The Lck Paradox: Because of Inconsistent Experimental Evidence, the Role of the Protein Tyrosine Kinase p56lck in Early Thymic Development Remains Poorly Defined

Since this article was written, publications by several groups of investigators shed additional light on the lck paradox: Firstly, as considered in this mini-review, two papers now show that fyn can partially substitute for Ick in pre-T cell receptor dependent proliferation and differentiation. (T. Groves, P. Smiley, M.P. Cooke K. Forbush, R.M. Perlmutter, and C.J. Guidos. (1996). fyn can partially substitute for lck in T lymphocytes development. Immunity 5: 417; N.S.C. van Oers, B. Lowin-Kropf, D. Finlay, K. Connolly, and A. Weiss. (1996). cq3 T cell development is abolished in mice lacking both lck and fyn protein tyrosine kinases. Immunity 5" 429). This work implies that the dominant negative mutant of lck competes with both Ick and fyn and thus partially resolves the lck paradox. Secondly, concerning TCR/3 locus allelic exclusion, it was shown that components of the CD3 complex are necessary for the exclusion of endogenous TCR/3 genes by a rearranged TCR/3 transgene (L. Ardouin, J. Ismaili, B. Malissen, and M. Malissen. (1998) The CD3-y6 and CD3-/7 modules are each essential for allelic exclusion of the T cell receptor/3 locus but are both dispensable for the initiation of V to (D)J recombination at the T cell receptor-/3, -% and -6 loci. J. Exp. Med. 187:5). However, these as well as many previous studies on TCR/3 transgenemediated exclusion of endogenous TCR/3 genes may have been misleading as recent work also suggested that this approach may generate transgenic artefacts (I. Aifantis, J. Buer, H. von Boehmer, and O. Azogui. (1997) Essential role of the pre-T cell receptor in allelic exclusion of the T cell receptor /3 locus. Immunity 7: 601). Since much of the data on the role of lck in TCR/3 allelic exclusion has been generated by this approach, it is possible that such transgenic artefacts have contributed to the inconsistencies concerning the role of lck in TCR/3 locus allelic exclusion.

abolished in mice lacking both lck and fyn protein tyrosine kinases. Immunity 5" 429). This work implies that the dominant negative mutant of lck competes with both Ick and fyn and thus partially resolves the lck paradox. Secondly, concerning TCR/3 locus allelic exclusion, it was shown that components of the CD3 complex are necessary for the exclusion of endogenous TCR/3 genes by a rearranged TCR/3 transgene (L. Ardouin, J. Ismaili, B. Malissen, and M. Malissen. (1998) The CD3-y6 and CD3-/7 modules are each essential for allelic exclusion of the T cell receptor/3 locus but are both dispensable for the initiation of V to (D)J recombination at the T cell receptor-/3, -% and -6 loci. J. Exp. Med. 187:5). However, these as well as many previous studies on TCR/3 transgenemediated exclusion of endogenous TCR/3 genes may have been misleading as recent work also suggested that this approach may generate transgenic artefacts (I. Aifantis, J. Buer, H. von Boehmer, and O. Azogui. (1997) Essential role of the pre-T cell receptor in allelic exclusion of the T cell receptor /3 locus.
Immunity 7: 601). Since much of the data on the role of lck in TCR/3 allelic exclusion has been generated by this approach, it is possible that such transgenic artefacts have contributed to the inconsistencies concerning the role of lck in TCR/3 locus allelic exclusion. BACKGROUND During development in the thymus, cq3 lineage thymocytes proceed through two main developmental checkpoints: First, cells with a double coreceptor negative (DN) phenotype differentiate to double coreceptor positive (DP) cells; second, DP thymocytes develop into single coreceptor positive (SP) cells (reviewed in Godfrey and Zlotnik, 1993). Progression through either of these checkpoints is the result of a process of selection (reviewed in Robey and Fowlkes, 1994). Differentiation of DN to DP thymocytes depends on the expression of the pre-TCR, consisting of the TCR/3 chain, a surrogate TCRce polypeptide called pre-Tce, and components of the CD3 complex (Groettrup et al., 1993;Saint-Ruf et al., 1994). Differentiation of DP to SP cells depends on expression of the mature TCR, consisting of the TCR/3 chain, together with a mature TCRce chain forming the TCRcq3 heterodimer, and the CD3 complex (reviewed in von Boehmer, 1990). The TCR/3 genes rearrange during the DN stage (Godfrey et al., 1994) and expression of the pre-TCR selects for the approximetely 56% of rearranging cells that statisticially succeed in generating a productive TCR/3 gene (Mallick et al., 1993;Dudley et al., 1994). Selection by the pre-TCR, therefore, generates the repertoire of TCR/3 chains (reviewed in Levelt and Eichmann, 1995). The TCRce genes rearrange during the DP stage (Petrie et al., 1993). DP cells expressing a mature cq3TCR are selected, positively or negatively, by the ability of the cq3TCR to interact with MHC/peptide ligands, thus generating the self-tolerant, self-MHC-restricted repertoire of mature cq3 T cells (reviewed in Rothenberg, 1994).
