Department of Immunology, Jerome H. Holland Laboratory for the Biomedical Sciences and George Washington University School of Medicine, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA
1 Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
Correspondence to: Correspondence to: L. M. Spain
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Abstract |
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Keywords: assembly, mutagenesis, signaling, TCR, transmembrane domain
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Introduction |
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To fully understand the signaling mechanism of the TCRCD3 complex, it is imperative to understand the structurefunction relationship of the TCR subunits. Different domains of the TCR chains not directly participating in antigen binding may nevertheless play roles in inter-subunit associations and in signal transduction. The presence of conserved, oppositely charged, amino acids in the transmembrane domains of all the subunits of the TCRCD3 complex predicted that these residues may have a critical function in the assembly and/or stabilization of the complex (10). Indeed, it has been shown that at least one of the two positively charged residues in the transmembrane domains of the TCR antigen binding chains is required for assembly and surface expression of the TCR complex (9). Deletion of a transmembrane leucine (L) alters the spacing of these charges and also prevents the assembly of the TCR complex (11).
Extracellular domains also are involved in inter-subunit associations and signaling. For example, a conserved domain within the TCR chain constant region connecting-peptide domain controls antigen responsiveness (12,13), while a single amino acid residue in TCR ß within the constant region was shown to be required for efficient signaling in response to superantigens (14). Confirming the importance of these sequences for the association of CD3 components, the TCR
mutation weakened the association of the
in the TCR complex in T hydridomas and in mature T cells (12), while preventing CD3
association and hence ERK activation during positive selection in thymocytes (13,15). An evolutionarily conserved serine in the extracellular E strand of Cß has been shown to be required for correct folding of the surrounding region, and for efficient assembly of the
ß dimer and surface TCR complex (16). Mutation of the interchain disulfide bond between
and ß did allow the surface expression of the complex but weakened the interactions with
without altering any signaling (17). The less conserved elbow-loop region of Cß was dispensable for development and function of most
ß T cells, but required for the subset of NK T cells which uses a canonical V
domain (18,19).
The TCR ß chain transmembrane domain contains several polar residues which have been highly conserved through evolution (20). We and others have investigated the importance of the well-conserved tyrosine (Y) residues in the transmembrane domain using site-directed mutagenesis. Previously, we found that mutation of Y to phenylalanine (F) at position 275 (YF275) of the transmembrane domain of the ß chain led to severe reduction in function including the dramatic reduction in cytokine production and reduced apoptosis in response to antigen (21). Apoptosis was more severely affected compared to cytokine production. Interestingly, this conservative mutation did not prevent the formation of TCR multimeric complex, which was expressed very well on the surface and found to be associated with . However, a study by another group using the human Jurkat thymoma line saw that mutation of Y to L at position 275 in the human TCR ß chain dramatically affected surface expression, association with
and apoptosis, but not cytokine production in response to stimulation (22). These differences in phenotype between two independent mutations at the same residue could be because of differences in cell lines (Jurkat versus murine hybridoma), or differences in ß chains (human versus murine), due to the presence of endogenous ß chain in the hybridoma cell line or lack of it, or different amino acid replacements.
In the present report, we extend our investigations of the function of TCR ß chain transmembrane Y by making non-conservative amino acid replacements and by expressing mutant receptors in primary splenic T cells as well as three different T hybridoma lines.
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Methods |
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Construction of TCR ß mutants
The basic protocol used for the site-directed mutagenesis was as described previously (21). In brief, site-directed mutagenesis of the target amino acids was carried out in pAlter plasmid vector using Altered sites II kit from Promega (Madison, WI). Mutant oligonucleotides were synthesized at the Oligonucleotide Synthesis Laboratory, Wistar Institute or Cybersyn (Lenni, PA). Oligonucleotides used for mutations, the amino acids targeted and the resulting amino acid residues are outlined in Table 1, where altered nucleotides are typed in bold face. The double mutants were generated by subjecting pAlter plasmid with a single mutation to a second round of mutagenesis using the appropriate oligonucleotide or with the oligonucleotide spanning both the desired mutations. Mutations were confirmed by sequencing double-stranded plasmid DNA in pAlter using a SP6 primer.
