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Blood, 15 November 2006, Vol. 108, No. 10, pp. 3420-3427. Prepublished online as a Blood First Edition Paper on August 3, 2006; DOI 10.1182/blood-2006-03-010850.
IMMUNOBIOLOGY
Overlapping functions of human CD3
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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subunit of the pre-TCR and TCR complexes exhibit a mild 
T lymphopenia, but have normal T cells. By contrast, CD3-deficient mice are almost devoid of mature T cells due to an early block of intrathymic development at the CD4–CD8– double-negative (DN) stage. This suggests that in humans but not in mice, the highly related CD3
chain replaces CD3 during T-cell development. To determine whether human CD3 (hCD3) functions in a similar manner in the mouse in the absence of CD3, we introduced an hCD3 transgene in mice that were deficient for both CD3 and CD3, in which thymocyte development is completely arrested at the DN stage. Expression of hCD3 efficiently supported pre-TCR–mediated progression from the DN to the CD4+CD8+ double-positive (DP) stage. However, TCR-mediated positive and negative thymocyte selection was less efficient than in wild-type mice, which correlated with a marked attenuation of TCR-mediated signaling. Of note, murine CD3-deficient TCR complexes that had incorporated hCD3 displayed abnormalities in structural stability resembling those of T cells from CD3-deficient humans. Taken together, these data demonstrate that CD3 and CD3 play a different role in humans and mice in pre-TCR and TCR function during T-cell development. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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TCR are multimeric protein complexes expressed on the surface of developing T cells at different stages of their ontogeny in the thymus. The pre-TCR complex consists of a disulfide-linked heterodimer of an invariant pre-T (pT) and a variable TCR chain noncovalently associated with the CD3, , 
, and 
polypeptides.1 The TCR contains an identical subunit composition but with a variable TCR chain in lieu of pT. Within the pre-TCR and TCR, the CD3 subunits are assembled as , , and dimers, with all but CD3 being structurally related.2 Functionally the CD3 proteins play a role in the assembly, surface transport, and signal capacity of the pre-TCR and TCR complexes.3,4
During thymocyte development, the pre-TCR and TCR control discrete developmental checkpoints through which T-cell precursors have to pass to mature successfully.5,6 Signals initiated at the pre-TCR trigger progression from a CD44–CD25+ CD4–CD8– double-negative (DN) to a CD4+CD8+ double-positive (DP) differentiation stage, through CD44–CD25– DN intermediates. Thus, only cells with a productive TCR gene rearrangement undergo this transition (a checkpoint termed selection). Subsequently, signals emanating from the TCR determine the positive (survival and differentiation) and negative (clonal deletion) selection of immature DP thymocytes, processes that shape the repertoire of mature CD4+CD8– and CD4–CD8+ single-positive (SP) T cells that exit from the thymus and populate the peripheral lymphoid organs. The position of the CD3 proteins at the initiation of the pre-TCR and TCR-mediated signaling cascade implies a critical role of these molecules in alternate signal transduction during thymic selection.
The overall structure and functioning of the pre-TCR and TCR complexes in mice and humans are quite similar. Despite this similarity, ablation of the highly related CD3 and CD3 subunits has markedly different effects in mice and humans. In mice made deficient for CD3, thymocyte development is completely blocked at the DP stage due to defective TCR expression and function,7,8 but pre-TCR function is normal. On the other hand, CD3-deficient humans exhibit an earlier developmental block at the pre-TCR–controlled checkpoint.9,10 Loss of CD3 in the mouse results in a severe block at the DN-to-DP transition, indicating that pre-TCR signaling is defective.11 In particular, the majority of CD3-deficient thymocytes cannot pass the CD44–CD25+ DN checkpoint. In contrast, CD3-deficient humans show only slightly reduced numbers of peripheral T cells, which display decreased TCR surface expression, and functional defects that lead to immunodeficiency with a variable spectrum of clinical signs.12 Since CD3-deficient mice possess a different phenotype, a mouse model for this human disease is lacking to date.
