likely to account for the selective V{alpha} down-regulation.



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  		FIGURE 6. TCR{alpha} chain protein exclusion requires the presence of the coreceptor molecule CD8 and might be linked to positive selection. A, Disengaging thymocytes from their normal microenvironment (disrupting the TCR:MHC contact) results in the expression of both TCR{alpha} chains. Freshly isolated or overnight cultured suspensions of thymocytes from OT-1/H-Y TCRdTg mice were stained in a four-color protocol for the expression of CD4, CD8, and each of the TCR{alpha} chains. Expression of TCR{alpha} chains on electronically gated DN or DP cells is shown in upper and lower panels, respectively. B, Cell surface expression of the V{alpha} chains in TCRdTg (OT-1/H-Y) and TCRsTg (OT-1 and H-Y) thymocytes in the presence and absence of the coreceptor molecule CD8. Left panels show a representative increase in expression of the excluded TCR{alpha} chain in CD8–/– animals. Right panels show the cumulative percentage of cells expressing each TCR{alpha} (five mice per group, mean ± SD) in CD8-wild-type and CD8-KO TCRdTg thymocytes.

 
Phenotypic and functional dominance of one Tg TCR{alpha}{beta} in peripheral CD8+ T cells of TCRdTg mice

The net result of the above dominance of one TgTCR over the other was that most TCRdTg thymocytes expressed high levels of only one of the two TCR{alpha}{beta} heterodimers. Because the allelic exclusion at the tcr-a locus is, at best, very weak, the observed dominance appeared to be part of the postrearrangement mechanism(s) that ensures monospecificity of most T cells. If so, and if this down-regulation is physiologically relevant, one would expect to find it among the positively selected peripheral CD8+ T cells. Indeed, both {alpha}{beta} heterodimers were unequally expressed in peripheral T cells of all three Tg combinations, and the high expression was reserved for only one of them (inset to Table I and not shown). For example, in a typical experiment with OT-1/2CdTg mice shown in Table I, 88 ± 9% of CD8+ cells expressed high levels of 2C Id 1B2 (compared with 96 ± 3% in sTg2C), while only 13 ± 6% expressed clearly detectable levels of the V{alpha}2 of the OT-1 (as opposed to 97 ± 3% in OT-1sTg mice). Thus, TCR dominance in TCRdTg mice ensured that very few positively selected peripheral CD8+ T cells (<3% according to direct TCR{alpha}1/TCR{alpha}2 double-expressor analysis; not shown) would express high levels of both TCR{alpha} chains.


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  		Table I. T cell proliferation in doubly {alpha}{beta}TCRTg mice

 
What were the functional consequences of TCR{alpha} down-regulation? This issue was addressed by testing functional reactivity of TCRdTg cells to cognate Ags recognized by each of the two Tg receptors. In such an experiment, we observed functional monospecificity of TCRdTg cells. For example, dTg OT-1/2C splenocytes responded vigorously to p2Ca + Ld, and did so indistinguishably from 2C TCRsTg splenocytes (Table I). The same cells did not respond to OVA + Kb any better than 2C TCRsTg cells (Table I). Comparable results were obtained in the other two TCRdTg combinations, but the results were less remarkable owing to a weak response of the female H-Y splenocytes to male Ag levels physiologically present on splenic APCs (usually three to four times over the background–Table I). Thus, as assessed by in vitro responsiveness, TCR dominance in TCRdTg mice ensured that of the two known TCR specificities only one was functionally expressed.

Toward the mechanism of TCR{alpha} exclusion

To assess at which level TCR{alpha} expression may be regulated, we first studied the expression of TCR{alpha} and TCR{beta} mRNA and protein levels in TCRsTg and TCRdTg mice. Results shown in Fig. 2A show that comparable levels of both TCR chain mRNA were present in TCRdTg thymocytes. We further determined whether TCRdTg cells expressed similar TCR{alpha} protein levels and whether these proteins stably incorporated into TCR complexes. FCM analysis of intracellular TCR{alpha} protein levels in TCRdTg thymocytes revealed comparably lower H-Y TCR{alpha} content, compared with the OT-1 TCR{alpha} chain in OT-1/H-Y TCRdTg thymocytes: these cells exhibited 10% lower MFI of the dominant V{alpha}2 chain, but 40% lower MFI of the suppressed T3.70 chain than comparable TCRsTg thymocytes (Fig. 2B). Consistent with our proposed model, these results indicate that the TCR dominance operates at the protein level.



