Subunit Composition of Pre-T Cell Receptor Complexes Expressed by Primary Thymocytes: CD3delta Is Physically Associated but Not Functionally Required

By Marc A. Berger, Vibhuti Davé, Michele R. Rhodes, Gayle C. Bosma, Melvin J. Bosma, Dietmar J. Kappes, and David L. Wiest

From the Division of Basic Sciences, Immunobiology Working Group, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

SUMMARY
Materials and Methods
Results
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
References


Summary

Maturation of immature CD4-CD8- (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre-T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-Talpha (pTalpha ). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pTalpha -containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pTalpha -containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pTalpha -TCR-beta heterodimer associated not only with CD3-gamma and -epsilon , as previously reported, but also with zeta  and delta . Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta -deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma , delta , epsilon , and zeta ), possibly because pTalpha itself possesses signaling capabilities.


Maturation of immature thymocytes from the CD4- CD8- (DN)1 to the CD4+CD8+ (DP) stage of development requires productive rearrangement of the gene segments encoding TCR-beta (1). The fidelity with which TCR-beta gene rearrangement has occurred is thought to be assessed using a surrogate receptor complex termed the pre-TCR, which is defined by a novel 33-kD pre-Talpha (pTalpha ) subunit (4). Upon productive rearrangement of TCR-beta , the pre-TCR complex transduces signals through CD3 (and possibly also pTalpha ) that direct allelic exclusion at the TCR-beta locus and promote maturation of DN thymocytes to the DP stage (5); however, the molecular details of how pre-TCR complexes discriminate between aberrant and productive beta  gene rearrangements remain unclear.

Progress towards a deeper understanding of pre-TCR complex function is hampered by the absence of a precise description of pre-TCR subunit composition. At present, the pre-TCR complex is thought to consist of a disulfide-linked pTalpha -TCR-beta (pTalpha -beta ) heterodimer associated with an inadequately defined complement of signaling molecules (5). Indirect support for the presence of particular TCR- CD3 chains within the pre-TCR complex comes from gene-targeted mice, which reveal that the DN to DP transition is attenuated by elimination of TCR-beta , pTalpha , or TCR-zeta alone, or by simultaneous elimination of CD3-gamma /delta /epsilon (2, 6). Direct biochemical analysis of pre-TCR composition has yielded only incomplete and contradictory results, particularly regarding the CD3-delta and TCR-zeta subunits, possibly because of the idiosyncrasies of thymic lymphoma lines used in these analyses (4, 9). These discrepancies could be resolved, if it were possible to analyze pre-TCR structure using normal primary thymocytes; however, thus far, attempts to do so have been unsuccessful (4, 12).

This study comprises a rigorous biochemical analysis of surface TCR expression that clarifies these issues. We provide the first demonstration that pTalpha -containing pre-TCR complexes are actually expressed at low levels on the surface of normal thymocytes in vivo; moreover, we have defined their subunit composition. They consist of a disulfide-linked pTalpha -beta heterodimer associated not only with CD3-gamma /epsilon as was previously thought, but also with TCR-zeta and CD3-delta . In addition, we demonstrate that pre-TCR function is not attenuated in CD3-delta -deficient (CD3-delta 0) mice (13). Thus, while CD3-delta is a pre-TCR component, it is not required for pre-TCR function.


Materials and Methods

Animals. Mice lacking TCR-alpha expression (TCR-alpha 0) due to gene targeting (2, 14) were obtained from The Jackson Laboratory (Bar Harbor, ME) and then maintained in our colony. CD3-delta 0 mice were generated as previously described (13). Production of TCR-alpha 0CD3-delta 0, and TCR-alpha 0CD3-delta + mice was achieved by intercrossing the F1 progeny of a TCR-alpha 0 × CD3-delta 0 mating and then screening F2 mice by Southern blot as previously described (15).

Cell Lines and Antibodies. The DP alpha /beta -TCR+ thymic lymphoma VL3-3M2 (obtained from Dr. C. Guidos, Hospital for Sick Children, Toronto, Canada) and a spontaneous thymic lymphoma from SCID mice, SL-12 (TCR-; reference 16) were maintained in RPMI supplemented as previously described (17). The following mAbs were used: anti-TCR-beta (H57-597); anti- CD3-gamma /epsilon (7D6); and anti-TCR-alpha (H28-710). The following polyclonal rabbit Abs were used: anti-TCR-zeta (551; reference 17); anti-CD3-delta (R9; gift of Dr. Lawrence Samelson, National Institutes of Health [NIH], Bethesda, MD); and anti-pTalpha . The anti-pTalpha Ab was raised against a GST (glutathione-S-transferase) fusion protein encompassing the cytoplasmic domain of pTalpha as previously described (17).