To further dissect the roles of CD3 and CD3 in mouse and human T-cell development, we crossed mice lacking CD3 and CD3, in which thymocyte development is completely arrested at the DN stage,13 with mice transgenic for a human CD3 (hCD3) chain,14 resulting in the – x hTg mouse strain. These mice incorporated the hCD3 protein in surface pre-TCR and TCR complexes, and displayed a nearly normal-sized population of TCR-expressing DP cells, indicative of normal pre-TCR function. In contrast, – x hTg TCR complexes displayed an impaired capability to signal for efficient positive as well as negative selection and differentiation of DP thymocytes into mature T cells. Of importance, the T-cell compartment of – x hTg mice, in contrast to the previously reported mice lacking CD3,11 resembled that of CD3-deficient humans. Therefore, this "humanized" mouse strain might be an adequate model system for the human CD3 immunodeficiency.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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CD3-deficient mice expressing a human CD3 chain (hereafter referred to as – x hTg mice) were generated by breeding CD3 doubly-deficient mice13 with a strain of transgenic mice, termed Tg4, which carries a low copy number of a human CD3 transgene.14 Double mutant mice were identified by Southern blot and polymerase chain reaction (PCR). CD3 doubly-deficient mice expressing a mouse CD3 transgene (– x mTg) will be described in detail elsewhere. In all experiments, C57BL/6 mice were used as wild-type (WT) controls. Mice were analyzed at 6 to 8 weeks of age. CD3-deficient and HY, OT-I, and AND TCR transgenic mice have been described elsewhere.11,15-17
Antibodies
For flow cytometry, the following biotinylated, FITC-, PE-, or PE-Cy5–conjugated antibodies were obtained from BD Biosciences Pharmingen (San Jose, CA): anti-CD3/ (clone 145-2C11), anti-CD3 (clone 17A2), anti-CD4 (clone RM4-5), anti-CD5 (clone 53-7.3), anti-CD8 (clone 53-6.7), anti-CD69 (clone H1.2F3), anti-TCR (clone H57-597), anti-TCRV2 (clone B20.1), anti-TCRV3 (clone KJ25), and anti-TCRV8 (clone MR5-2). Streptavidin-PE-Cy5 was used as second-step reagent. Immunoblottings were performed using the following antibodies: anti–phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY), anti–ZAP-70 (Zap4; a gift from S. Ley), anti–Cbl-1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-LAT (Upstate Biotechnology), anti-CD3 antibody 448,18 anti–phospho-MAPK (ERK, JNK, and p38), and anti–total MAPK (New England Biolabs, Beverly, MA).
Flow cytometry
Cells were stained with specific fluorochrome-conjugated or biotinylated antibodies and analyzed by 2- or 3-color flow cytometry in a FACSCalibur flow cytometer using the CellQuest software (Becton Dickinson, San Jose, CA).
TCR stimulation, immunoprecipitation, and immunoblotting
Thymocytes were resuspended in PBS and incubated for 10 minutes at 4°C in the presence or absence of biotin-conjugated anti-CD3 (145-2C11) and anti-CD4 (RM4-5) antibodies. After removal of unbound antibody, the cells were incubated at 37°C for varying periods of time with 100 µg/mL streptavidin. After stimulation, cells were lysed in a buffer containing 1% Brij-96, 20 mM Tris-HCL (pH 7.8), 150 mM NaCl, and a cocktail of protease and phosphatase inhibitors. For immunoprecipitations, lysates were incubated overnight with specific antibodies preadsorbed to protein G–sepharose beads. The beads were washed 4 times in lysis buffer and resuspended in SDS sample buffer. Total cell lysates or immunoprecipitates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Immobilon-P (Millipore) membranes, and immunoblotted with specific antibodies. Bound antibodies were detected using an enhanced chemiluminescence system (Amersham).