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  		FIGURE 2. mRNA and protein analysis of TCR{alpha} expression in TCRsTg and TCRdTg thymocytes. A, RT-PCR analysis of TCR{alpha} mRNA expression. Total mRNA from indicated thymocytes was analyzed by RT-PCR/Southern, using the probe indicated on the top and the left of each set. To make the assay semiquantitative, the template titration over serial 1/10 dilutions for each sample was performed using the cycle number within the linearity range. B, The low intracellular level of the second TCR{alpha} chain suggests cytosolic degradation. Intracellular staining of permeabilized TCRsTg and TCRdTg thymocytes was performed as in Materials and Methods. Numbers represent the MFI of intracellular staining with the indicated Ab. Results are representative of three experiments and were also reproduced in the other two TCR dTg strains. C, TCR{alpha} protein expression in H-Y TCRsTg and H-Y/OT-1 TCRdTg thymocytes was analyzed by [35S]methionine pulse (30 min) chase (2 h) metabolic labeling and immunoprecipitation using H-Y-specific TCR{alpha} (T3.70, specific for the V{alpha} Id of the H-Y) mAb. Radiographic analysis of samples using a nonreducing SDS-PAGE is shown, with numbers representing the percentage of precipitated TCR{alpha} chain remaining within the TCR{alpha}{beta} complex at the end of the chase.

 
We next investigated relative stabilities of different TCR chains. [35S]Methionine metabolic labeling of TCRdTg thymocytes followed by immunoprecipitation with chain-specific mAbs revealed that TCRsTg and TCRdTg thymocytes contained comparable levels of free TCR{alpha} chain (Fig. 2C, time 0, lower bands) and of TCR{alpha} complexed to TCR{beta} (Fig. 2C, time 0, upper bands) immediately after pulse labeling. Two hours after labeling (chase), TCRsTg thymocytes retained ~75% (compared with time 0 = 100%) of the TCR{alpha} chain in complex with the TCR{beta} chain. However, in TCRdTg thymocytes, only a small fraction of the initially labeled suppressed chain (20%) was present in the TCR{alpha}{beta} complex (Fig. 2C, time 2, arrow). This was consistent with an initial formation of proper TCR{alpha}{beta} complexes followed by a subsequent selective loss of the suppressed TCR{alpha}.

TCR dominance in TCRdTg mice requires PTK signaling

Lck was shown to constitutively down-regulate TCR expression in DP thymocytes of normal mice before positive selection, by phosphorylation which induces TCR{alpha} internalization and lysosomal degradation (27). We therefore tested whether Lck inhibition may lead to increased cell surface expression of the suppressed TCR{alpha}. Overnight treatment with both the specific PTK Lck inhibitor, PP1 (33) and a less specific inhibitor, herbimycin A (34), led to increased expression (up to 3-fold) of the suppressed H-Y TCR{alpha} (Fig. 3, top two panels) in a dose- and time-dependent manner (not shown). By contrast, this same treatment had essentially no influence upon the expression of both TCR chains in TCRsTg cells (not shown) nor upon the expression of the dominant TCR{alpha} (Fig. 3) or both TCR{beta} chains in TCRdTg thymocytes (not shown). A proteasomal inhibitor, lactacystine, had no effect on TCR expression, suggesting that ubiquitination and proteasomal degradation were not involved in this allelic exclusion. Comparable results were obtained in the other two types of TCRdTg thymocytes (not shown). Of interest, no up-regulation was observed in thymic or peripheral mDN cells, where both receptors are coexpressed at identical levels. These results suggest that the TCR dominance in TCRdTg cells is an active process that depends on intracellular signaling via PTKs.