Plasmid Construction. The pTalpha cDNA was cloned by performing reverse transcriptase-PCR on total RNA from day 15 fetal thymocytes (gift of Dr. Paul Love, National Institute of Child Health and Human Development [NICHD], Bethesda, MD) using standard methodology (15). First, strand cDNA was synthesized using the Superscript Preamplification System (GIBCO BRL, Bethesda, MD), and then the pTalpha cDNA was amplified by PCR (MJ Research, Inc., Watertown, MA) using primers flanking the coding region. The resultant fragment was cloned into pCR2.1 (Invitrogen, San Diego, CA) and verified by automated sequencing in our facility. The pTalpha cytoplasmic tail-GST fusion protein was generated by PCR amplification of the pTalpha cDNA using primers that appended SmaI and EcoRI linkers onto the cytoplasmic tail fragment. The fragment was ligated into pCR2.1, excised using SmaI and EcoRI, and directionally cloned in frame into pGEX-2T (Pharmacia Biotech Inc., Piscataway, NJ). After transformation into BL21pLysS cells, expression of the pTalpha cytoplasmic tail-GST (glutathione-S-transferase) fusion protein was induced, after which it was purified from bacterial extracts using glutathione-Sepharose (Pharmacia Biotech Inc.) according to the manufacturer's recommendations (18).

A 1.1-kb XhoI-XbaI cDNA fragment encoding the TCR-beta subunit of the 2B4 TCR (pCDMB-2B4-beta ; provided by Dr. Juan Bonifacino, NICHD, Bethesda, MD) was subcloned into the pXS expression vector (gift of Dr. Juan Bonifacino) using standard methodology (15).

Transfection. pXS-2B4-beta and the neomycin resistance plasmid pFneo were linearized with HindIII and EcoRI, respectively, and then transfected into SL-12 SCID thymic lymphoma cells by electroporation (300 V, 400 µFa) using a BTX electroporation unit (BTX, Inc., San Diego, CA). Clones that were resistant to G418 (Boehringer Mannheim, Indianapolis, IN) were analyzed for TCR-beta expression by flow cytometry.

Biotin Labeling, Immunoprecipitation, and Electrophoresis. Biotin surface labeling, after which cell viability was consistently >98%, was performed as previously described (17, 18). Cell lysis, immunoprecipitation, and electrophoresis were as previously described (17, 18). Electrophoretically separated surface-biotinylated proteins were transferred to membranes and visualized with horseradish peroxidase-conjugated streptavidin (HRP-Av; Southern Biotechnology Associates, Inc., Birmingham, AL) as previously described (17).

Recapture Assay. Recapture assays were performed as previously described (17) using the anti-pTalpha Ab. The resultant immune complexes were resolved either on two- or one-dimensional nonreducing SDS-PAGE gels, after which the surface-labeled proteins were visualized as above.

Flow Cytometry. Flow cytometry was performed as previously described (18).


Results

Expression of pTalpha -containing Pre-TCR Complexes on the Surface of Primary Thymocytes.

It is well established that immature thymocytes express surface TCR complexes containing TCR-beta without TCR-alpha ; however, it remains unclear whether these complexes comprise TCR-beta homodimers or, alternatively, heterodimers of TCR-beta and pTalpha , the hallmark of the pre-TCR complex (4, 12, 19). This issue remains unresolved because previous attempts to demonstrate expression of pTalpha -containing pre-TCR complexes on the surface of primary thymocytes have been unsuccessful (4, 12, 19). Consequently, we wished to determine if pTalpha -containing pre-TCR complexes were actually expressed on the surface of primary thymocytes. Thymocytes from TCR-alpha 0 mice were used because they lack alpha /beta -TCR complexes which might otherwise complicate analysis of pre-TCR structure, yet they still exhibit normal pre-TCR function (2, 14). Detergent extracts of surface biotin-labeled thymocytes were immunoprecipitated either with control hamster IgG or with an anti-TCR-beta mAb after which the resultant immune complexes were resolved by two-dimensional nonreducing × reducing (2D NR×R) SDS-PAGE (Fig. 1). The anti-TCR-beta mAb immunoprecipitation contained associated CD3-gamma /epsilon heterodimers as well as disulfide-linked pTalpha -beta heterodimers. Curiously, neither surface-labeled TCR-zeta nor CD3-delta was visible. Thus, primary thymocytes do indeed express surface pre-TCR complexes in which pTalpha -beta and CD3-gamma /epsilon heterodimers are evident.