Protein analysis
For analysis of the TCR organization under nondenaturing conditions, blue native–polyacrylamide gel electrophoresis (BN-PAGE)19,20 was performed. Briefly, the TCR complex from digitonin lysates of pervanadate-stimulated thymocytes was immunoprecipitated with antiphosphotyrosine-coupled protein G–sepharose and eluted with phenylphosphate. After separation of the samples by BN-PAGE,21,22 the proteins were transferred to nitrocellulose membranes and detected by immunoblotting with anti-CD3 antiserum 448. Ferritin was used as the marker protein in its 24-mer, 48-mer, and 72-mer forms (440 kDa, 880 kDa, and 1320 kDa, respectively). For antibody-shift assays, the antibody amounts indicated were added to the samples and incubated for 30 minutes on ice before BN-PAGE loading, as described.22
TCR-induced thymocyte death assays
Mice were injected with either vehicle alone (PBS) or anti-TCR antibody (H57-597). After 48 hours, thymi were removed for analysis of thymocyte numbers and CD4/CD8 expression. For in vitro thymocyte death assay, cells were cultured in plastic wells precoated with the indicated amount of anti-TCR antibody. After 48 hours, thymocytes were harvested and cell death was assessed by propidium iodide (PI) staining followed by flow cytometry. Specific cell death (%) denotes the percentage of dead (PI positive) cells in antibody-coated wells, normalized to control wells without antibody as follows:
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Calcium mobilization
Calcium mobilization was measured by flow cytometry in Fluo-3-AM–loaded thymocytes upon addition of biotin-conjugated antibodies to CD3 and CD4 followed by cross-linking with streptavidin.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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doubly-deficient mice expressing a human CD3 transgene
– x hTg mice were generated by crossing mice lacking both CD3 and CD3, in which thymocyte development is completely arrested at the CD44–CD25+ DN stage, with mice carrying a low copy number of a complete human CD3 transgene controlled by its own regulatory elements. The size of the thymi and total thymocyte number of these – x hTg mice were relatively normal compared with control WT mice. Total thymocyte numbers (n = 10) were as follows: WT, 88 ± 31 x 106; – x hTg, 74 ± 17 x 106. Total splenocyte numbers (n = 9) were as follows: WT, 48 ± 15 x 106; – x hTg, 25 ± 6 x 106. Further, all thymocyte subsets defined by expression of CD4 and CD8 coreceptors were present in – x hTg mice (Figure 1A).
More detailed analysis of the DN population revealed that progression beyond the CD44–CD25+ DN stage, severely impaired in CD3-deficient and CD3 doubly-deficient mice,11,13 proceeds efficiently in the thymi of – x hTg mice (Figure 1B upper panel). In contrast to DP cells in CD3-deficient mice,11 numbers of DP thymocytes were comparable between – x hTg and WT mice (Figure 1A). Further, – x hTg DP cells lacked surface CD25 (Figure 1B lower panel). Together, these data indicate that expression of a human CD3 transgene is able to restore pre-TCR function in the absence of CD3 in the mouse, resulting in the generation of DP thymocytes that were phenotypically normal. However, the numbers of CD4+ SP and CD8+ SP thymocytes in – x hTg mice were reduced on average to 36% and 42%, respectively, of those in WT mice (Figure 1A). These mature thymocytes were able to migrate to and accumulate in peripheral lymphoid organs, as shown by the presence of CD4+ and CD8+ SP cells in the spleens of – x hTg mice, in numbers averaging 50% of those in WT mice (Figure 1A).
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TCR expression on the surface of mutant thymocytes was indistinguishable from that of control WT thymocytes (Figure 1C upper panel), despite the specific absence of dimers in the TCRs of these cells (Figure 1C lower panel). Furthermore, the up-regulation of – x hTg TCR expression was normal during intrathymic development in the absence of CD3 (Figure 1D). Yet, TCR surface expression in thymic and splenic SP cells from – x hTg mice was reduced in comparison with their WT counterparts (Figure 1D), although the extent of decrease was less than that reported for CD3-deficient mice23 or humans.24 Also, the impairment of TCR expression in peripheral mature T cells from – x hTg mice was more prominent in CD4+ SP than in CD8+ SP cells (Figure 1D). This was in contrast with either CD3-deficient mice or humans, where TCR expression is more markedly diminished in the CD8+ lineage.23,24 Collectively, these data confirm previous findings indicating that CD3 is not essential for TCR assembly and surface expression in mouse T cells.11 Further, these results demonstrate that a human CD3 transgene can effectively support the DN-to-DP and, albeit with lesser efficiency, the DP-to-SP transition in mice lacking both CD3 and CD3. Rescue of intrathymic T-cell development does not appear to be related to simply transgene overexpression, since a mouse CD3 transgene was unable to restore thymocyte cellularity and maturation in CD3 doubly-deficient mice (Figure 1E). Accordingly, thymic development in this – x mTg strain resembled that of CD3-deficient mice (Figure 1E), with most of the thymocytes arrested at the DN stage.
