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BRIEF DEFINITIVE REPORT |
T cell receptor explains different phenotypes of CD3 and CD3 immunodeficiencies
CORRESPONDENCE Wolfgang Schamel: schamel{at}immunbio.mpg.de
 Top ABSTRACT RESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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T cell receptor for antigen (TCR) comprises the clonotypic TCR, the CD3 (CD3
and/or CD3), and the 
dimers. T cells do not develop in CD3-deficient mice, whereas human patients lacking CD3 have abundant peripheral blood T cells expressing high TCR levels. In an attempt to identify the molecular basis for these discordant phenotypes, we determined the stoichiometries of mouse and human TCRs using blue native polyacrylamide gel electrophoresis and anti-TCR–specific antibodies. The TCR isolated in digitonin from primary and cultured human T cells includes CD3, with a TCRCD322 stoichiometry. In CD3-deficient patients, this may allow substitution of CD3 by the CD3 chain and thereby support T cell development. In contrast, the mouse TCR does not incorporate CD3 and has a TCRCD3222 stoichiometry. CD3-deficient mice exhibit a block in T cell development. A human, but not a mouse, CD3 transgene rescues T cell development in mice lacking both mouse CD3 and CD3 chains. This suggests important structural and/or functional differences between human and mouse CD3 chains during T cell development. Collectively, our results indicate that the different T cell phenotypes between CD3-deficient humans and mice can be explained by differences in their TCR composition. The
TCR is a multimeric complex consisting of a clonotypic TCR heterodimer, the CD3 and/or CD3 dimer, and the dimer. Because TCR signaling regulates the commitment of double-negative (CD4–CD8–) cells to the T cell lineage and is required for their subsequent differentiation into mature T cells, the development of T cells depends on the expression of the TCR. Indeed, neither CD3- nor CD3-deficient mice have T cells (1–3). Although the overall structure and function of the TCRs in mice and humans are quite similar, ablation of the highly related CD3 and CD3 subunits has markedly different effects in these two species. Hence, T cell development is severely impaired in CD3-deficient mice but not in their human counterparts (3, 4). Conversely, CD3 deficiency results in a block in human, but not mouse, T cell development (5, 6).
In contrast to the TCR, the 
ß TCR has been studied extensively and its minimal stoichiometry is proposed to be TCRßCD322 in mice and humans (7, 8). Unlike the mouse ß TCR, mouse TCR does not incorporate CD3 even if this subunit is expressed intracellularly (9, 10), explaining why T cells develop normally in CD3-deficient mice (6). Interestingly, the composition of the mouse TCR complex changes in activated cells: a differentially glycosylated form of CD3 becomes incorporated into the receptor (9) and can be substituted by the FcR chain (10).
Contradictory findings have been reported concerning human TCR stoichiometry. Primary human T cells were found to incorporate little or no CD3 into their surface TCRs (10). In contrast, human T cell clones and lines were found to possess both CD3 and CD3 dimers (11, 12). In light of the reported activation-induced changes in mouse TCR composition, it is possible that although CD3 is not incorporated into TCRs of naive human T cells, this chain becomes part of the receptor on T cell clones that have undergone activation and expansion.
In this study, we use blue native PAGE (BN-PAGE) and specific anti-CD3 antibodies to determine the stoichiometries of human and mouse TCRs. These data are complemented by studies on the human CD3 (hCD3) deficiency phenotype, as well as those of CD3-deficient mice supplemented with mouse or hCD3 transgenes. In conclusion, we show that there are differences in the stoichiometries and, thus, subunit requirements for the assembly of mouse and human TCRs.
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RESULTS AND DISCUSSION |
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TopABSTRACTRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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T cells with high levels of TCR are present in CD3-deficient patients knockout (CD3–/–) mice, T cell development is blocked (3); however, this is not the case in CD3-deficient humans. We have studied four CD3-deficient patients (13, 14), including one >20 yr old, and consistently found that T cells are present in their peripheral blood (Fig. 1 A).
