Nature Immunology
3, 564 - 569 (2002)
Published online: 20 May 2002; | doi:10.1038/ni800
Early TCR expression generates TCR complexes that signal the DN-to-DP transition and impair developmentBatu Erman1, Lionel Feigenbaum2, John E. Coligan3
& Alfred Singer11 Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2 SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA. 3 Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
Correspondence should be addressed to Alfred Singer Singera@nih.govClonotypic T cell receptor (TCR) genes undergo ordered rearrangement and expression in the thymus with the result that TCR and TCR proteins are not expressed in the same cell at the same time. Such "TCR/ exclusion" is a feature of normal thymocyte differentiation, but it is abrogated in TCR-transgenic mice, which prematurely express transgenic TCR proteins in early double-negative (DN) thymocytes. We report here that early expression of TCR proteins results in the formation of TCR complexes that efficiently signal the differentiation of DN into double-positive thymocytes independently of pre-TCR and TCR expression. Thus, abrogation of TCR/ exclusion by early TCR expression results in the formation of isotypically mixed TCR complexes whose in vivo signals circumvent TCR selection and redirect thymocyte development along an aberrant developmental pathway.T cell receptor  (TCR) proteins are unique among clonotypic TCR chains because they are excluded from early double-negative (DN) CD4-CD8-thymocytes. The genes encoding TCR , TCR and TCR all rearrange in thymocytes at the DN stage of development, so an individual DN thymocyte might contain one or more of these clonotypic protein chains1,
2,
3,
4. In contrast, TCR gene rearrangement is delayed until thymocytes have been signaled to differentiate into CD4+CD8+ double positive (DP) cells, at which stage TCR expression is transcriptionally silenced5,
6,
7. As a result, TCR and TCR proteins are not normally present in the same cell at the same time. Whether such "TCR/ exclusion" is of developmental significance is not known, but it is abrogated in TCR-transgenic mice, which prematurely express TCR-transgenic proteins in DN thymocytes that might also contain TCR proteins.
At the DN stage of differentiation, developing thymocytes commit to either the or T cell lineages. The nature of the intrathymic signals that dictate the - lineage choice remains uncertain, but signaling by TCR complexes is associated with commitment to the T cell lineage, whereas signaling by pT-TCR complexes is associated with commitment to the T cell lineage. It is not clear whether the - lineage choice is dictated by the identity of the TCR signaling complex or whether it is dictated by other factors, such as the timing, intensity or duration of the transduced TCR signals. The concept that the - lineage decision is dictated by factors other than the identity of the TCR signaling complex is supported by observations that TCR signals at the DN2 stage of development may drive differentiation into -lineage T cells, even when the stimulatory signals are transduced by transgenic TCR complexes8,
9. Similarly, TCR signals at the DN3 stage of development appear to drive development of -lineage T cells that differentiate into CD4+CD8+ DP thymocytes, even when the stimulatory signals are transduced by TCR complexes composed of pre-T (pT) and transgenic TCR chains7. Thus, the timing of TCR signaling in developing thymocytes appears to influence lineage choice and developmental fate.
Here, we specifically considered whether coexpression of TCR and TCR proteins in the same DN thymocyte alters thymocyte development. It seems that coexpression of TCR and TCR proteins in the same DN thymocytes would be unlikely to have much impact, as TCR and TCR proteins are of different TCR isotypes and so might be constrained from assembling into a functional TCR complex. However, the rules of TCR assembly, as they are currently understood, do not preclude the formation of mixed isotype TCR complexes. Indeed, isotypically mixed TCR complexes have been observed, although only in a human leukemia cell line10. We show here that early expression of TCR in immature DN thymocytes does result in formation of isotypically mixed TCR complexes, and that these TCR complexes efficiently signal the differentiation of DN into DP thymocytes independently of the pre-TCR, generating DP cells that are TCR- and therefore unable to further differentiate into mature T cells. These results indicate that abrogation of TCR exclusion by transgenic TCR expression in DN thymocytes results in the formation of TCR complexes whose in vivo signals circumvent TCR selection and redirect thymocyte differentiation along an aberrant developmental pathway.