Human CD3 substitutes both mouse CD3 and CD3 in the – x hTg TCR
To determine the native structure of the TCR in – x hTg T cells, and to characterize the impact of the CD3-deficiency and incorporation of a human CD3 on the composition and stability of the TCR complex, we performed blue native (BN)–PAGE analysis of the TCR expressed by WT and – x hTg thymocytes (Figure 2A). The digitonin-extracted native TCR/CD3 complex isolated from WT thymocytes was visualized with anti-CD3 as a single band with mobility just below the 440-kDa ferritin marker. It had the stoichiometry of as reported before.22,25 In contrast, anti-CD3 detected 3 bands in the – x hTg complexes. The largest 2 bands had a size similar to the WT TCR complex. The highest mobility band consisted of free CD3 as a band with similar mobility appeared upon SDS treatment of the WT TCR (data not shown). SDS is a harsh detergent that disrupts all TCR complexes.
To characterize the subunit composition and stoichiometry of the TCR in – x hTg T cells, we used a novel technique combining antibody-mediated gel-shift and BN-PAGE, termed native antibody-based mobility-shift (NAMOS) assay22 (Figure 2B). Digitonin lysates from WT and – x hTg splenocytes were used to purify the TCR complex. Subsequently, the purified receptor was incubated with different amounts of the anti-CD3 antibody 2C11 and then subjected to BN-PAGE. The WT TCR was shifted to 2 new bands (Figure 2B left). Since the complete () TCR complex has 2 binding sites for 2C11 ( and ), and since each antibody molecule bound to the complex produces a discrete change in electrophoretic mobility, the band appearing at the lowest amount of antibody tested is consistent with one antibody molecule bound to one TCR complex. The highest antibody amount generates a band likely corresponding to 2 antibody molecules associated with a single TCR complex, with an intermediate amount generating a mixture of the products observed at the highest and lowest amounts of shifting antibody (Figure 2B left). These data show that the WT TCR complex has 2 binding sites for 2C11 and, therefore, possesses 2 CD3 dimers ( and ).
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– x hTg TCR complex was analyzed (Figure 2B right). This indicated that the mutant receptor contained 2 binding sites for 2C11 and corresponds therefore to a (h)2 complex. The shift pattern of the band migrating immediately below the predominant TCR complex (Figure 2A arrowhead) was consistent with a partial TCR complex containing only one h dimer (h) (data not shown). As expected, the TCR of – x hTg T cells was not shifted by the anti-CD3 antibody 17A2 (Figure 2B right), whereas its WT counterpart was (Figure 2B left). Collectively, these data indicate that the majority of TCRs in – x hTg T cells incorporates 2 h dimers per complex, thus keeping with the typical architecture of the WT TCR.22 Further, a minor fraction of partial TCR containing only one CD3 dimer (h) within the complex was detected. To date, it is unclear whether these complexes existed on the cell surface or were generated upon detergent lysis. Both cases could indicate that the chimeric TCR is less stable than the WT TCR.
Inefficient positive selection of thymocytes in – x hTg mice
The decrease in the number of mature CD4+ and CD8+ SP thymocytes observed in – x hTg mice suggested an impairment of positive selection. To test this, 3 transgenic TCRs were independently introduced onto the – x hTg background: HY, OT-I, and AND. The HY TCR recognizes a male-specific peptide in the context of MHC class I H-2b. Thymocytes expressing the HY TCR are positively selected as CD8+ SP cells in female H-2b mice.15 Consequently, 20.7% ± 7.6% of total thymocytes are CD8+ SP (Figure 3A upper panel and 3C). In contrast, only 2.3% ± 1.7% CD8+ SP cells were present in female HY – x hTg mice (Figure 3A upper panel and 3C). Numbers of CD8+ SP thymocytes were also reduced by 90% in the HY TCR mutant mice (Figure 3C). Positive selection of another MHC class I–restricted TCR (OT-I,16 specific for an ovalbumin peptide in the context of H-2b) was also affected in – x hTg mice, albeit to a lesser degree (Figure 3A middle panel). In this case, the number of CD8+ SP cells was reduced by only 45% in OT-I transgenic – x hTg compared with WT mice (Figure 3C).