Fig. 1. Expression of pTalpha -containing pre-TCR complexes on the surface of primary thymocytes. Surface biotin-labeled thymocytes from TCR-alpha 0 mice were solubilized in Brij 96, immunoprecipitated with anti-TCR-beta Ab (H57-597; right), or control hamster IgG (left) and resolved on 2D NR×R SDS-PAGE gels. Surface-labeled proteins were visualized by HRP-Av and chemiluminescence. Migration positions of individual subunits are indicated.
[View Larger Version of this Image (44K GIF file)]

The apparent absence of CD3-delta and TCR-zeta from pre-TCR complexes does not exclude them as potential pre-TCR components, as subunit visibility in this assay is dependent upon accessibility during surface labeling. Accordingly, proteins that did not label efficiently would be missed, providing an inaccurate view of pre-TCR subunit composition. In particular, surface-labeled TCR-zeta molecules were conspicuously absent from alpha /beta -TCR complexes expressed by the VL3-3M2 thymic lymphoma (Fig. 2 A), despite the fact that these alpha /beta -TCR complexes do contain TCR-zeta (data not shown). Likewise, our inability to identify labeled CD3-delta subunits in the pre-TCRs expressed by primary thymocytes may result from inefficient biotin-labeling of CD3-delta and/or from comigration with a contaminating protein that obscures CD3-delta .



Fig. 2. pTalpha -beta heterodimers are specifically recaptured by Abs reactive with the cytoplasmic domain of pTalpha . (A) Recapture assay. Recapture assay is schematized in the top panel. (Bottom) Brij 96 detergent extracts of surface-labeled VL3-3M2 and SL-12beta .12 cells were immunoprecipitated with anti-TCR-beta . After elution by boiling in SDS, the anti- TCR-beta immune complexes were quenched with NP-40 and split into three equal parts which were either: (a) held on ice; (b) immunoprecipitated with anti-pTalpha Ab; or (c) immunoprecipitated with control rabbit IgG. The samples were then resolved on 2D NR×R SDS-PAGE gels, after which the surface-labeled proteins were visualized with HRP-Av and chemiluminescence. (B) Anti-pTalpha Ab specifically recaptured pTalpha -beta heterodimers from detergent extracts of surface-labeled pre-TCR+ (SL-12beta .12) but not from alpha /beta -TCR+ (VL3-3M2) cells. A recapture assay was performed as above except that the samples were resolved on one-dimensional SDS-PAGE under nonreducing conditions.
[View Larger Versions of these Images (50 + 36K GIF file)]

Recapture Assay for Analyzing Pre-TCR Composition.

To circumvent the limitations outlined above, we used a coimmunoprecipitation strategy to rigorously determine the subunit composition of the pre-TCR complex. A subunit was deemed part of the pre-TCR complex if Abs reactive with that subunit coprecipitated surface-labeled pTalpha , the hallmark of the pre-TCR complex. Coimmunoprecipitated pTalpha molecules were identified using the recapture assay, which occurs in three phases (Fig. 2 A, top): (a) Ab reactive with TCR subunits were used to coimmunoprecipitate pTalpha molecules (if associated) from cell extracts made with detergents selected for their ability to maintain the noncovalent interactions between subunits; (b) noncovalent interactions were disrupted by boiling the immune complex in 1% SDS; and (c) coimmunoprecipitated pTalpha -beta heterodimers were identified by using anti-pTalpha Abs to recapture them from among the SDS-solubilized proteins. To verify that the anti-pTalpha Abs specifically recaptured pTalpha -beta heterodimers, the recapture assay was performed on anti- TCR-beta immunoprecipitates from both alpha /beta -TCR-expressing (VL3-3M2) and pre-TCR-expressing (SL-12beta .12) thymic lymphoma cells (Fig. 2 A, bottom). Parallel anti- TCR-beta immunoprecipitates were resolved on 2D NR×R SDS-PAGE gels to reveal the protein composition of the original immunoprecipitation. In the anti-TCR-beta immunoprecipitations from pre-TCR+ cells, heterodimers of pTalpha -beta and CD3-gamma /epsilon were evident, whereas in those from alpha /beta -TCR+ cells, we observed heterodimers of TCR-alpha and -beta in association with CD3-gamma /delta /epsilon , but not pTalpha (Fig. 2 A, bottom left). The anti-pTalpha Ab recaptured pTalpha -beta heterodimers from pre-TCR-expressing SL-12beta .12 cells, but not from cells expressing only alpha /beta -TCR complexes (VL3-3M2; Fig. 2 A, bottom center), demonstrating that the recapture assay specifically identifies pTalpha -beta heterodimers. This was also evident in a similar experiment in which the samples were resolved on one-dimensional gels in which the interchain disulfide bonds between pTalpha and TCR-beta were maintained (Fig. 2 B).

