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Silencing during 
T Cell Development Depends upon Pre-TCR-Induced Proliferation
* Ludwig Institute for Cancer Research, Lausanne Branch, and
Institute for Biochemistry, University of Lausanne, Lausanne, Switzerland; and
Centre d’Immunologie de Marseille Luminy, Parc Scientifique de Luminy, Marseille, France
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Abstract |
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or 
TCR. In the lineage, developing thymocytes progressively extinguish transcription of the TCR genes by a poorly understood process known as silencing. We show that lineage thymocytes in mice lacking a functional pre-TCR undergo limited proliferation and fail to silence TCR genes during development. Stimulation of pre-TCR-deficient immature thymocytes with anti-CD3 Abs does not directly down-regulate TCR transcription but restores TCR silencing following proliferation. Collectively our data reveal an important role for pre-TCR induced proliferation in activating the TCR silencer in lineage thymocytes, a process that may reinforce or lineage commitment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-chain and TCR -chain, whereas the lineage expresses a TCR:TCR heterodimer. During development in the thymus TCR, TCR, and TCR genes are rearranged early at the CD4–CD8– double-negative (DN)2 stage. Productive rearrangement of TCR and TCR genes in DN thymocytes gives rise to a TCR, whereas a productively rearranged TCR pairs with an invariant protein called pT to form a pre-TCR. These immature lineage thymocytes then proliferate extensively and proceed to the CD4+CD8+ double-positive (DP) stage in which TCR rearrangement occurs. Displacement of pT by TCR allows the formation of an TCR that can undergo positive and negative selection to yield the mature T cell population.
Several different models of or lineage commitment have been proposed (reviewed in Refs. 1, 2, 3). The instructive model proposes the existence of a common precursor that will be directed to the lineage if it expresses a pre-TCR, or to the lineage if it expresses a TCR. In contrast, the stochastic or separate lineage model proposes that and T cells develop from different precursors that are committed before TCR, TCR, and TCR rearrangement. According to this model, the TCR would be irrelevant for lineage specification, and the main developmental function of the TCR or pre-TCR would be to ensure correct maturation of lineage-committed and precursor cells. Recently, a new model has been proposed in which TCR signal strength determines or lineage commitment (4, 5).
It has been known for some time that TCR expression is very low in cells of the lineage, despite the fact that TCR rearrangements are not inhibited. The extinction of TCR transcripts in T cell precursors might be required to prevent inappropriate pairing between TCR and either pT (6) or TCR (7). Based on comparative studies with TCR -chain transgenic mice it was concluded that TCR expression in lineage cells is suppressed via a cis-acting transcriptional silencer present in the flanking region of the TCR locus (8). No information about the precise genomic localization or mechanism of activation of this putative silencer is currently available. In this report we have used real-time RT-PCR to further analyze TCR silencing in lineage thymocytes rescued by various alternative pre-TCR, or by signals that mimic pre-TCR-induced differentiation and proliferation.
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Materials and Methods |
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C57BL/6 wild-type (WT) mice were purchased from Harlan Netherlands. C57BL/6 TCR–/–, C57BL/6 TCR–/–, and C57BL/6 TCR–/–TCR–/– mice were originally obtained from The Jackson Laboratory. C57BL/6 pT–/– mice were provided by Dr. H.-J. Fehling (University Clinics, Ulm, Germany). C57BL/6 pT–/–TCR–/– mice were obtained by crossing C57BL/6 pT–/– mice with C57BL/6 TCR–/– mice. All mice were used at 4–8 wk of age.