As is the case with ß T cells, the number of T cells in these patients was at or just below the lower limit (P5) of healthy CD3-sufficient controls. In the absence of CD3, CD3 expression by ß T cells is reduced to 
20% of that of healthy controls (4). However, when we analyzed T cells from these patients by flow cytometry using anti-CD3 antibodies, we found that the amount of TCR per T cell was only reduced to 30–55% of healthy individuals, depending on the antibody used (Fig. 1, B and C). These data show that hCD3 can compensate, at least partially, for the lack of hCD3 in assembly and surface transport of the human TCR. In fact, in the absence of CD3, these processes appear to occur more efficiently in T cells than in ß T cells. As a consequence, T cells can develop in CD3-deficient patients, indicating that hCD3 can functionally replace hCD3 to promote T cell development. In conclusion, the human TCR can assemble and signal for selection efficiently without hCD3.
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TCR includes CD3 T cell development in mice and humans could reflect distinct TCR subunit composition in these species. To clarify the composition of the human TCR, we used established human T cell clones as well as primary T cells. Because our T cell clones contained 5% residual irradiated feeder cells expressing the ß TCR, we depleted ß TCRs after cell lysis by immunopurification with anti-TCRß antibodies (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20070782/DC1). This was done for all experiments in which T cell clones were used. In the first experiment, we lysed human ß as well as T cell clones and immunopurified the TCRs with anti- antibodies. After nonreducing SDS-PAGE, purified proteins were detected using anti-CD3 and anti- antibodies (Fig. 2 A).
The ß TCR of the ß T cell line Jurkat and the ß clones ßB6 and ßPA (lanes 2–4), as well as the TCR of clones 19 and 46 (lanes 5 and 6), all contained CD3. The reduced electrophoretic mobility of CD3 associated with the TCR could be caused by more complex glycosylation (11). To test this, we treated purified TCRs with N-glycosidase F, which cleaves N-linked carbohydrate moieties. Indeed, the deglycosylated forms of ß TCR– and TCR–associated CD3 are the same size (Fig. 2 B, lanes 2, 4, and 6). This clearly indicates that the TCR expressed on human clones contains CD3.
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ß TCR–deficient variant of Jurkat stably expressing transfected TCRV9 and V2 chains, named J92 (15). The CD3 of J92 had a similar mobility to CD3 in the clones (Fig. 2 B, lanes 5 and 7). Therefore, the complex CD3 glycosylation is intrinsic to the TCR and not caused by different cellular environments of ß and T cells. Incorporation of CD3 into the TCRs of human clones and cell lines was confirmed by anti-CD3 immunoprecipitation and subsequent anti- Western blotting (Fig. S1 and not depicted). This is in line with earlier reports using T cell clones and lines (11, 12). When primary human T cells were used, CD3 could not be detected (10); however, the composition of the mouse TCR changed upon cultivation of primary T cells such that was replaced by FcR (10). Likewise, the TCR of primary human T cells might not contain CD3 but may incorporate it during cultivation.
To determine whether CD3 is present in the TCR of primary human T cells, we lysed PBMCs from a healthy donor and purified ß and TCRs with anti-TCRß and anti-TCR antibodies, respectively. Purified proteins were left untreated or deglycosylated and separated by SDS-PAGE (Fig. 2 C). Jurkat cells were used as a control. Indeed, the TCR and ß TCR roughly contained equal amounts of CD3 (lanes 4 and 8, when normalized to ). The CD3 double band from T cells exhibited a slower electrophoretic mobility than that from ß T cells (lanes 3 and 7). In addition, shorter chains, which probably represent differential mRNA splicing, were incorporated into the TCR (lanes 7 and 8). In an earlier study, CD3 was not found associated with the TCR from primary human T cells (10). It is likely that, because of its different glycosylation, the TCR–associated CD3 had similar mobility to CD3 and, therefore, could not be resolved when biotinylated proteins were detected by SDS-PAGE and streptavidin Western blotting (10). In conclusion, our data show that the human TCR contains CD3 in cultured as well as in primary T cells.
The human TCR has a stoichiometry of TCRCD322
BN-PAGE is a method used to study the native structures of multiprotein complexes (16). In our experiments, we used this technique to analyze the size of the human TCR compared with that of the ß TCR. After digitonin lysis of Jurkat and the human T cell line Peer, as well as T cell clones 19 and 46, TCRs were purified, separated by BN-PAGE, and detected by immunoblotting with an anti- antibody (Fig. 3 A).
The ß TCR, with a stoichiometry of TCR ß CD322 (7, 8), had the same size as the TCR, suggesting a similar stoichiometry for the TCR. Similar results were obtained from nonpurified TCRs (unpublished data).