Composition of Pre-TCR Complexes Expressed on the Surface of Primary Thymocytes.

Having established that the recapture assay was specific, we next wished to evaluate subunit composition of pre-TCR complexes expressed on the surface of primary thymocytes. Abs reactive with TCR-beta , CD3-gamma /epsilon , TCR-zeta , and CD3-delta coprecipitated pTalpha -beta heterodimers from detergent extracts of surface-labeled thymocytes (Fig. 3 A). Thus, according to the above definition, our analysis demonstrates that the pre-TCR complexes expressed by primary thymocytes in vivo comprise pTalpha -beta heterodimers associated with CD3-gamma /delta /epsilon , and TCR-zeta .



Fig. 3. Subunit composition of pre-TCR complexes expressed by primary explanted thymocytes. (A) Digitonin extracts of biotin-labeled TCR-alpha 0 thymocytes were immunoprecipitated using Abs reactive with the following subunits: TCR-beta (H57-597), CD3-gamma /epsilon (7D6), CD3-delta (R9) or TCR-zeta (551). SDS eluates of the resultant immune complexes were either resolved directly on SDS-PAGE gels under nonreducing conditions (Total) or after neutralization and immunoprecipitation with either anti-pTalpha (pTalpha ) or control rabbit IgG (rIgG). Control anti-TCR-alpha Ab did not coprecipitate pTalpha -beta heterodimers from TCR-alpha 0 thymocytes (data not shown). (B) CD3-gamma /delta /epsilon and TCR-zeta are all associated with pTalpha -beta heterodimers within the same pre-TCR complex. Digitonin extracts of biotin-labeled TCR-alpha 0 thymocytes were either immunoprecipitated directly with anti-TCR-beta or after the detergent extracts had been depleted of CD3-delta , -gamma /epsilon , or -zeta with Abs reactive with those proteins. Surface-labeled proteins were visualized by HRP-Av and chemiluminescence.
[View Larger Versions of these Images (30 + 34K GIF file)]

To determine if CD3-gamma /epsilon , CD3-delta , and TCR-zeta were all associated with pTalpha -beta within the same complex, we performed a series of sequential immunoprecipitations to see if all of the pTalpha -beta heterodimers could be depleted from the detergent extracts by exhaustive preclearing using Abs reactive with those proteins (Fig. 3 B). Indeed, virtually all of the pTalpha -beta heterodimers expressed on the surface of TCR-alpha 0 thymocytes could be precleared using Abs reactive with either CD3-gamma /epsilon or -delta . In contrast, anti-TCR-zeta Ab was able to preclear only ~50% of the pTalpha -beta heterodimers, indicating that either zeta is associated with only half of the pre-TCR complexes expressed by primary thymocytes or, alternatively, that zeta  association with the pre-TCR is weak. We favor the latter interpretation because zeta  association with the pre-TCR complex, unlike that of the other pre-TCR subunits, was easily disrupted by solubilization in harsh detergents (data not shown). Taken together, these data demonstrate that all pre-TCR complexes expressed on the surface of primary thymocytes in vivo contain pTalpha -beta heterodimers associated with CD3-gamma /delta /epsilon , and of these at least half are also associated with TCR-zeta .

Loss of CD3-delta Does Not Affect Development of Thymocytes from the DN to the DP Stage.