Cell preparation, flow cytometry, and sorting
For the isolation of DN2, DN3, and DN4 immature thymic subsets, DN-enriched thymocyte cell suspensions (9) were incubated with a mixture of FITC-conjugated Abs to CD4, CD8, CD3
, TCR, TCR, B220, CD11c, Gr-1, and F4/80, together with anti-CD44 PE Cy5, anti-CD117 PE Cy7, and anti-CD25 PE. DN2, DN3, and DN4 cells (FITC mixture– CD25+CD44+CD117+, FITC mixture–CD25–CD44+CD117–, and FITC mixture–CD25–CD44–CD117–, respectively) were isolated by electronic sorting. Thymic T cells were isolated by incubating DN-enriched cell suspension with anti-CD3 PE Cy5, anti-TCR PE, and anti-TCR FITC and subsequent electronic sorting of CD3+TCR–TCR+ cells. For DP and immature single-positive (ISP) isolation, total thymocyte suspension was three-color stained with anti-CD4 PE Cy5, anti-CD8 FITC, and anti-TCR PE or anti-TCR PE. DP (CD4+CD8+TCR–) and ISP (CD4–CD8+TCR–) cells were isolated by electronic sorting.
Cells were analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). Dead cells were gated out by their forward and side scatter profile.
Peripheral B cells and T cells were isolated from spleen. Spleen suspensions were three-color stained with anti-TCR PE, anti-CD3 FITC, and anti-CD19 PE Cy5. B cells (CD3–CD19+TCR–) and T cells (CD3+CD19–TCR+) were isolated by electronic sorting. All sortings were performed on a FACSAria flow cytometer (BD Biosciences). Cell cycle analysis and intracellular TCR staining were performed as previously described (9).
Isolation of nuclear RNA
Cell nuclei were isolated as described by Masternak et al. (10). Briefly, after washing three times in PBS, 106 cells were resuspended in 100 µl of hypotonic buffer (HB) 0.3 M sucrose (10% glycerol, 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 15 mM HEPES (pH 7.9), 0.5 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM PMSF, and 1 mM DTT) and lysed by the addition of 100 µl of HB 0.3 M sucrose containing 0.8% Nonidet P-40. Nuclei were pelleted by overlaying the cell lysate onto 600 µl of HB 0.9 M sucrose and centrifugation at 1500 x g. All steps were performed at 4°C. RNA from the purified nuclei was extracted using TRIzol (Invitrogen Life Technologies).
Real-time PCR
Real-time PCR using SYBR was performed on a LightCycler (Roche) according to the manufacturer’s instructions. Total RNA from cell samples was purified using TRIzol. Total or nuclear RNA was reverse-transcribed using random nonamers and AMV reverse transcriptase (Roche). For the PCR, the LightCycler FastStart DNA Master SYBR Green I (Roche) was used following the instruction manual. V1.1-C4, V2-C1, and inducible cAMP early repressor (ICER) transcripts were normalized to TATA-binding protein, whereas V1.1-J4 and V2-J1 rearrangements were normalized to Thy-1. All primer sequences are available upon request. Amplification plots were analyzed using the second derivative method with LightCycler data analysis software version 3.5 (Roche) and the relative quantification was determined using the LightCycler relative quantification software version 1.0 (Roche). Sextuplet analysis showed that measurement errors were always <9%.
Run-off analysis of V1.1-J4 and V2-J1 rearrangements
Genomic DNA extracted from sorted thymic populations was PCR amplified using primers specific for the V1.1 and V2 gene segments in combination with primers situated downstream of J4 and J1, respectively. PCR was performed as follow: 5 min at 94°C, followed by 40 cycles consisting of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, and finally 5 min at 72°C. PCR products were purified using the QIAquick gel extraction kit (Qiagen) and subjected to primer extension in run-off reactions. Infrared dye IRD-700-labeled primers specific for J4 (5'-GGG GAA TTA CTA CGA GCT TTG-3') and J1 (5'-CAG AGG GAA TTA CTA TGA GC-3') were used for the extension of V1.1-J4 and V2-J1 rearrangements, respectively. The run-off reactions were performed as follow: 3 min at 94°C, followed by 10 cycles consisting of 1 min at 94°C, 1 min at 60°C, 2 min at 72°C. The products were loaded onto a 6% acrylamide/8 M urea gel and run on a Long Readir 4200 sequencer (LI-COR). The size of the products was determined by comparison with the microSTEP-20a DNA size standard (Microzone Products), and quantitation was performed using the AIDA software (Raytest Schweiz).