Results
Paradoxical effect of early TCR expression
To examine the consequences of early TCR expression in DN thymocytes, we constructed a transgene encoding 2B4 TCR V 11 cDNA driven by human CD2 promoter and enhancer elements, and used this transgene to generate TCR-transgenic mice11,
12,
13. Assessment of TCR protein expression in thymocytes from TCR transgenic mice revealed profound but unanticipated effects of TCR transgene expression on T cell development (Fig. 1a). In normal C57BL/6 (B6) mice, DP thymocytes are the progeny of DN thymocytes that expressed TCR proteins and so could be signaled by pre-TCR complexes to differentiate into DP cells14 (Fig. 1a). As a result, essentially all DP thymocytes are TCR+ by intracellular staining, although a significant minority of DP thymocytes do not express TCR on their cell surfaces because of a failure to productively rearrange and express TCR (Fig. 1a). We anticipated that the introduction of an already rearranged TCR transgene, such as the 2B4 transgene, would promote TCR surface expression on 100% of DP thymocytes; what we observed was almost the exact opposite. Instead of DP thymocytes that were 100% surface TCR+, expression of the 2B4 TCR transgene resulted in DP thymocytes that were almost all TCR-, by both intracellular and extracellular staining (Fig. 1a). The generation of TCR- DP thymocytes was not limited to mice that expressed the 2B4 TCR V11 transgene, as identical results were obtained with mice that expressed a different TCR (TCR V2) transgene, whose expression was also driven by human CD2 control elements (data not shown). This did not result from some complex interaction between transgenic and endogenous TCR proteins, as TCR- DP thymocytes also appeared upon introduction of the TCR transgene into TCR-/- mice that did not express endogenous TCR proteins (Fig 1a).
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The disappearance of TCR+ DP thymocytes in TCR transgenic mice may result in part from the early expression of TCR complexes that arrest thymocyte development at the DN stage of differentiation9,
15,
16. In contrast, the replacement of TCR+ DP thymocytes with TCR- DP thymocytes in TCR-transgenic mice has not previously been appreciated and cannot be explained by any currently known mechanism for DP thymocyte generation. As a result, we wished to verify that DP thymocytes that were TCR- by staining were not synthesizing TCR proteins. To do so, we purified surface TCR- DP thymocytes and metabolically labeled their nascent proteins in vitro (Fig. 1b). In fact TCR- DP thymocytes from TCR-transgenic mice were not synthesizing TCR proteins (Fig 1b, lane 2), even though they were synthesizing transgenic 2B4 TCR proteins (Fig 1b, lane 1).
Early TCR expression induces TCR complexes
To explain the generation of TCR- DP thymocytes, we speculated that premature expression of TCR proteins in DN thymocytes might have resulted in the formation of an unknown TCR receptor complex that signaled the DN to DP transition before TCR rearrangements, thereby circumventing -selection and promoting the generation of TCR- DP thymocytes. If such a TCR complex were formed in DN thymocytes, we thought it most likely to be a TCR complex, as all known TCR complexes consist of paired TCR chains, one of which has two positive transmembrane charges (for example TCR, TCR, pT) and the other of which has only one positive transmembrane charge (for example TCR, TCR)7,
10,
17,
18,
19.
To determine whether TCR expression in early DN thymocytes actually resulted in formation of TCR complexes that signal the DN to DP transition, we introduced the TCR transgene into recombination-activating gene 2−deficient (referred to hereafter as RAG-/-) and TCR-/-TCR-/- (referred to hereafter as TCR-/--/-) mice. Thymocytes from both RAG-/- and TCR-/--/- mice were arrested at the DN stage of development (Fig. 2a, groups 1, 2). Introduction of the TCR transgene into RAG-/- mice (which potentially express only pT chains) did not result in any DP thymocytes, showing that TCR did not associate with pT chains to signal the DN to DP transition (Fig. 2a, group 1). However, introduction of the TCR transgene into TCR-/--/- mice (which potentially express both pT and TCR chains) resulted in the generation of >12  106 DP thymocytes (Fig. 2a, group 2), indicating that TCR and TCR chains do associate to form TCR complexes that signal the DN to DP transition. Unlike DP thymocytes from normal B6 mice, which are CD25-negative20, DP thymocytes from TCR-transgenic TCR-/--/- mice were CD25+ (Fig. 2b). This suggested that they were the progeny of CD25+ DN thymocytes that had not extinguished CD25 expression prior to differentiating into DP cells. CD25+ DP thymocytes have been observed21,
22, but only in settings in which pre-TCR signals are circumvented.
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Consistent with this perspective, DN4 (CD25-CD44-) thymocytes were markedly under-represented in TCR-transgenic TCR-/--/- compared to DN thymocyte populations from normal B6 mice (Fig. 2c). Thus, TCR expression in DN thymocytes appeared to result in formation of TCR complexes that signaled CD25+ DN thymocytes to differentiate into TCR- DP cells.