We next used mice expressing the pigeon cytochrome-C–specific TCR AND17 crossed with – x hTg mice to assess whether positive selection of class II–restricted CD4+ SP thymocytes was also affected. In AND – x hTg mice, the percentage and number of CD4+ SP cells were reduced to 32% and 43%, respectively, of those in WT mice (Figure 3A lower panel and 3C). This result demonstrated that the efficiency of positive selection of CD4+ T cells was also decreased in – x hTg mice. Thus, positive selection of both class I– and class II–restricted T cells appeared to proceed less efficiently in – x hTg mice. Yet, surface up-regulation of CD526 (data not shown) and the TCR (Figure 3B) along the DP-to-SP transition was comparable in WT and – x hTg TCR transgenics, suggesting that positive selection is not qualitatively affected in the mutant mice.
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– x hTg mice result from a decrease in surface TCR expression on the DP population (Figure 3B), which is the one subjected to this selection process. To address this issue further, we performed functional assays of TCR signaling using the AND.DP.–/– cell line. This cell line was established from a thymic tumor spontaneously arising in a mouse from our – x hTg colony bearing the AND TCR. It expresses surface CD4 and CD8, and TCR, but not CD3 (Figure 4A). Expression of the transgenic hCD3 was detected by PCR (data not shown). Of interest, surface TCR expression in AND.DP.–/– was even higher than that of DP cells from AND TCR transgenics (Figure 4B). Despite this high TCR expression, both CD69 and CD5 upregulation in response to TCR ligation with antibody or peptide-MHC,27,28 an event that correlates with positive selection,29 was clearly impaired in the DP thymocyte line when compared with its counterparts from WT AND TCR transgenics (Figure 4C). To exclude that this defect was entirely due to the transformed state of the cell line, we analyzed activation marker up-regulation in primary DP thymocytes from – x hTg mice, in which TCR surface expression is comparable with that of WT DP cells (Figure 1D). TCR-mediated up-regulation of CD69 and CD5 expression was also reduced in primary – x hTg DP thymocytes compared with their WT counterparts (Figure 4D), although to a lesser extent than in the thymocyte line. Together, these data suggest that TCR complexes incorporating a human CD3 chain and lacking CD3 have an impaired signaling capability, and that this defect is not related to the TCR density on the thymocyte surface.
Impaired negative selection and TCR-induced thymocyte death in – x hTg mice
To study negative selection in – x hTg mice, we analyzed male HY TCR transgenics. In male (but not in female) H-2b mice, thymocytes expressing the HY TCR are negatively selected as a result of extensive apoptotic cell death at the DP stage upon recognition of male-specific peptide and MHC.15 Indeed, only 3% DP cells were detected in WT HY TCR mice (Figure 5A top panel). In contrast, HY – x hTg mice exhibited a partial but clear defect in negative selection, demonstrated by the persistent DP population that constituted 56% of the thymocytes (Figure 5A top panel). Moreover, splenic CD8+ SP T cells that escaped intrathymic clonal deletion in – x hTg mice were CD8high, in contrast to WT mice where they expressed characteristically low levels of surface CD830 (Figure 5A bottom panel). Furthermore, thymocyte death induced by anti-TCR antibody in vivo31 (Figure 5B), and in vitro (Figure 5C), was markedly reduced in – x hTg mice. Collectively, these data indicate that effective coupling of the TCR to death-inducing pathways in thymocytes, and resulting negative selection, is impaired in – x hTg mice.
Reduced strength of TCR-initiated signals in – x hTg thymocytes
To determine whether thymocytes from – x hTg mice exhibited a TCR signaling defect, we analyzed several intracellular events associated with TCR signal transduction. Early biochemical events elicited upon TCR engagement,32 namely tyrosine phosphorylation of src- and syk-kinase substrates including ZAP-70, Cbl-1, and LAT, were markedly attenuated but not completely abrogated in – x hTg thymocytes, and also in AND TCR transgenic – x hTg thymocytes compared with their WT counterparts (Figure 6A). Although a reduced level of tyrosine phosphorylation of key signal-transduction molecules was found in anti-TCR–activated thymocytes (Figure 6A), it would appear though that no single pathway was affected specifically. TCR-induced calcium mobilization32 was also decreased in – x hTg thymocytes compared with their WT counterparts (Figure 6B).