Injection of anti-CD3 Abs in TCR–/––/– mice
Eight- to 10-wk-old mice were injected i.p. with 30 or 100 µg of anti-CD3 (1452C11) and analyzed 7 days or 1 day later, respectively.
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Results |
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silencing in immature T cells
To investigate the timing of TCR silencing, immature thymocytes at DN2, DN3, DN4, ISP, and DP stages from WT mice were purified by cell sorting, and the expression level of V1.1-C4 (V1.1) and V2-C1 (V2) (the most commonly expressed thymic TCR transcripts) in each population was compared by real-time RT-PCR. As shown in Fig. 1A, both V1.1 and V2 transcripts could already be detected at the DN2 stage and increased to maximal levels in the DN3 population, although these levels are significantly lower than levels found in mature T cells (Fig. 1B). TCR expression progressively declines from the DN3 stage to the DP stage, at which a 12-fold decrease in V1.1 and a 90-fold decrease in V2 expression are observed when compared with DN3. To compare these levels with those of mature lymphocytes, the same analysis was performed in thymic T cells and peripheral T and B cells. As shown in Fig. 1B, TCR transcripts are present in both mature T cells and B cells, though at very low levels. Compared with mature T cells, V1.1 expression is 
40- and 80-fold reduced in mature T and B cells, respectively, whereas V2 expression is 90- and 450-fold reduced in these cells.
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transcripts in mature T cells shows that the silencing of TCR is not complete in the T cell lineage. Moreover, as the level of TCR expression found in mature T cells is comparable with that found in DP thymocytes, our results suggest that maximal TCR silencer activity in the T cell lineage already occurs at the DP stage.
Increased TCR expression in DP cells in the absence of pre-TCR signaling
As the kinetics of silencing of TCR transcription in developing thymocytes correlates with the onset of pre-TCR expression, we considered the possibility that the pre-TCR could play a role in activation of TCR silencing. To test this hypothesis, we took advantage of mutant mice in which pre-TCR signaling cannot occur due to the absence of essential components of the pre-TCR, either the pT molecule (pT–/– mice) or the TCR -chain (TCR–/– mice). As shown previously (11, 12), the absolute number of thymocytes in pT–/– and TCR–/– mice is notably reduced (50-fold) compared with WT mice (Fig. 2A). Nevertheless, as already reported, a limited development of DP cells is reproducibly observed in both mutant mice (Fig. 2A).
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T cells because they express lineage-restricted genes such as pT (for TCR–/– mice) and TCR (Fig. 2B) and fail to express lineage-restricted genes such as ICER (13). Therefore rescued DP cells represent an ideal population to test the hypothesis that the pre-TCR plays a role in TCR silencing. To this end, purified DP thymocytes from WT, pT–/–, and TCR–/– mice were isolated, and real-time RT-PCR analysis was performed to quantify the presence of TCR transcripts. As shown in Fig. 2D, DP cells that develop in pT–/– and TCR–/– mice contain 10- to 20-fold higher amounts of V1.1 and V2 transcripts compared with WT DP cells when total RNA was analyzed. This increase in TCR expression very likely reflects de novo transcription rather than mRNA stabilization because analysis of nuclear RNA (greatly enriched for nascent transcripts) demonstrated much higher levels of V1.1 and V2 expression in pT–/– DP thymocytes than in WT controls (Fig. 2E). These results indicate that DP cells that have differentiated from DN precursors in the absence of a pre-TCR are unable to shut down the expression of TCR, suggesting a relationship between pre-TCR signaling and the activation of the TCR silencer.