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  		FIGURE 3. The role of PTK activation and lysosomal degradation in TCR{alpha} down-regulation. TCRdTg thymocytes were incubated for 17 h in the absence or the presence of 3 µM herbimycin A, 20 µM PP1, 10 µM lactacystin, or 20 mM NH4Cl. After this period, cells were washed, stained, and analyzed by flow cytometry. Results are shown for gated DP cells and were similar for the other two TCRdTg combinations. Numbers represent the MFI of each histogram, and are representative of four experiments. No selective TCR up-regulation was seen in gated DN cells in any of these experiments (not shown). All agents showed a dose-dependent effect, except for lactacystin, which failed to show any effect, even when replenished in culture every 6 h (not shown).

 
The above results would suggest that different TCR proteins in TCRdTg mice might occupy different subcellular compartments. We therefore performed confocal microscopy experiments to track all four TCR chains (H-Y{alpha}, H-Y{beta}, OT-1{alpha}, and OT-1{beta}) and found that both TCR{beta} chains, as well as the dominant TCR{alpha} chain, reside mainly at the cell surface of TCRdTg cells, as indicated by a ring-shaped staining that delineated the cell boundaries (Fig. 4A). In contrast, the suppressed TCR{alpha} chain showed a cytosolic distribution resembling perinuclear internalization vesicles in TCRdTg thymocytes. This was not the case in TCRsTg cells, where the same TCR{alpha} chain was found at the cell surface (Fig. 4A). This pattern was similar to the one described for non-Tg TCR{alpha} chains (27) suggesting lysosomal localization of down-regulated TCR{alpha}. We next performed colocalization studies using TCRdTg thymocytes stained for LAMP-2, a lysosomal marker (Ref. 35 ; green color in Fig. 4B), and for TCR{alpha} chains (red color in Fig. 4B). Despite the limitation of the high nucleus to cytosol ratio in T cells, our analysis indicated that the TCR{alpha} chain in TCRsTg thymocytes (Fig. 4B) and the dominant TCR{alpha} chain in TCRdTg thymocytes (Fig. 4B) remained on the cell surface (nuclei were positively identified by 4',6'-diamidino-2-phenylindole staining, not shown). The distribution pattern of the down-regulated TCR{alpha} in TCRdTg thymocytes suggested an intracellular distribution in addition to a cell membrane staining. Furthermore, only the down-regulated TCR{alpha} chain colocalized with LAMP-2 in TCRdTg mice (Fig. 4B). These results are consistent with the possibility that TCR protein exclusion involves targeting of one TCR{alpha} chain to the lysosome, although, at the present, we still lack the complete mechanistic picture of this process.



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  		FIGURE 4. Subcellular localization of TCR proteins in TCRdTg thymocytes. A, Of the four TCRTg chains, only the down-regulated TCR{alpha} can be found intracellularly. TCRsTg and TCRdTg thymocytes were fixed, permeabilized, stained with mAbs specific for V{alpha} or V{beta} TCR chains (T3.70, B20.1, F23.1, or MR9.4 mAbs biotinylated + streptavidin Alexa 568 conjugate), and analyzed by confocal microscopy. Note that all four chains can be found at the surface in TCRsTg cells (no chain is localized in the cytosol); by contrast, in TCRdTg cells, H-Y TCR{alpha}, but no other chain, can be found in the cytosolic vesicles (*). B, Accumulation of the down-regulated TCR{alpha} in the lysosomal compartment. Staining for TCR chains was performed exactly as above; cells were also stained with anti-Lamp-2 FITC (detecting the marker for the lysosomes). Only in dTg OT-1/H-Y cells was there evidence for colocalization of the down-regulated {alpha}-chain with the lysosomes in the scant cytosol; * indicates nuclear localization. The down-regulated, but not the dominant TCR{alpha}, colocalized with the lysosomes. The dominant V{alpha} chain was distributed mainly on the cell membrane.