Pre-TCR-dependent maturation from DN to DP thymocytes is associated with a complex cluster of differentiation events (reviewed in Tanaka et al., 1995;Zunicka-Pflticker and Lenardo, 1996). To discuss all of its components would go far beyond the scope of a minireview. I will therefore concentrate on the two main developmental responses most relevant to the scope of this paper: (1) The burst of cell divisions: Induction of proliferation generates a large number of DP cells from each original TCR/3-positive DN cell, thus giving rise to a diverse set of TCR/3 clones for further diversification by combination with TCRa chains (Falk et al., 1996). (2) The transition from TCR/3 to TCRc locus rearrangement: Once a DN cell has produced a TCR/3 chain, signals through the pre-TCR arrest further rearrangements in the TCR/3 locus (Levelt et al., 1995a), a process called allelic exclusion and believed to account for the fact that most T cells express only one of two possible TCR/3 loci (Malissen et al., 1992). Shutdown of rearrangement is accompanied by transient downregulation in the expression of the recombination activating genes RAG1 and RAG2 (Wilson et al., 1994), a response also induced through a pre-TCRmediated signal (Levelt et al., 1995a). This is followed by initiation of TCRce locus germline transcription and initiation of rearrangement of the TCRce genes (Capone et al., 1993), a late consequence of pre-TCR-induced differentiation (Levelt et al., 1995a).
The function of the pre-TCR depends on intracellular signal transmission as suggested by the involvement of the p21ras/MAP kinase pathway (Crompton et al., 1996). Moreover, the pre-TCR transmits its messages into the cell by signal transduction through the CD3 complex. This has first been suggested by experiments involving cross-linking of CD3e on immature thymocytes by anti-CD3 antibodies (Levelt et al., 1993a). It was found that essentially all developmental responses ascribed to the pre-TCR could be induced by anti-CD3 (reviewed in Levelt and Eichmann, 1995). However, and unexpectedly, the immature thymocytes responding to anti-CD3 were in a developmental stage prior to expression of the TCR/3 chain (Levelt et al., 1993a(Levelt et al., , 1993b). This suggested that CD3 components were expressed on thymocytes prior to the complete pre-TCR, but also raised doubts whether or not the antibody-induced signals were indeed a faithful reflection of the pre-TCR signals. Nevertheless, the involvement of CD3 signaling in the function of the pre-TCR has been further corroborated by the phenotype of mice genetically deficient in the CD3e6y components . Thymocyte development in these mice is arrested at exactly the same stage as in mice that are unable to rearrange their TCRfl genes owing to deficiency in RAG1 or RAG2 (Mombaerts et al., 1992a;Shinkai et al., 1992). Signaling through the CD3 complex critically depends on phosphorylation of immune-receptor tyrosine-activation motifs (ITAMs) (reviewed in Samelson and Klausner, 1992). ITAMs are associated with the cytoplasmic tails of CD3 components as well as of other receptors such as the lgce/ components of the B-cell receptor complex, and multiple forms of Fc receptors. The main family of protein tyrosine kinases (PTKs) involved in ITAM phosporylation in hemopoetic cells is the src family, consisting of nine members (reviewed in Cooper, 1990). As a general rule, members of the src family are in physical contact with cytoplasmic tails of members of the lymphocyte receptor complexes ((reviewed in Bolen et al., 1992;Chan et al., 1994;Chow and Veillette, 1995): The hemopoetic isoform of 59 fy" (f-ynT) is associated with members of the CD3 complex, and p56 lck (Ick) is associated with the coreceptors CD4 and CD8 on T cells. Similarly, p53/yn (lyn) and p55 b/k (blk), to a lesser extent also lck and fynT, are associated with the lgce/fi components of the B-cell receptor. Upon receptor ligation, these PTKs phosporylate the ITAMs, which then act as docking sites for SH2 domains, thus recruiting PTKs belonging to the ZAP-70/Syk family into the signaling process. Src-family PTKs show preferential tissue-specific expression (reviewed in Chow and Veillette, 1995): The aforementioned lck, fynT, lyn, and blk are predominantly expressed in lymphocytes, p58 c-fgr and p56/59 hck are predominantly expressed in cells of the myeloid lineage, and p60c-sr, p62c-yes, and perhaps also p60 yr are reported as ubiquitously expressed. Both lymphoid and myeloid src-PTKs have also been implicated in Fc-receptor and cytokine-receptor signal transduction.