The development of DP thymocytes in pT–/– and TCR–/– mice is dependent upon signals from an alternative TCR that partially mimics the pre-TCR. In the case of TCR–/– mice it is clearly the TCR that rescues DP thymocytes because rescue is abolished in TCR–/–TCR–/– mice (12). Moreover the rescued DP cells are enriched for productive TCR and TCR rearrangements (14). The situation for pT–/– mice is less clear because genetic evidence suggests that both and TCR can rescue DP thymocytes in pT–/–TCR–/– and pT–/–TCR–/– mice, respectively (15). Nevertheless only 25% of DP thymocytes in pT–/– mice express intracellular TCR protein (16) (Fig. 2A), indicating that there is no selection for productive TCR rearrangements (and hence for TCR) in this population. Moreover productive V1.1-J4 and V2-J1 rearrangements are significantly over-represented in DP thymocytes from pT–/– mice (Fig. 2F), suggesting that the TCR is mainly responsible for the rescue of DN thymocytes to the DP stage in the absence of pT. Because DP thymocytes in both TCR–/– and pT–/– mice are primarily selected by a TCR, it is possible that the defect in TCR silencing in these cells is due to the presence of a TCR (rather than the absence of a pre-TCR). In this case, one might expect that the residual TCR expression in DP thymocytes of WT mice is due to the presence of a subpopulation of cells bearing a TCR. However TCR expression in DP thymocytes of TCR–/– mice is not reduced compared with WT controls (Fig. 2D), suggesting that a TCR does not contribute to the residual TCR expression in WT DP thymocytes. To address this issue more directly, we analyzed TCR expression in DP thymocytes from pT–/– TCR–/– mice. In agreement with previous reports (15), a small population of DP cells was observed in these mice (Fig. 2A). Most of these DP thymocytes express intracellular TCR (Fig. 2A) and exhibit properties of normal lineage DP thymocytes such as the expression of C, RAG2, and pT (Fig. 2B) and notably do not express lineage-specific genes such as ICER (Fig. 2C). These results are consistent with the assumption that DP thymocytes emerging in pT–/–TCR–/– mice are bona fide T cells rescued by premature TCR signaling in DN cells (15). Interestingly DP thymocytes from pT–/–TCR–/– mice expressed similarly high levels of V1.1 and V2 transcripts as those from pT–/– or TCR–/– mice (Fig. 2D). Taken together, these results show that DP thymocytes rescued by either or TCR do not silence TCR transcripts, suggesting that the activation of TCR silencing depends on correct pre-TCR signaling.
Anti-CD3 induced proliferation of precursors restores TCR silencing in DP thymocytes
At least two hypotheses could be proposed to explain the correlation between TCR silencing and pre-TCR signaling during T cell development. One possibility would be that pre-TCR signaling directly activates the TCR silencer. Alternatively pre-TCR signaling may only be required to induce proliferation, and TCR transcripts may subsequently disappear as dividing immature thymocytes progress to the DP stage. To distinguish between these possibilities, we devised a system in which TCR expression in synchronized precursors could be measured either as a direct consequence of pre-TCR signaling (in DN3 thymocytes) or following pre-TCR induced proliferation (in DP thymocytes). It is known that in vivo treatment of RAG2–/– mice with anti-CD3 mAb promotes both proliferation and differentiation of DN3 thymocytes to the DP stage (17) in the absence of any TCR rearrangement. We therefore performed a similar experiment in TCR–/–TCR–/– mice that have a comparable block in T cell development (due to combined absence of , , and pre-TCR) but nevertheless are able to rearrange and express TCR. This model system, although formally independent of the pre-TCR, most likely recapitulates physiological pre-TCR induced differentiation and proliferation because recent studies have demonstrated that cross-linking of CD3 is the key molecular event initiating pre-TCR signaling (18).
As previously described for RAG2–/– thymocytes (19) anti-CD3 treatment of TCR–/–TCR–/– DN3 thymocytes for 24 h induces down-regulation of CD25 and initiation of cell cycling without any increase in cell number (Fig. 3A and data not shown). However, only a very slight decrease in TCR transcripts was observed (Fig. 3B), indicating that pre-TCR signaling does not directly activate TCR silencing. After 7 days anti-CD3 treatment of TCR–/–TCR–/– mice induces a notable proliferation of thymocytes, and differentiation to the DP stage (Fig. 4A). DP cells that develop after anti-CD3 injection in TCR–/–TCR–/– mice exhibit properties of normal DP thymocytes as shown by the expression of high levels of TCR, RAG2, and pT (Fig. 4B) and low levels of the lineage-specific gene ICER (Fig. 2C). In addition, TCR rearrangement in this population occurs to a similar extent as in WT DP cells or mature T cells (Fig. 4C). Importantly the levels of TCR transcripts in these anti-CD3 rescued DP cells were as low as those detected in WT mice (Fig. 4D) demonstrating directly that proliferation of precursors during the transition to the DP stage restores TCR silencing.