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TCR, we made use of the native antibody mobility shift (NAMOS) assay that we previously developed to determine the ß TCR stoichiometry (8, 17). The digitonin-extracted TCR was incubated with different amounts of an anti-CD3 antibody, UCHT1, and then subjected to BN-PAGE (Fig. 3 B, lanes 2–4). At the highest antibody concentration, the TCR shifted twice (lane 4, arrowhead labeled TCR+2Ab). This indicates that the TCR has two binding sites for UCHT1, because each antibody molecule bound to the complex produces a discrete change in electrophoretic mobility. At nonsaturating antibody concentrations, a partial shift, indicating TCR bound to only one antibody (TCR+Ab) and cross-linked products, in which one antibody bound to two TCRs (marked with X), were observed (lanes 2 and 3). These data show that the human TCR incorporates two CD3 dimers.
To verify the specificity of anti-CD3 and anti-CD3 antibodies, we expressed individual mouse and human TCR subunits in Drosophila S2 cells and performed subsequent immunopurifications, verifying antibody specificity for HMT3.2 (anti-hCD3) and APA1/2 (anti-hCD3; Fig. S2 A, available at http://www.jem.org/cgi/content/full/jem.20070782/DC1). Using these antibodies in the NAMOS assay revealed that one copy each of CD3 and CD3 are present in the human TCR (Fig. 3 B, lanes 5–8). This is in agreement with the fact that CD3 pairs with either CD3 or CD3 (18). An anti-TCR antibody produced only one shift, whereas anti- produced two shifts (lanes 9–14). Although is a homodimer, the antibody could not bind twice to most TCRs (lane 12). This is caused by steric hindrance, because is very small (16 kD) (8). The same band patterns were observed for several other human T cell clones analyzed (unpublished data). In conclusion, the digitonin-solubilized human TCR has a stoichiometry of TCRCD322.
The mouse TCR has a stoichiometry of TCRCD3222
The mouse TCR was reported to lack CD3 (10). We aimed to study mouse TCR stoichiometry using our reagents and methods. Splenocytes from TCRß–/– mice (19) carrying transgenes for the TCRV1.1 and TCRV6 chains (TCRß–/–16tg) (20) served as a source of primary T cells. Initially, we compared the ß TCR from wild-type Bl/6 mice with the TCR from TCRß–/–16tg mice by anti- immunopurification and anti-CD3 Western blotting (Fig. 4 A).
As expected, the TCR did not contain CD3 (lanes 3 and 4). BN-PAGE showed that the digitonin-solubilized TCR has a similar mobility to the ß TCR for which the stoichiometry has been determined to be TCRßCD322 (Fig. 4 B, lanes 1,4, 6, and 8) (8). To ascertain mouse TCR stoichiometry, the NAMOS assay was applied using antibodies that had been controlled for specificity (Fig. S2 B). Anti-CD3, as well as anti-CD3, antibodies produced two shifts, indicating that the mouse TCR contains two CD3 dimers (Fig. 4 B, lanes 6–11). Because CD3 always pairs with either CD3 or CD and because the mobility of the TCR in BN-PAGE was the same as that of the ß TCR, we concluded that the digitonin-solubilized mouse TCR has a stoichiometry of TCRCD3222. This stoichiometry is in agreement with the conserved charge distributions in the transmembrane segments of the ß and TCR subunits and with the 1:2 ratio of TCR/CD3 in primary mouse T cells (21).
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can bind to mouse CD3 (mCD3) but not to mCD3 (9, 10, 21), whereas human TCR binds both hCD3&egr; and hCD3 (15). These results are in agreement with the different stoichiometries determined in our experiments for human and mouse TCRs.
Human, but not mouse, CD3 can restore T cell development in CD3/CD3 double-deficient (CD3–/–) mice
We asked whether the different subunit requirements for mouse versus human TCR formation were caused by sequence differences in their respective CD3 subunits. As expected, our CD3–/– and CD3–/– mice both lack T cells (Fig. 5 B and not depicted) (3, 22).