TCR-dependent transient activation of mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38 has been shown to affect thymocyte selection.33 We therefore examined the phosphorylation levels of these enzymes as an indication of their activation state. Upon in vitro TCR ligation with antibody, phosphorylation of the ERK, JNK, and p38 kinases was diminished in – x hTg thymocytes compared with control WT cells (Figure 6C). Thus, – x hTg mice display a general and quantitative defect in the TCR-induced activation of all MAPK pathways in the thymus. Taken together, these data demonstrate that proximal as well as distal signaling events coupled to TCR stimulation in thymocytes are markedly attenuated in – x hTg mice.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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, due to targeted gene disruption, the DN-to-DP transition is severely impaired,11 most likely as a consequence of defective pre-TCR function. Thus, mature T cells are practically absent in the peripheral lymphoid organs of CD3-deficient mice. In contrast, T lymphocyte numbers are grossly normal or only slightly diminished in CD3-deficient humans,12 suggesting that in humans, but not in mice, the highly related CD3 chain is capable of functionally replacing CD3 during thymopoiesis. Here, we show that human CD3 could restore mouse T-cell development in the absence of CD3 by expressing a hCD3 transgene in CD3 doubly-deficient mice.
In the – x hTg mouse strain, the pre-TCR–mediated DN-to-DP transition was efficiently restored by hCD3 expression. It is rather intriguing that human but not mouse11 CD3 can replace CD3 for pre-TCR–mediated thymocyte development. In the absence of CD3, the amount of CD3 could be limiting with regard to pre-TCR expression. In this scenario, enhanced expression of transgenic (relative to endogenous) CD3 would overcome that limitation, allowing the expression of CD3-lacking pre-TCR complexes. This appears unlikely as the relative amount of intracellular CD3-containing dimers in DN thymocytes was comparable in WT, CD3-deficient, and – x hTg mice (data not shown). Also, CD3 dimers present on the surface of CD3-deficient DN thymocytes are sufficient to mediate developmental progression when engaged with anti-CD3 antibodies,11 or to permit the expression of a transgenic TCR and rescue of the DN-to-DP transition.34 Finally, early thymocyte development was not restored in CD3 doubly-deficient mice expressing a mouse CD3 transgene. In line with this, a human CD3 transgene that encodes full-length CD3 and a truncated but functional form of CD3 restored the defective pre-TCR function in CD3- and CD3-deficient mice.35 Therefore, in the absence of CD3, human but not mouse CD3 appears to endow the pre-TCR complex with specific properties essential for pre-TCR function. Whatever these properties might be, they appear to be independent of the signal-transducing ITAM motif, as early thymocyte development proceeded unaffected in mice expressing a CD3 chain lacking the ITAM motif.34 Accordingly, the differential capability of human versus mouse CD3 to functionally replace CD3 within the pre-TCR must map in the extracellular and/or transmembrane domain, as it has previously been shown for CD3 regarding TCR function and thymocyte selection.8 In the extracellular region, there are several amino acid positions where human but not mouse CD3 matches mouse CD3. Whether these residues contribute specific structural and/or functional features to the pre-TCR complex deserves further investigation.