 
Evidence for PTK-mediated TCR{alpha} protein exclusion in non-Tg thymocytes

We next sought to address the physiological relevance of these observations. We reasoned that if the described protein exclusion occurs in normal thymocytes, it should exhibit the same PTK dependence demonstrated in Fig. 3. We therefore quantified the percentage of double-TCR{alpha} expressors among thymocytes of B6 and BALB/c mice by assessing how many V{alpha}2+, V{alpha}8+, or V{alpha}11+ cells also express V{alpha}3.2. Consistent with the results of Alam et al. (20), under physiological conditions such double expressors could be found mostly among TCRlow cells, and they made up to 4% of total thymocytes positive for V{alpha}2, 8, and 11 (Fig. 5, left panels). Importantly, this percentage doubled in the presence of PTK inhibitors (Fig. 5, middle panels). Although these percentages are small and the TCR levels low, the staining was both reproducible and specific (FcR blocking applied to all samples; moreover, staining could be blocked with excess unlabeled V{alpha}-specific Abs–Fig. 5, right panels). As we saw that the percentage of TCRhigh cells increased by 5-fold following PTK inhibitor treatment (data not shown), we believe it likely that the same PTK-mediated mechanism that lowers TCR expression by destabilizing TCR{alpha} also actively participates in reducing the percentage of thymocytes that express two TCR{alpha} chains.



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  		FIGURE 5. Allelic exclusion of TCR{alpha} chains in the normal repertoire. Total thymocytes from wild-type mice (B6 and BALB/c) were incubated in the absence (left panel) or the presence (middle and right panels) of the PTK inhibitor PP1 at 37°C for 18 h, washed and blocked with excess of anti-FcR mAb. Cells were then stained with the four available directly conjugated mAbs against the TCR V{alpha}2, V{alpha}3.2, V{alpha}8, and V{alpha}11, as indicated (left and middle panels); or were preincubated with an excess unlabeled anti-V{alpha}2, V{alpha}3.2, V{alpha}8, and V{alpha}11 mAb before staining (right panel) as a specificity control. The percentage of thymocytes expressing two V{alpha}s on the cell surface within the box is shown. This staining was specific, inasmuch as incubation with excess unlabeled TCR{alpha}-specific mAb resulted in >95% blocking of staining (right panel). Results are representative of three experiments.

 
The role of TCR/CD8/MHC contact and positive selection in TCR{alpha} protein exclusion

TCR{alpha} protein exclusion spares one TCR{alpha} chain, while permitting down-regulation of the other via Lck activation, by an unknown mechanism. Because Lck functionally couples to TCR signaling via CD4 and CD8 coreceptors, the above data raised the possibility that coreceptor-dependent contacts may play a role in TCR{alpha} protein exclusion, consistent with the model proposed by Alam et al. (20). Indeed, as mentioned in Fig. 1D, DN thymocytes, that lack coreceptor molecules, do not show TCR{alpha} exclusion, and express both chains at the cell surface (Fig. 6A). Moreover, when we disrupted intrathymic TCR:pMHC interactions by culturing thymocytes in suspension at 37°C, we found that such treatment had no influence on DN cells (which mostly already expressed two TCR{alpha} chains), but lead to an increase in levels of the suppressed TCR{alpha} chain on most DP cells (Fig. 6A, lower right panel–38% cells in the upper right quadrant; however, in fact most cells express both chains at similar levels but did not fall into the upper right quadrant due to lower overall TCR levels). Finally, the role of coreceptor molecules was demonstrated genetically. We crossed the targeted disruption of the CD8 coreceptor into our TCRdTg mice, and found that this manipulation increased the expression of the excluded TCR{alpha}, so that all thymocytes expressed both receptors (Fig. 6B), supporting the role of the CD8 coreceptor, most likely via lck activation, in this process.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results described in this study document the existence of a powerful TCR protein exclusion mechanism that operates at the TCR{alpha} protein level. Because the allelic exclusion of tcr-a rearrangement is virtually nonexistent, it is perhaps not surprising that other mechanisms must operate to reduce the production of dual reactivity T cells. TCR protein exclusion described here clearly provides this function. In fact, this mechanism was powerful enough to lead to a near-complete functional exclusion of the TCR composed of a pair of rearranged and overexpressed Tg chains encoding a well-paired, positively selected heterodimer. Analysis of normal thymocytes suggested that this mechanism may also be active under physiological conditions, consistent with the previous work of Gascoigne and colleagues (20, 23, 32). However, it also must be stressed that this mechanism (as well as any other mechanism operating to maximize monoclonality of TCR{alpha} chains) is leaky, as the cases of expression of two TCR{alpha} chains can readily be detected.

