Progress in understanding the molecular details of how the pre-TCR regulates early thymocyte development has been hampered by the absence of a precise description of pre-TCR subunit composition. This report addresses this problem. We provide the first demonstration that primary thymocytes express pTα-containing pre-TCR complexes on the cell surface and we have elucidated their subunit composition. They consist of pTα–β heterodimers associated not only with CD3-γ/ε as was previously thought, but also with TCR-ζ and CD3-δ subunits. Finally, we found that despite being a component of the pre-TCR, CD3-δ is dispensable for the biological role of the pre-TCR complex.
Before our study, it was unclear whether the TCR-β complexes (without TCR-α) that were expressed on developing thymocytes contained TCR-β homodimers or, alternatively, disulfide-linked pTα–β heterodimers, the hallmark of the pre-TCR complex (4
). Moreover, because of previous failures to demonstrate surface expression of pTα in vivo, it was proposed that pre-TCR complexes evaluate TCR-β protein structure not through interactions with an extracellular ligand at the cell surface, but rather from the cell interior (5
), possibly during subunit assembly within the endoplasmic reticulum. Consistent with this viewpoint, a TCR-β transgene lacking the variable domain is able to allelically exclude endogenous TCR-β rearrangement and promote the DN to DP transition, both hallmarks of pre-TCR function (20
). Moreover, analysis of the efficiency with which TCR-β transgenic DN precursors differentiate to the DP stage suggests that this transition is not constrained by a limiting number of intrathymic “niches” or extracellular ligands, as is true of the antigen-driven selection events that promote maturation of DP thymocytes to the CD4+
). Thus, if pre-TCR complexes do evaluate the fidelity of TCR-β gene rearrangement by interacting with an extracellular ligand, then that ligand does not absolutely require the TCR-β variable domain, nor is it present in limiting quantities. While we provide the first compelling demonstration that primary thymocytes express pTα-containing pre-TCR complexes on the cell surface (Figs. and ), this does not rule out the possibility that pre-TCR complexes might function from the cell interior.
Previous analyses of pre-TCR structure were consistent in indicating that pre-TCRs contained pTα, TCR-β, CD3-γ, and CD3-ε; however, there were conflicting data regarding CD3-δ and TCR-ζ (4
). CD3-δ was found to be a component of pre-TCR complexes in some tumor lines, but not in others (9
). Furthermore, TCR-ζ association with the pre-TCR has been implicated by functional criteria but not by physical association (11
). These discrepancies might result either from idiosyncrasies of the lymphoma cell lines used or from the experimental conditions (9
). In particular, the detergent used in cell lysis can markedly affect association of individual subunits with the pre-TCR. We found that association of TCR-ζ with the pre-TCR complex could be more easily disrupted by lysis in harsh detergents than that of CD3-γ/δ/ε (data not shown). This was not true for ζ association with the α/β-TCR complex (data not shown). Finally, while we have elucidated the subunit composition of pre-TCRs expressed by TCR-α0
thymocytes, this population consists primarily of DP thymocytes and so it remains possible that distinct subpopulations of DN thymocytes might express alternative forms of the pre-TCR complex. Experiments are currently in progress to investigate this possibility.
The ability of TCR and pre-TCR complexes to transduce signals resides in their invariant CD3-γ/δ/ε and TCR-ζ subunits. While both receptors carry the same array (γ, δ, ε, and ζ), the requirements of these receptors for individual signaling subunits differ (Fig. ), raising the fundamental question of whether the different subunits subserve redundant or unique roles in receptor function. If CD3-γ/δ/ε and TCR-ζ subunits are redundant and function to amplify signals, then it is surprising that the pre-TCR complex can tolerate the loss of CD3-δ, given that surface expression levels of the pre-TCR complex are so low (Figs. and ). In that regard, the pre-TCR may be able to tolerate loss of CD3-δ because pre-TCR signals need not be as quantitatively intense or because pre-TCRs have a lower signaling threshold (than α/β-TCR complexes). In support of the latter possibility, pre-TCR complexes function before expression of surface molecules that can be inhibitory, such as CD4, which we have shown can decrease signaling competence of the α/β-TCR on DP thymocytes by sequestering p56lck
protein tyrosine kinase (22
). It is also possible that the individual CD3-γ/δ/ε and TCR-ζ signaling subunits perform unique functions. Consistent with this hypothesis, it has been shown that immunoreceptor tyrosine-based activation motifs of different signaling subunits are able to interact with different cytoplasmic signaling effector molecules and induce phosphorylation of different substrates (23
). Finally, it is possible that pre-TCR complexes can tolerate loss of CD3-δ because in the absence of CD3-δ the pTα subunit itself is also able to function as a signaling subunit. Murine pTα has a cytoplasmic tail of ~30 amino acids which contains consensus motifs for phosphorylation by protein kinase C and for docking of SH3 domain containing proteins (12
); however, the functional importance of these motifs is unclear as there is little sequence conservation between the cytoplasmic tails of the murine and human pTα homologues (5
). Recently, the role of the cytoplasmic tail of pTα was tested by reconstitution of pTα-deficient mice with a pTα transgene lacking the cytoplasmic tail (24
). While tailless pTα compensated for pTα deficiency, it did so only partially, leaving open the possibility that the cytoplasmic tail of pTα does function as a signaling domain within the pre-TCR complex and might underlie the pre-TCR's ability to tolerate loss of the CD3-δ subunit. In that regard it would be informative to analyze the function of pre-TCR complexes in CD3-δ0
thymocytes expressing tailless pTα molecules.
Figure 5 Schematic of the role of the pre-TCR in thymocyte development. After β gene rearrangement, immature thymocytes express surface pre-TCR complexes comprising pTα–β heterodimers associated with CD3-γ/δ/ε (more ...)
In summary, this study not only provides the first demonstration that pTα-containing pre-TCRs are expressed on the surface of primary thymocytes in vivo, but also provides a precise description of pre-TCR subunit composition. Contrary to previous reports, we found that pre-TCR complexes contained the CD3-δ subunit, but, importantly, did not require CD3-δ to fulfill their biological role in regulating early thymocyte development. Curiously, loss of CD3-δ does interfere with function of the α/β-TCR complex (13
), illustrating that while both receptors possess the same array of TCR signaling components (γ, δ, ε, and ζ), their dependence on individual subunits differs. A deeper understanding of how individual signaling subunits function in the pre-TCR and α/β-TCR complexes must await the generation of new strains of transgenic mice bearing signaling subunits with mutated immunoreceptor tyrosine-based activation motifs.