This was not caused by limiting amounts of CD3, because a mCD3 transgene (mCD3tg) could not rescue T cell development (Fig. 5 C). In contrast, the CD3–/– mouse strain carrying an hCD3 transgene (CD3–/–hCD3tg) (23) has as many T cells as wild-type mice (Fig. 5, A and D). These cells could be detected in the thymus, spleen, lymph nodes, and blood (Fig. 5 and not depicted), indicating that hCD3 can functionally replace mCD3 in the mouse TCR, whereas mCD3 cannot. Sequence- and structure-wise, hCD3 is more related to mCD3 than to mCD3 (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20070782/DC1) (24, 25). The property of hCD3 to replace mCD3 is probably independent of the signal-transducing immunoreceptor tyrosine-based activation motif sequence of the cytoplasmic tail, because T cell development is unaffected in mice lacking the CD3 immunoreceptor tyrosine-based activation motif (26). Thus, the functional differences between hCD3 and mCD3 might map to the extracellular region, as the transmembrane regions are highly conserved between the different CD3 chains (Fig. S3). Indeed, the ectodomains are critical for TCR assembly, suggesting that hCD3 can assemble within mouse TCR to form a functional TCR, whereas mCD3 cannot. This conclusion is in line with the finding that mouse TCR can assemble with hCD3 (15), in contrast to mCD3.
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ß TCR expression was reduced to 60%, whereas that of TCR was 80–100% of wild-type TCR levels. As in hCD3-deficient patients (Fig. 1), in CD3–/–hCD3tg mice, the TCR expression level was less affected by the absence of CD3 than that of the ß TCR (Fig. 5, E and F) when compared with wild-type mice.
The stoichiometries of human and mouse TCRs correlate well with the phenotypes for human and mCD3 deficiencies. Mice lacking CD3 exhibit normal T cell development (6), consistent with the finding that mCD3 is not part of the mouse TCR (9, 10). In contrast, CD3–/– mice do not contain T cells (3), because CD3 is an obligatory subunit of the mouse TCR. In humans, both CD3 and CD3 are part of the TCR (Figs. 2 and Figs.3; and Fig. S1). CD3-deficient TCRs are still able to support T cell development in humans (Fig. 1), likely because hCD3 can partially substitute for hCD3. Remarkably, hCD3 is also able to rescue T cell development in CD3–/– mice, indicating its ability to substitute for mCD3 in the mouse TCR as well. In contrast, CD3-deficient patients do not develop T cells (Fig. 1 A) (5). Presumably, hCD3 cannot substitute for hCD3 in human TCR formation and function. Along this line, replacement of the mTCR connecting peptide by the one of mTCR promotes the exclusion of mCD3 from the complex (27), suggesting that the connecting peptide of mTCR is involved in the association with CD3 but does not permit the assembly of CD3. A difference in the connecting peptide sequences of human and mouse TCR could be responsible for the differential involvement in TCR assembly of CD3 in both species.
Conclusions
Using both conventional immunopurification followed by Western blotting and our novel NAMOS assay, we have determined the human digitonin-solubilized TCR stoichiometry to be TCRCD322. The CD3 chain is differentially glycosylated depending on its association with the ß or the TCR (11), likely accounting for contradictory results previously reported (10, 12). We show that the mouse TCR stoichiometry is TCRCD3222, as proposed by Hayes and Love (21). Clarification of both mouse and human TCR stoichiometries finally explains the different phenotypes observed in CD3-deficient humans and mice. We show that, in contrast to mCD3, an hCD3 transgene is able to rescue T cell development in mice lacking both mCD3 and mCD3. This indicates important structural and functional differences between hCD3 and mCD3 chains, as already suggested from the analysis of ß T cells (23). Indeed, the phenotype of CD3–/–hCD3tg mice (Fig. 5) resembles that of CD3-deficient humans (4, 23), as opposed to that of CD3–/– mice (3). This is true for ß (23) as well as for T cells (Fig. 5). Thus, this humanized CD3-deficient mouse strain may be a valuable tool to further study the impact of CD3-deficiency in ß as well as T cell pathophysiology in humans.
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MATERIALS AND METHODS |
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TopABSTRACTRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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ß and T cell clones were generated as previously described (28). TCRß–/– Jurkat cells transfected with V92 (J92) were previously described (15). Human PBMCs were isolated from a healthy donor using a Ficoll gradient. TCRß–/– (19) and V1.1V6tg mice (a gift of P. Pereira, Institut Pasteur, Paris, France) (20), both on a C57BL/6 background, were mated, generating the TCRß–/–16tg strain. CD3–/– (3), CD3–/–mCD3tg, and CD3–/–hCD3tg (23) mice were previously described (D. Kappes [Fox Chase Cancer Center, Philadelphia, PA] and C. Terhorst [Beth Israel Deaconess Medical Center, Boston, MA] provided the mCD3tg and CD3–/– mice, respectively). Mice were killed between 6 and 12 wk of age, and lymphocytes were isolated from tissues indicated in the figures using standard protocols. Animal research was approved by the Regierungspräsidium-Freiburg (G.02/84) and the local animal care commission. Antibodies are described in Supplemental materials and methods (available at http://www.jem.org/cgi/content/full/jem.20070782/DC1).