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– x hTg mice suggested a defect in TCR-mediated thymocyte selection. This was confirmed by using 3 different transgenic TCRs expressed in the – x hTg background. Positive selection of both these class I– or class II–restricted TCR transgenic T cells was markedly reduced in the mutant mouse, but without any sign of alteration in lineage commitment. Negative selection was similarly impaired in – x hTg mice. Further, these defects were not likely related to decreased TCR surface expression in mutant DP cells, as pointed out by our studies with the AND.DP.–/– thymocyte line, which presented defects in TCR signaling despite expressing high levels of receptor on the cell surface. Collectively, our results demonstrate that positive and negative selection proceeded less efficiently in – x hTg mice, probably because signals from CD3-lacking TCR incorporating human CD3 are weaker. The latter is supported by the observation that signaling events proximal to TCR stimulation were markedly attenuated, but not totally abrogated, in thymocytes from – x hTg mice. More distal TCR-triggered events were also quantitatively but not qualitatively affected. A recent study further supports this notion by showing that the strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes,36 a phenotype similar to that observed in – x hTg mice. Our results are also consistent with the impairment in TCR-mediated tyrosine phosphorylation and calcium mobilization found in T cells from CD3-deficient patients.37,38 Similarly to mature T cells in CD3-deficient humans,24,37 but in contrast to CD3-null mutant mice,23 splenocytes from – x hTg mice showed normal up-regulation of CD69 and CD25 in response to anti-TCR antibody stimulation (data not shown). Further, like human T cells lacking CD3,37 – x hTg splenocytes exhibited defective phorbol ester–stimulated but only slightly reduced anti-TCR antibody-induced TCR down-regulation (data not shown). Taken together, these results indicate that TCR signaling is grossly similar in – x hTg mice and CD3-deficient humans.
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TCR complex expressed by thymocytes from – x hTg mice retained epitopes recognized by antibodies to murine TCR (H57-597) and CD3/ dimers (145-2C11). Further, the mutant TCR complex displayed the same stoichiometry as that of the WT TCR. Together, these data indicate that the – x hTg TCR topologically resembles CD3-sufficient TCR in some aspects. Of importance, abnormal glycosylation of hCD3 (data not shown) was observed for the – x hTg TCR. This might explain its slightly reduced electrophoretic mobility under native conditions compared with the WT TCR, and its inability to trigger late T-cell activation in response to the lectin concanavalin A (data not shown). In addition, a weak association of CD3 chains, including hCD3/CD3 and CD3, to the TCR heterodimer was observed for the – x hTg TCR complex. These alterations in glycosylation and stability have also been reported for T cells from human CD3-deficient individuals,39 and they could be partly underlying the impaired signaling capability of the TCR in – x hTg mice.40,41 Whether CD3 provides specific functions to the TCR that are essential for thymocyte selection34 remains an open question, awaiting the analysis of more CD3-deficient TCR transgenic mice.
CD3 and CD3 appeared for the first time in mammals as a result of the duplication of an ancestral gene found in birds with properties of both CD3 chains.42 The evolution of CD3 and CD3 as discrete subunits in mammals suggests that they might have some nonredundant but essential functions within the pre-TCR and TCR complexes. The present study further demonstrates that CD3 and CD3 play different roles in pre-TCR and TCR function in mice and humans.
The gross phenotype of – x hTg mice, in contrast to previously reported CD3-deficient mice,11 resembled that of humans who lack expression of the CD3 protein.43 Thus, this humanized CD3-deficient mouse strain may constitute a valuable tool44 to analyze the consequences of the CD3 deficiency in a more detailed way than is now possible in human patients, and for the design and preclinical evaluation of therapeutic strategies to correct human disorders associated with CD3 deficiencies.45
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Footnotes |
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Prepublished online as Blood First Edition Paper, August 3, 2006; DOI 10.1182/blood-2006-03-010850.
Supported by grants BMC2002-01431 from the Spanish Ministry of Science and Education (MEC) (E.F.-M.), GR/SAL/0168/2004 from Community of Madrid (E.F.-M.), and Areces Foundation (to the Centro de Biología Molecular Severo Ochoa [CBMSO]). E.F.-M. is supported by the "Ramón y Cajal" Program of MEC. M.P. was supported by a BEFI fellowship from the Fondo de Investigaciones Sanitarias (FIS). W.W.A.S. was supported by the Deutsche Forschungsgemeinschaft through the Emmy Noether program (SCHA 976/1) and through SFB620.
The authors declare no competing financial interests.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
Reprints: Edgar Fernández-Malavé, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain; e-mail: efernandez{at}cbm.uam.es.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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-deficient TCR/CD3 complex in immortalized mature CD4+ and CD8+ T lymphocytes. J Immunol. 1998;161: 3152-3160.
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| Copyright © 2006 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
and mouse CD3
in 
T-cell development revealed in a humanized CD3
) and 
) mice. Five to 8 mice per group were analyzed. Error bars indicate the mean plus or minus standard deviation.
) and 
) thymocytes cultured for 48 hours in plastic wells precoated with the indicated amount of anti-TCR