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Discussion |
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lineage development. In addition to the well-characterized roles of the pre-TCR in inducing selective proliferation of immature thymocyte precursors during the DN to DP transition and contribution to the establishment of TCR allelic exclusion and lineage commitment (reviewed in Ref. 16), we now show that the pre-TCR is also required to down-regulate TCR expression during the maturation of lineage precursors. This ability of the pre-TCR to suppress TCR expression is not an immediate consequence of pre-TCR signaling in DN3 thymocytes but rather directly depends upon pre-TCR induced proliferation that occurs between the DN3 and DP stages of development. Indeed, TCR expression is extinguished progressively during the DN4 and ISP stages where extensive cell division is taking place.
An important issue raised by our results is whether TCR silencing is a consequence of immature thymocyte proliferation per se, or alternatively a mechanism that is lineage specific. In this regard it has generally been assumed that T cells, in contrast to their counterparts, undergo very limited proliferation during development. However this concept has been challenged very recently by the identification of a small subset of rapidly proliferating DN3 precursors of T cells (20). Thus, it appears that immature T cells in fact undergo significant proliferation and, consequently, that the role of proliferation in TCR silencing is intrinsic to the lineage.
The concept of a TCR silencer in T cells was first established by studies of TCR and TCR transgenic mice. By comparing the expression of full-length and truncated TCR transgenes in lineage cells of these mice, it was concluded that the TCR locus contains in its flanking region a cis-acting DNA silencer element that down-regulates TCR transcription selectively in T cells (8). In the context of the silencer model, one interpretation of our data is that pre-TCR signaling in immature thymocytes leads to the production (or degradation) of a protein (or proteins) that is implicated in the activation (or repression) of the TCR silencer element. The progressive nature of the repression of TCR expression during the DN to DP transition could then imply that activation of the TCR silencer may depend critically upon the concentration of such a protein, which would accumulate (or be diluted out) during subsequent cell divisions. According to this hypothesis the failure to activate TCR silencing in cells would be explained by lineage-specific expression of this putative regulatory protein. Clearly the identification and characterization of proteins that bind selectively to the TCR DNA silencer element will be required to test this hypothesis. In addition, comparison of DP thymocytes from WT and pT–/– (or TCR–/–) mice by microarray analysis may help to identify candidate genes that could be involved in TCR silencing.
An alternative explanation for the strong correlation between TCR silencing and proliferation in immature lineage thymocytes would be that TCR expression is progressively modified by chromatin remodeling during subsequent cell divisions. According to this scenario, increased accessibility of the TCR silencer or decreased accessibility of positive regulatory elements (such as promoters and enhancers) at the TCR locus could explain the progressive decline in TCR expression between the DN3 and DP stages. Lack of information concerning the precise localization and composition of the TCR silencer as well as other TCR regulatory sequences make it very difficult to directly test this hypothesis.