Flow cytometry.
Normal distributions for ß and T cell numbers were obtained from the literature (29, 30). The normal ranges (which include 90% of the data) were depicted in a logarithmic scale as median values (dashed line) between the 5th and 95th percentiles (P5 and P95). In CD3-deficient patients, ß T cells were defined as CD4+ and CD8+ or CD8bright, thus excluding most T cells (<8%). T cells were defined as surface TCR+ using the antibodies 11F2 or Immu510. In patients, T cell counts may be underestimated because of the TCR expression defect.
Mouse cells were stained with PE-conjugated GL3, FITC-conjugated H57-597, and biotinylated 145-2C11 antibodies. Streptavidin–PE-Cy5 was used as a second-step reagent. Stained cells were analyzed in a flow cytometer (FACSCalibur) using CellQuest software (both purchased from Becton Dickinson).
Cell lysis, TCR purification, and deglycosylation.
Cells were lysed using 1% digitonin or 0.5% Brij96V, and immunoprecipitations were performed using the antibodies 448, Jovi1, and 5A6.E9, as previously described (17). TCRs bound to the beads were treated with 1 U N-glycosidase F (Roche Diagnostics).
For TCR immunopurifications used in BN-PAGE, 107 cells were incubated with 200 µM pervanadate and lysed, and phosphorylated proteins were purified with 2 µg 4G10 and 5 µl protein G–coupled sepharose (GE Healthcare). Native elution was done in BN buffer including 50 mM phenylphosphate, the detergent indicated in the figures, and phosphatase to dephosphorylate the TCR (16, 17). In experiments in which T cell clones were used, ß TCRs were depleted by two sequential immunodepletions using Jovi1 and ßF1 bound to protein G–coupled sepharose (Fig. S1).
Gel electrophoresis and Western blotting.
SDS- and BN-PAGE were performed using standard protocols (16). Ferritin in its 24- and 48-meric forms was used as the marker protein (f1 and f2, 440 and 880 kD, respectively). In brief, for the NAMOS assay (unpublished data), antibodies were added to 10 µl of eluted purified TCR before separation by BN-PAGE (4–9%). Western blotting was performed according to standard protocols using 448 (1:5,000), M20, and M20 (both 1:1,000) antisera.
Online supplemental material.
Supplemental materials and methods provides the specificities and sources of the antibodies. Fig. S1 A shows flow cytometric analysis of a human T cell clone stained for ß and TCR. Fig. S1 B shows Western blotting of anti–ß TCR depletion of a human T cell clone lysate. Fig. S1 C shows immunopurification of the TCR from a human T cell clone using an anti-CD3 antibody. Fig. S2 (A and B) shows that anti–human and anti–mouse CD3 and CD3 antibodies are specific for their respective chains by immunoprecipitation of individually expressed CD3 chains in Drosophila S2 cells. In Fig. S3, a sequence alignment of hCD3 and mCD3 is discussed. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20070782/DC1.
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Acknowledgments |
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This work was supported by an Emmy Noether Fellowship to W.W. Schamel from the Deutsche Forschungsgemeinschaft (SCHA 976/1), Ministerio de Educacion y Cultura (MEC) grant BMC2002-01431 to E. Fernández-Malavé, MEC grant BFU2005-01738/BMC to J.R. Regueiro, the European Union–funded grant EPI-PEP-VAC to S. Minguet, a University of Freiburg Wiedereinstiegsstipendium to G.M. Siegers, grant R05/01 from the Deutsche Jose Carreras Leukämie-Stiftung to P. Fisch, and grants SFB620 B6 and Z2 from the Deutsche Forschungsgemeinschaft to W.W. Schamel and P. Fisch. The support of the Fundacion Ramon Areces to the Centro de Biologia Molecular is acknowledged.
The authors have no conflicting financial interests.
Submitted: 17 April 2007
Accepted: 17 August 2007
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TopABSTRACTRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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