To further characterize the signaling complex that induced the DN to DP transition in TCR-transgenic mice, we introduced the TCR transgene into pT-/-, TCR-/- and CD3 -/- mice (Fig. 2a, groups 3−5)14,
23,
24. In the absence of the TCR transgene, pT-/- and TCR-/- thymi both contained  1 106 DP thymocytes that resulted from TCR signaling23,
25. In contrast, introduction of the TCR transgene resulted in markedly greater numbers of DP thymocytes in both pT-/- and TCR-/- mice (Fig. 2a, groups 3, 4). Note that most of the DP thymocytes generated in TCR-transgenic pT-/- mice were TCR- and so had been generated by putative TCR complexes (Fig. 2d). Thus, signaling by putative TCR complexes proceeded independently of both pT and TCR chains. On the other hand, CD3-/- mice were devoid of DP thymocytes, regardless of the presence or absence of the TCR transgene (Fig. 2a, group 5). This indicated that TCR complexes resemble all other TCR complexes in their requirement for CD3 chains to transduce intracellular signals24,
26.
TCR complexes consist of TCR and TCR C 4 chains
To characterize TCR complexes biochemically, thymocyte lysates were immunoprecipitated with TCR antibodies, run on reducing SDS−polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with a TCR-specific antibody that detects all TCR proteins. If TCR complexes exist in TCR-transgenic thymocytes, transgenic TCR proteins should be coprecipitated by TCR antibodies. We found that transgenic TCR proteins were not only immunoprecipitated by the TCR clonotypic antibody A2B4 (Fig. 3a, lane 3), but they were also coprecipitated from thymocyte lysates by anti-TCR specific for the TCR C 4 chain (referred to hereafter as C4) (Fig. 3a, lane 1). Transgenic TCR proteins were not coprecipitated by TCR antibody that binds all other TCR molecules, C1 C2 and C3 (C123) (Fig. 3a, lane 2), nor were they coprecipitated by TCR antibody (Fig. 3a, lane 4). Thus, TCR complexes consisted of TCR and C4 chains.
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Next, we compared thymocyte lysates from TCR-transgenic and nontransgenic mice, as TCR complexes should only be formed in TCR-transgenic thymocytes. In nontransgenic mice, TCR and TCR proteins are not expressed in the same cell. We found that C4 antibody only coprecipitated TCR proteins from transgenic thymocyte lysates (Fig. 3b, lanes 5, 6; Fig. 3c, lanes 3, 7), even though the antibody precipitated C4 molecules from both transgenic and nontransgenic thymocyte lysates (Fig. 3b, lanes 7, 8). TCR complexes were almost exclusively endoglycosidase H (EndoH)-sensitive (Fig. 3c, lanes 7, 8), indicating that too few TCR complexes were transported beyond the medial Golgi to be detectable by protein immunoblotting. In contrast, conventional TCR complexes in nontransgenic B6 thymocytes had both EndoH-sensitive and -resistant components (Fig. 3c, lanes 1, 2), with the resistant components mainly due to surface TCR complexes. Thus, TCR complexes are present in thymocyte lysates from TCR-transgenic mice, are composed of transgenic TCR proteins associated with endogenous C4 chains and do not appear in detectable quantities on the cell surface (a characteristic reminiscent of pre-TCR complexes).
Genetic reconstruction of TCR complexes
To verify that the TCR complexes we detected biochemically were actually responsible for signaling the generation of TCR- DP thymocytes, we genetically reconstructed these complexes in DN thymocytes by introducing both TCR and TCR transgenes into RAG-/- mice (Fig. 4). The TCR transgenes we used (encoding either C1 or C4) were both driven by endogenous TCR control elements so that expression of these TCR transgenes paralleled expression of endogenous TCR molecules during intrathymic development27,
28. Confirming published data7, introduction of TCR transgenes into RAG-/- mice resulted in the generation of small numbers (1 106−2 106) of DP thymocytes as a consequence of signals derived from pT-TCR complexes (Fig. 4). Introduction of the 2B4 TCR transgene into these TCR-transgenic RAG-/- mice created the genetic potential for formation of either TCR-C4 signaling complexes (which we had detected biochemically) or TCR-C1 signaling complexes (which we had not detected biochemically). Concordant with our biochemical analyses, we found that coexpression of TCR and C4 chains in RAG-/- mice signaled the generation of over 57 106 DP thymocytes, whereas coexpression of the TCR and C1 chains did not signal the generation of any DP thymocytes in RAG-/- mice (Fig. 4).