In TCRdTg thymocytes and T cells, both TCR{beta} chains were expressed equally, and the TCR{beta} chain whose {alpha} partner was down-regulated remained at the cell surface (Fig. 1A), likely in complex with the dominant TCR{alpha} chain (30). This indicated that TCR{alpha} chain exclusion operates at the protein, and not at the gene rearrangement, level. In an intriguing earlier study using MHC class II-restricted TCRTg, Sant’Angelo et al. (31) reported protein down-regulation of both TCR{alpha} and TCR{beta} in most, but not all, Tg combinations. However, Dave et al. (30) used a mixture of class I- (H-Y, also used in our study) and class II-restricted TCRTg strains (AND, also used by Sant’Angelo et al.) to generate TCRdTg mice, and found selective TCR{alpha} dominance but no TCR{beta} down-regulation, similar to our findings. It is possible that differences in TCR{beta} down-regulation are linked to different CD4 and CD8 engagement, or to other, unknown factors. Still, the fact that the TCR{beta} chain is not down-regulated, but rather persists at the surface (Figs. 1 and 4 and Ref. 30) is somewhat puzzling, given the disulfide bond between the two chains. However, this is consistent with findings that TCR{alpha} has an internalization signal, is unstable, and is targeted by Lck for down-regulation, features not shared by TCR{beta} (27, 36, 37, 38). Perhaps TCR{beta} is retrieved from down-regulated TCR complexes, an issue currently under investigation. Regardless, the TCRdTg model characterized in the present study is representative of dual TCR{alpha} expression, because under physiological conditions thymocytes very rarely (~1%) express two TCR{alpha} chains, and there is therefore no need for exclusion of TCR{beta} at the protein level.

We found that both TCR{alpha} chains were transcribed and expressed in the cytosol, yet only one was expressed at the surface at high levels. Importantly, the TCR{alpha} protein that was poorly expressed at the surface was selectively excluded via PTK Lck activation and lysosomal degradation. These results suggest that phosphorylation of an unknown substrate(s) selectively targets the excluded protein for degradation. In an interesting case of biological economy, Lck, a kinase decisively involved in tcr-b locus allelic exclusion, also appears to be involved in TCR{alpha} protein exclusion by an entirely different mechanism. This is consistent with the data of Boyd et al. (22), who found that the tyrosine phosphatase CD45 increases internalization of one TCR{alpha} in normal thymocytes which express two TCR{alpha} chains. CD45, a potentiator of TCR signaling, operates in concert with the Src family PTKs to enhance the TCR selection in the thymus (reviewed in Ref. 39). The possible downstream mediators of PTK-mediated TCR{alpha} down-regulation include the TCR signaling adaptor GRB2 (40) and the associated GTPases such as Rab5 (41) or dynamin (22), all implicated in TCR internalization.

Two groups reported that two TCR{alpha} chains can be relatively frequently detected at the surface of normal or TCRsTg DP thymocytes, but rarely at the surface of murine mature SP and peripheral T cells (20, 22, 23), and one group recently reported that in TCRdTg mice one TCR{alpha} is selectively down-regulated (30). These groups hypothesized that differential {alpha}{beta} abundance and pairing mediates this phenotypic allelic exclusion (20, 23, 30), while the fourth group (31) proposed problems in TCR assembly. Our results, however, cannot be explained by differential TCR chain pairing. First, each of the TCR{alpha}{beta} pairs in TCRdTg mice has been pretested for pairing in the cell that was the original source of the transgenes. Second, and most importantly, if the pairing affinity was the determining factor of TCR expression, then, regardless of the stage of differentiation, every cell expressing the receptors should exhibit the same pattern of dominance by one TgTCR. Our results clearly show that neither mDN cells in TCRdTg mice (Figs. 1 and 6) nor the DP and SP thymocytes in TCRdTg mice lacking CD8 (Fig. 6) exhibit TCR dominance, but rather coexpress both TCRs. We conclude that TCR{alpha} dominance is an active, signaling-dependent process. This view is supported by recent findings in normal mice (32).