Irrespective of the silencing mechanism, it is interesting to speculate on the relevance of TCR silencing during lineage development. In this context several surrogate pre-TCR have been shown to promote progression of some DN thymocytes to the DP stage in different model systems, including (14, 21, 22, 23), (24, 25, 26, 27), (7), and pT/ (6) heterodimers. As shown in our study and elsewhere (14, 21, 22, 23), the TCR is able to promote significant development of DP thymocytes in pT–/– or TCR–/– mice. In contrast the TCR is only capable of very limited DP development (10-fold lower than TCR) in pT–/– TCR–/– mice, most probably because TCR rearrangements are very infrequent at the DN3 stage (28). Moreover the putative and pT/ TCR, although capable of promoting development of DP cells in transgenic models (6, 7), do not participate in normal thymus development because DP thymocytes are essentially absent in TCR–/–TCR–/– mice (12) despite the fact that TCR or pT heterodimers could theoretically be present. In light of these considerations it seems clear that the TCR is potentially the most physiologically relevant surrogate pre-TCR. Viewed from this perspective the primary role of pre-TCR-induced TCR silencing may be to reinforce or lineage commitment by suppressing TCR transcription in those immature thymocytes that happen to express productively rearranged TCR and TCR genes.
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Acknowledgments |
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ugich and Walter Reith for helpful discussions, and Queralt Seguin for help with the nuclear RNA isolation. |
Disclosures |
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Footnotes |
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1 Address correspondence and reprint requests to Dr. H. Robson MacDonald, Ludwig Institute for Cancer Research, Lausanne Branch University of Lausanne, CH-1066 Epalinges, Switzerland. E-mail address: hughrobson.macdonald{at}isrec.unil.ch 
2 Abbreviations used in this paper: DN, double negative; DP, double positive; WT, wild type; ISP, immature single positive; ICER, inducible cAMP early repressor.
Received for publication July 14, 2006. Accepted for publication August 18, 2006.
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References |
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TCR+ and TCR+ T cells from a common precursor. Semin. Immunol. 9: 171-179. [Medline]/ lineage commitment: clues from intracellular TCR staining. Immunol. Rev. 165: 87-94. [Medline]/ lineage commitment in the thymus of normal and genetically manipulated mice. Adv. Immunol. 71: 1-76. [Medline]/ lineage fate. Immunity 22: 583-593. [Medline] TCR signaling efficiently diverts thymocytes to the lineage. Immunity 22: 595-606. [Medline] gene regulatory sequences prevent the function of a novel TCR/pT pre-T cell receptor. Immunity 8: 713-721. [Medline] expression generates TCR complexes that signal the DN-to-DP transition and impair development. Nat. Immunol. 3: 564-569. [Medline] and transgenic mice suggest a role of a gene silencer in the generation of T cells. Proc. Natl. Acad. Sci. USA 87: 3067-3071. and TCR proteins during adult thymus development. Int. Immunol. 11: 1641-1650. gene in development of but not T cells. Nature 375: 795-798. [Medline] and block thymocyte development at different stages. Nature 360: 225-231. [Medline]+ and + cells. Nat. Immunol. 4: 991-998. [Medline] lineage-committed thymocytes can be rescued by the T cell receptor (TCR) in the absence of TCR chain. Eur. J. Immunol. 27: 2948-2958. [Medline] versus T lineage commitment. Immunity 9: 649-655. [Medline]-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2–/– mice in the absence of TCR chain expression. Int. Immunol. 6: 995-1001. expression promoting thymocyte survival at the transition from the double-negative to double-positive stage. Eur. J. Immunol. 33: 1968-1977. [Medline] T cell development in the adult thymus. Nat. Immunol. 7: 995-1003. [Medline] lineage thymocytes to the CD4+CD8+ stage in the absence of TCR- rearrangement. J. Immunol. 154: 5706-5714. [Abstract] enhancer-deleted mice: implications for T cell lineage commitment and differentiation. J. Immunol. 165: 1364-1373. / cell receptor heterodimer induces the expression of CD4 and CD8 in thymocytes. J. Exp. Med. 174: 293-296. and TCR are not interchangeable partners of TCR during T lymphocyte development. J. Exp. Med. 199: 607-615. chain on thymocyte development. Eur. J. Immunol. 34: 1532-1541. [Medline] T cell receptors with distinct ligand specificities do not require those ligands to signal double negative thymocyte differentiation. J. Exp. Med. 199: 1719-1724. gene rearrangement and transcription in adult thymic cells. Eur. J. Immunol. 27: 389-396. [Medline]This article has been cited by other articles:
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