Since we had demonstrated CD3-delta was a component of the pre-TCR complex, it was important to determine if delta  were critical to pre-TCR function. Thus, we analyzed TCR-alpha 0CD3-delta 0 mice to determine if the loss of the CD3-delta signaling component affected pre-TCR expression as well as two manifestations of pre-TCR function, thymic cellularity and maturation of DN thymocytes to the DP stage (5, 6). Thymocytes from TCR-alpha 0CD3-delta 0 mice expressed surface pre-TCRs comprising pTalpha -beta heterodimers associated with CD3-gamma /epsilon and TCR-zeta , indicating that CD3-delta deficiency does not prevent assembly and surface expression of the remaining pre-TCR subunits (Fig. 4, A and B, and data not shown). Likewise, delta  deficiency did not attenuate pre-TCR function. Flow cytometric analysis of thymocytes from TCR-alpha 0CD3-delta + and TCR-alpha 0CD3-delta 0 mice revealed that each contained 96% DP thymocytes (Fig. 4 B). Furthermore, total thymic cellularity in TCR-alpha 0CD3-delta 0 mice was equivalent to that in TCR-alpha 0CD3-delta + mice (2.80 × 108 versus 3.31 × 108, respectively; P <0.01). Thus, the absence of CD3-delta from the pre-TCR complex does not adversely affect the progression of immature thymocytes from the DN to the DP stage of development, demonstrating that CD3-delta is not necessary for pre-TCR function.



Fig. 4. The CD3-delta deficiency does not attenuate the DN to the DP transition. (A) The absence of CD3-delta does not affect pre-TCR expression. Digitonin extracts of surface biotin- labeled TCR-alpha 0CD3-delta 0 thymocytes were immunoprecipitated with anti- CD3-gamma /epsilon or anti-CD3-delta Abs after which the resultant immune complexes were analyzed by recapture assay. Immune complexes were resolved by one-dimensional SDS-PAGE under nonreducing conditions. The absence of any recaptured pTalpha -beta heterodimers in the anti-CD3-delta immunoprecipitations demonstrates the specificity of the anti-CD3-delta Ab. (B) Thymocytes from both TCR-alpha 0CD3-delta + and TCR-alpha 0CD3-delta 0 mice have the same percentage of CD4+CD8+ cells (top) and express similar levels of TCR-beta on the cell surface (bottom; thick line), as measured by flow cytometry with fluorochrome conjugated mAbs. The thin line represents control staining with anti-human CD3-epsilon Ab. The mean number of cells per thymus for each strain is indicated at the bottom of the figure.
[View Larger Versions of these Images (33 + 20K GIF file)]


Discussion

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 pTalpha -containing pre-TCR complexes on the cell surface and we have elucidated their subunit composition. They consist of pTalpha -beta heterodimers associated not only with CD3-gamma /epsilon as was previously thought, but also with TCR-zeta and CD3-delta subunits. Finally, we found that despite being a component of the pre-TCR, CD3-delta is dispensable for the biological role of the pre-TCR complex.