Although the src-PTK fynT as well as ZAP-70 are intimately involved in CD3 signaling of mature thymocytes and T cells, targeted mutation of either PTK did not affect pre-TCR-dependent thymocyte differentiation (Appleby et al., 1992;Stein et al., 1992;Negishi et al., 1995). In contrast, genetic manipulations concerning lck had profound effects on several developmental responses ascribed to the pre-TCR. However, the defects observed in different genetic experiments were not always consistent with one another and with findings from other types of experiments. These inconsistancies give rise to the lck paradox and concem the two main components of pre-TCR-dependent differentiation: the burst of all divisions and the regulation of TCR gene rearrangements.

Lck INVOLVEMENT IN PRE-TCR-INDUCED PROLIFERATION
Mice genetically unable to synthetize a complete pre-TCR fall into two different categories with respect to their impairment in pre-TCR-induced proliferation. One group, consisting of scid mice (Schuler et al., 1986), RAG1/2-deficient mice (Mombaerts et al., 1992a;Shinkai et al., 1992), and CD3y6e-deficient mice , is virtually completely defective, that is, its thymic cellularity remains at 1-2 X 10 6 with almost no DP cells detected. The other group, consisting of TCR/3-deficient mice (Mombaerts et al., 1992b), CD3deficient mice (reviewed in Tanaka et al., 1995), and pre-Tce-deficient mice (Fehling et al., 1995), has a residual proliferative activity generating 10-20 106 thymocytes and about 10-20% of the normal number of DP cells. Some of the latter phenotypes have been explained by a positive feedback between thymic stroma and CD3positive cells, that is, 3/6 T cells (Shores et al., 1990(Shores et al., , 1991Ritter and Boyd, 1993). However, it is also possible that incomplete versions of the pre-TCR excert some residual signaling activity.
Two different genetic approaches toward the deletion of functional Ick have been taken: Targeted mutation to delete the functional lck gene (Molina et al., 1992), and introduction of a transgene encoding a dominant negativ lck mutant that competes with the product of the wildtype Ick gene . Paradoxically, mice carrying a dominant negative mutant lck gene fall into the category of strains with completely defective proliferation, whereas lck-deficient mice fall into the group with residual proliferative and differentiating activity.
Using the anti-CD3e cross-linking approach, we have previously examined the proliferation defects in newborn RAG1/lck single-and double-deficient mice (Levelt et al., 1995b). RAG1-deficient mice increase their thymic cellularity from 1-2 106 to 50 106 cells after injection of anti-CD3. Lck-deficient mice show no change in their thymocyte numbers (10 106) upon injection of anti-CD3. RAG1/Ick doubledeficient thymi increase from 1-2 106 to 10 106 cells after anti-CD3 treatment. These results suggest that the pre-TCR-dependent proliferative burst has two components; one appears to be lck-dependent, whereas the other may take place in the absence of lck. In RAG-deficient mice, CD3E stimulation in the presence of Ick initiates both components, whereas in the absence of lck, only the lck-independent component is stimulated. In lck single-deficient mice, the lck-independent component of pre-TCR-dependent proliferation takes place spontaneously, and anti-CD3 stimulation has no further effect. Although these results are consistent with one another, it remains obscure why a dominant negative mutant of lck apparently inhibits both the lck-dependent as well as the lck-independent component of pre-TCR-induced proliferation.

Lck INVOLVEMENT IN THE INDUCTION OF TCR/3 LOCUS ALLELIC EXCLUSION
Two experimental approaches have been used to assess competence of allelic exclusion of the TCR/3 locus in mice. The first is based on the expression of a rearranged TCR/3 transgene. In a normal mouse, this leads to the more or less complete inhibition of endogenous TCR/3 V to DJ rearrangements, so that a majority of the thymocytes and peripheral T cells express only the transgenic TCR/3 chain (Uematsu et al., 1988). The second approach to test competence of allelic exclusion is based on exposure of immature thymocytes to anti-CD3E. Since immature thymocytes express CD3e before they rearrange the TCR/3 chain (Levelt et al., 1993a(Levelt et al., , 1993b, CD3 mediated signals delivered at that stage are misinterpreted by the cell as indicating the presence of a complete pre-TCR, including a TCR/3 chain. As a result, the thymocyte arrests its rearrangement activity before it even started, resulting in a drastic reduction in the proportion of DP thymocytes expressing TCR/3 chains (Levelt et al., 1993a). Arrest of rearrangement concerns the V to DJ step, just as observed for TCR/3 transgenes (Levelt et al., 1995a).