How would one TCR{alpha} preferentially survive at the cell surface in the case where both TCR{alpha} can form TCR{alpha}{beta} heterodimers? We showed that removal of CD8 molecules abrogated TCR{alpha} protein down-regulation, indicating that CD8 recognition of the intrathymic MHC ligands plays a pivotal role in TCR{alpha} protein exclusion; moreover, TCR down-regulation does not occur in the coreceptor-negative cells (mDN). As discussed by Gascoigne and Alam (42), the observed down-regulation could be selective or stochastic, and our results, as well as those of others (30, 31) appear to favor the first possibility. Results from Fig. 6 provide insight into how this might occur–there, the removal of TCRdTg thymocytes from thymic microenvironment (release from intrathymic contacts, Fig. 6A) restored expression of the suppressed TCR{alpha} at the cell surface. Although these results are suggestive of the possible role for TCR:pMHC contact, at present we lack definitive evidence for its involvement. Clearly, further studies are needed to address this intriguing possibility.

Functional studies showed an essential monoreactivity of TCRdTg CD8 cells, with little to no response against the Ag that should be recognized by the down-regulated TCR. Thus, TCR down-regulation in TCRdTg T cells was capable of largelysuppressing or preventing dual reactivity of TCRdTg thymocytes in vitro, creating the effect of allelic exclusion. The surprising strength of this mechanism indicates that it may be of use in unmanipulated T cells. Although its in vivo effectiveness remains to be tested, we would expect that it could be significant. Indeed, there are many examples where cells were tolerant in vivo despite being reactive in the mixed lymphocyte reaction in vitro, suggesting that in vitro tests of reactivity may be more sensitive than those in vivo. Still, TCRdTg mice raise several issues related to T cell tolerance: if dual-TCR cells are allowed to exist, sporadically in normal mice and frequently in TCRdTg animals, they could be the source of autoimmunity (43, 44). Along these lines, it was recently shown that even the very low levels of autoreactive TCR expressed on transferred, dual-TCR T cells can lead to mild autoimmunity (44). But these cells could also be beneficial, as suggested by recent observations that dual-TCR cells may extend the immune repertoire (45). Experiments testing intrathymic and peripheral deletion and in vivo reactivity will be needed to address the above issues.


   Acknowledgments
 
We thank Drs. H. von Boehmer, H. S. Teh, D. Loh, and F. Carbone for mice and anti-TCR Abs; Dr. L. Denzin for advice on immunoprecipitation; Dr. J. Guevara for help with proliferation assays; D. Nikolich-Zugich for help with FCM analysis; Drs. S. Vukmanovic, H. Petrie, L. Denzin, and S. Murray for critical reading of the manuscript; A. Lang for critical reading of the manuscript; and A. Kennard for secretarial help.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by U.S. Public Health Service Grant AI-32064 (to J.N.-Z.) from the National Institutes of Health. Back

2 Current address: Department of Pathology, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX 77030. Back

3 Address correspondence and reprint requests to Dr. Janko Nikolich-Zugich, Vaccine and Gene Therapy Institute, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, OR 97006. E-mail address: nikolich{at}ohsu.edu Back

4 Abbreviations used in this paper: PTK, protein tyrosine kinase; Tg, transgenic; dTg, double Tg; sTg, single Tg; DP, double positive; SP, single positive; FCM, flow cytofluorometric; DN, double negative; mDN, mature DN; MFI, mean fluorescence intensity. Back

Received for publication May 25, 2004. Accepted for publication July 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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