Before our study, it was unclear whether the TCR-beta complexes (without TCR-alpha ) that were expressed on developing thymocytes contained TCR-beta homodimers or, alternatively, disulfide-linked pTalpha -beta heterodimers, the hallmark of the pre-TCR complex (4, 12, 19). Moreover, because of previous failures to demonstrate surface expression of pTalpha in vivo, it was proposed that pre-TCR complexes evaluate TCR-beta 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-beta transgene lacking the variable domain is able to allelically exclude endogenous TCR-beta rearrangement and promote the DN to DP transition, both hallmarks of pre-TCR function (20). Moreover, analysis of the efficiency with which TCR-beta 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+ or CD8+ stage (21). Thus, if pre-TCR complexes do evaluate the fidelity of TCR-beta gene rearrangement by interacting with an extracellular ligand, then that ligand does not absolutely require the TCR-beta variable domain, nor is it present in limiting quantities. While we provide the first compelling demonstration that primary thymocytes express pTalpha -containing pre-TCR complexes on the cell surface (Figs. 1 and 3), 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 pTalpha , TCR-beta , CD3-gamma , and CD3-epsilon ; however, there were conflicting data regarding CD3-delta and TCR-zeta (4, 9). CD3-delta was found to be a component of pre-TCR complexes in some tumor lines, but not in others (9, 10). Furthermore, TCR-zeta 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-zeta with the pre-TCR complex could be more easily disrupted by lysis in harsh detergents than that of CD3-gamma /delta /epsilon (data not shown). This was not true for zeta  association with the alpha /beta -TCR complex (data not shown). Finally, while we have elucidated the subunit composition of pre-TCRs expressed by TCR-alpha 0 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-gamma /delta /epsilon and TCR-zeta subunits. While both receptors carry the same array (gamma , delta , epsilon , and zeta ), the requirements of these receptors for individual signaling subunits differ (Fig. 5), raising the fundamental question of whether the different subunits subserve redundant or unique roles in receptor function. If CD3-gamma /delta /epsilon and TCR-zeta subunits are redundant and function to amplify signals, then it is surprising that the pre-TCR complex can tolerate the loss of CD3-delta , given that surface expression levels of the pre-TCR complex are so low (Figs. 1 and 4). In that regard, the pre-TCR may be able to tolerate loss of CD3-delta because pre-TCR signals need not be as quantitatively intense or because pre-TCRs have a lower signaling threshold (than alpha /beta -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 alpha /beta -TCR on DP thymocytes by sequestering p56lck protein tyrosine kinase (22). It is also possible that the individual CD3-gamma /delta /epsilon and TCR-zeta 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-delta because in the absence of CD3-delta the pTalpha subunit itself is also able to function as a signaling subunit. Murine pTalpha 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 pTalpha homologues (5). Recently, the role of the cytoplasmic tail of pTalpha was tested by reconstitution of pTalpha -deficient mice with a pTalpha transgene lacking the cytoplasmic tail (24). While tailless pTalpha compensated for pTalpha deficiency, it did so only partially, leaving open the possibility that the cytoplasmic tail of pTalpha 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-delta subunit. In that regard it would be informative to analyze the function of pre-TCR complexes in CD3-delta 0 thymocytes expressing tailless pTalpha molecules.


Fig. 5. Schematic of the role of the pre-TCR in thymocyte development. After beta  gene rearrangement, immature thymocytes express surface pre-TCR complexes comprising pTalpha -beta heterodimers associated with CD3-gamma /delta /epsilon and TCR-zeta signaling subunits. Pre-TCR complexes are thought to regulate development of thymocytes from the DN to the DP stage by evaluating the status of the TCR-beta protein product after beta  gene rearrangement. The molecular interactions underlying this evaluation process are currently unclear; however, development is blocked by elimination of pTalpha , TCR-zeta or CD3-gamma /delta /epsilon , but not CD3-delta . Curiously, elimination of CD3-delta does impair the ability of the alpha /beta -TCR to signal maturation of DP thymocytes to the CD4+ or CD8+ stage. Thus, while both pre-TCR and TCR complexes possess the same array of signaling subunits, they exhibit differential dependence on the individual components.
[View Larger Version of this Image (19K GIF file)]

In summary, this study not only provides the first demonstration that pTalpha -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-delta subunit, but, importantly, did not require CD3-delta to fulfill their biological role in regulating early thymocyte development. Curiously, loss of CD3-delta does interfere with function of the alpha /beta -TCR complex (13), illustrating that while both receptors possess the same array of TCR signaling components (gamma , delta , epsilon , and zeta ), their dependence on individual subunits differs. A deeper understanding of how individual signaling subunits function in the pre-TCR and alpha /beta -TCR complexes must await the generation of new strains of transgenic mice bearing signaling subunits with mutated immunoreceptor tyrosine-based activation motifs.


Footnotes

Address correspondence to Marc A. Berger, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Phone: 215-728-2968; FAX: 215-728-2412; E-mail: ma_berger{at}fccc.edu

Received for publication 14 July 1997 and in revised form 20 August 1997.

1   Abbreviations used in this paper: 2D NR×R, two-dimensional nonreducing × reducing; CD3-delta 0, CD3-delta -deficient; DN, CD4-CD8- double negative; DP, CD4+CD8+ double positive; HRP-Av, horseradish peroxidase-conjugated streptavindin; pTalpha , pre-Talpha ; pTalpha -beta , pTalpha -TCR-beta ; TCR-alpha 0, TCR-alpha -deficient.

We wish to thank Dr. Paul Love for assistance in cloning the murine pTalpha cDNA, Dr. Lawrence E. Samelson for anti-CD3-delta Ab, and Drs. Erica Golemis, Jennifer Punt, Alfred Singer, and Lisa Spain for critically reading the manuscript.

This work was supported by National Institutes of Health grant CA-73656-01 and by institutional funds.


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