Collectively, the data derived from TCR/3 transgenes and from anti-CD3e stimulation strongly support the notion that TCR/3 locus allelic exclusion is controlled by stimulation through the pre-TCR. A pivotal role for lck in this control is suggested by the results from mice carrying a dominant negative lck mutant transgene, in which inhibition of endogenous TCR/3 V to DJ rearrangements by a rearranged TCR/3 transgene is abrogated . Moreover, thymocytes from transgenic mice expressing a constitutively active mutant of lck showed a pronounced diminution of TCR/3 V to DJ rearrangements (Anderson et al., 1992). However, in contrast to these results, little or no defectiveness in the inhibition of endogenous TCR/3 V to DJ rearrangements by a rearranged TCR/3 transgene was seen in mice in which the Ick gene has been deleted by targeted mutation (Wallace et al., 1995). Upon quantitative analysis, the proportion of thymocytes with endogenous TCR/3 chains in TCR/3 transgenic lck-deficient mice did not exceed 10% of that seen in the absence of the TCR/3 transgene. Thus, more so than in pre-TCR-dependent proliferation, the in vivo effects of a dominant negative mutant of lck and of the deletion of the Ick gene are paradoxically discrepant.
An additional inconsistency becomes apparent on attempts to inhibit TCR/3 locus rearrangement in Ickdeficient mice by injection of anti-CD3e antibody. In multiple experimental attempts, no suppression of TCR/3 locus rearrangement could be induced (C.N. Levelt and K. Eichmann, unpublished observations). These findings tend to support the results obtained with the dominant negative lck mutant and contradict that of a TCR/3 transgene in lck-deficient mice.
The situation created by these conflicting data is truely paradoxical as no reasonably straightforward hypothesis can resolve all inconsistencies. For example, let us consider the hypothesis that the pre-TCR can signal along two different pathways, a major one involving CD3e and Ick, and a minor one utilizing CD3s c and other src PTKs, for example, fynT. If we assume that the dominant negative mutant lck successfully competes not only with lck, but also with fynT, the discrepancy between the drastic effects of the dominant negative mutant lck and the mild effects of lck deficiency could be readily explained. As far as allelic exclusion is concemed, the discrepancy between the effects of a TCR/3 transgene and of anti-CD3e stimulation in lck-deficient mice are resolved by this model if one considers that anti-CD3 stimulation triggers thymocytes before assembly of a complete pre-TCR, whereas a TCR/3 transgene exerts its effect through a complete pre-TCR. Before assembly of the pre-TCR, CD3 components are present as independent e6 and ey dimers, presumably not associated with CD3s c (Wiest et al., 1994). Thus, anti-CD3e cross-linking would activate the CD3e-lck pathway but not the CD3sC-fynT pathway, and therefore would be ineffective in lck-deficient mice.
Conversely, a complete pre-TCR is functionally associated with the CD3(chain (Vanoers et al., 1995) and therefore can induce allelic exclusion via fynT even in lck-deficient mice. So far, this hypothesis seems quite satisfactory. However, within this model, stimulation of proliferation of RAG1/lck doubledeficient thymocytes by anti-CD3e, which clearly takes place (Levelt et al., 1995b), should not be possible. The reader is encouraged to try alternative models; all of my own attempts have been unsuccessful.
It should be pointed out that even if the Ick paradox could be resolved, pre-TCR control alone seems insufficient to explain all present data on the regulation of allelic exclusion. In a RAG2 complementation approach, ES cells homozygously deleted for pre-Ta differentiated into thymocytes in which inhibition of endogeneous TCR/3 rearrangements by a TCR/3 transgene was fully maintained (Xu et al., 1996). This is strikingly reminiscent of IgH-chain allelic exclusion in B cells of mice deficient of the surrogate L chain of the pre-B-cell receptor: Although immature B cells in these mice were defective in allelic exclusion, mature B cells showed fully maintained allelic exclusion (L6ffert et al., 1996). Thus, allelic exclusion in either B or T cells may be protected by two safeguards, one provided by signaling through the preT/B-cell receptor complexes, the other by a downstream developmental checkpoint preventing the maturation of cells that have failed to become allelically excluded.