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These results demonstrate that TCR complexes that consist of TCR and C4 chains form when expressed in the same DN cell and that they are highly efficient in signaling the generation of DP thymocytes. Note that TCR C4-transgenic RAG-/- mice contained large numbers of DP thymocytes, but were devoid of single-positive (SP) T cells (Fig. 4). Because TCR expression is silenced in DP thymocytes5,
6,
7, these DP thymocytes did not express TCR complexes on their surface (data not shown); thus, they are dead-end cells that cannot be positively selected for further maturation into SP T cells.
Effect of TCR complexes on  T cell generation
Having documented that formation of TCR complexes in early DN thymocytes circumvents TCR selection and impairs subsequent TCR development, we next determined whether TCR complex formation also affected T cell development. Because TCR complexes are only formed with C4 chains, TCR complexes would only affect development of TCR thymocytes that use C4 chains. Unfortunately, monoclonal antibodies (mAbs) that specifically stain C4 do not yet exist, but TCR thymocytes expressing C4 chains can be identified by staining with V1.1 mAb, as V1 rearranges only with C4 and is expressed only on C4+ cells29,
30. Examination of DN TCR+ thymocytes from TCR-/- mice showed that overall numbers of TCR+ thymocytes were somewhat reduced by the addition of the TCR transgene (Fig. 5), but the reduction equally affected V1+ (that is, C4+) and V1- TCR thymocytes and so was unlikely to result from TCR complex formation (Fig. 5). We conclude that, unlike T cell development, T cell generation was not impaired by formation of TCR complexes in TCR-transgenic mice.
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Discussion
We have shown here that early TCR expression results in the formation of isotype-mixed TCR complexes that consist of TCR chains paired to C4 chains. These TCR complexes efficiently signal the DN to DP transition independently of the pT, TCR and TCR chains. DN thymocytes are signaled by TCR complexes at the CD25+ (that is, DN2 or DN3) stage of development to undergo a proliferative burst and to differentiate into DP cells. In this way, they circumvent the DN4 stage during which TCR selection occurs and CD25 expression is down-regulated. As a result, TCR signaled DN thymocytes differentiate into TCR- DP cells that still express CD25. Because TCR transcription is silenced at the DP stage of development5,
6,
7, such TCR- DP thymocytes do not express any surface TCR complexes; thus, they are dead-end cells that cannot undergo further maturation.
TCR exclusion is a feature of T cell development in normal animals, but it is abrogated in TCR-transgenic animals that prematurely express TCR in early DN thymocytes. An unexpected consequence of coexpressing TCR and TCR proteins in the same DN thymocyte is their pairing and assembly into isotypically mixed TCR complexes that are biologically functional in vivo. Although not previously described, the formation of TCR complexes is concordant with the known principles for TCR assembly17,
18,
19. Assembly of TCR components into receptor complexes is largely regulated by transmembrane charges, so that clonotypic TCR chains with one transmembrane-positive charge only associate with clonotypic TCR chains with two transmembrane-positive charges. TCR complexes conform to this basic paradigm. However, the TCR complexes we detected here were limited to C4 chains, even though, in theory, other TCR chains should also have been able to associate with TCR. Because V domains are constrained to specific C domains29,
30,
31, it is conceivable that TCR assembly may be limited by either C-region constraints or V-region incompatibilities that remain to be identified.
TCR is not unique in its ability to signal the DN to DP transition by circumventing TCR selection, as TCR and pT-TCR complexes also share this property5,
6,
7. However, TCR and pT-TCR signals do not induce DN thymocytes to undergo a proliferative burst and so they generate few TCR- DP thymocytes. In contrast, the large numbers of TCR- DP thymocytes generated by TCR signals indicate that TCR signals do induce DN thymocytes to undergo a proliferative burst, which appears to be as extensive as that signaled by the pre-TCR. As a result, aberrant TCR and pT-TCR signals can be tolerated during normal T cell differentiation, but TCR signaling must be avoided.
Although TCR complexes markedly altered T cell differentiation, TCR complexes did not appear to affect TCR development, as TCRV1+ thymocytes were not specifically depleted from the TCR pool. We assessed the representation of V1+ thymocytes among TCR+ cells as a measure of the frequency of C4+ thymocytes in the TCR pool, as reagents do not yet exist to directly examine C |
s.e.m. numbers of thymocytes for each strain (n = three mice for each group); numbers above the contour plots indicate the percentage of DP thymocytes. (Right panels) Surface and intracellular TCR