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Mutant and wild-type stable producer lines were made by standard calcium phosphate transfection of the MIN vector constructs into the retroviral producer cell line, 2 (24), followed by selection in G418 and pooling of resistant colonies. Retroviral producer lines were maintained in DME (Life Technologies, Gaithersburg, MD) supplemented with 10% calf serum (Cansera, Ontario, Canada) and antibiotics with G418. Titers of retrovirus produced by pooled populations of producer cells were high and it was not necessary to isolate individual producer clones.
Retroviral infection of T cell hybridomas and primary cells
2B4 cDNA was introduced to 58
cell line via MSCVpac retroviral-mediated gene transfer as described below for TCR ß. Puromycin-resistant colonies were pooled and maintained as 58
cell line in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, 20 µg/ml gentamycin, 50 µM 2-mercaptoethanol, 10% FCS (Cansera), 100 µM MEM non-essential amino acids and 10 mM HEPES (RP10). This
chain was found to be stable even after removal of the selection pressure.
58 or
28 T hybridomas were co-cultured with the MIN retroviral producer lines by incubating 5x105
2 producers with 2.5x104 58
T cell hybridomas in 5 ml RPMI containing 8 µg/ml polybrene (Aldrich, St Louis, MO) in one well of a six-well plate. After 48 h, the infected 58
hybridomas were removed from the surface of the producer monolayer and replated in RPMI containing 700 µg/ml G418. Spleen T cells from 2B4
TCR transgenic mice were plated at 2x106 cells/ml in 2 µg/ml Con A, in a 24-well dish, for 24 h. Retroviral supernatants (0.5 ml) plus polybrene (6 µg/ml) were added to each well and the plate spun at 700 r.p.m. for 50 min. Infections were repeated the next day and cultures were expanded 4-fold with IL-2 (10u/ml) for 7 days. Cells were harvested, re-stimulated with Con A and expanded into 0.5 mg/ml G418 for selection.
Antibody staining and flow cytometry
Approximately 15x105 T cell hybridomas were stained in round-microtiter wells in 20 µl volumes of HEPES-buffered saline/3% FCS or PBS/5% FCS. Background staining was blocked by pre-incubation and staining in the presence of 20% hamster serum (Cappel, Costa Mesa, CA). KJ25biotin (PharMingen) was added first and incubated for 20 min followed by washing 3 times, then streptavidinRed670 (Life Technologies, Gaithersburg, MD) plus A2B4FITC cocktail for 20 min. Cells were analyzed on a Becton Dickinson FACScan flow cytometer using CellQuest software. Similar staining procedures were used for other stains as needed.
T cell hybridoma stimulation assays
The MCC was added in serial dilutions as indicated. ß+ T hybridoma cells (5 x 104) expressing the 2B4 TCR were added together with 1x105 DCEK Hi7 L cells (APC) in 96-well flat-bottomed microtiter plates in 200 µl/well in triplicate. As controls, non-transduced 58
and wells containing no peptide or phorbol myristate acetate (10 ng/ml) plus ionomycin (0.35 µM) were included. Antibody cross-linking was performed by binding purified KJ25 antibody (PharMingen) to plates for 1 h at 37°C or overnight at 4°C, followed by 3 washes in PBS and subsequently seeding responder cells. Following a 24 h incubation, plates were frozen to kill the APC, thawed and the supernatants tested for cytokine concentrations. Then 5x103 indicator cells (CTLL-2) were cultured with 50 µl supernatants in 200 µl RPMI/well in 96-well microtiter plates. After 24 h, each well was pulsed with 1 µCi [3H]thymidine (Amersham, Piscataway, NJ) and incubated for a further 8 h before harvesting. Cells were harvested onto glass fiber filters, air dried and counted for 3 min using a direct ß-counter. For each population that induced a CTLL response, neutralizing antibodies against IL-4 were found to have no effect, while neutralizing IL-2 eliminated the response, confirming that the hybridomas produced only IL-2 (data not shown).
Apoptosis assays
After stimulation with either antigen or antibody, cells were collected from triplicate wells and stained with propidium iodide according to published procedures (27). DNA degradation was measured by FACS using doublet discrimination and the percentage of cells with less than 2N DNA content determined.
Immunoprecipitation and immunoblotting
Immunoprecipitation was carried out as described elsewhere (21). In summary, live T cell hybridoma cells were isolated by Ficoll gradient centrifugation followed by several washes in PBS. Cells (1x107) were lysed in lysis buffer containing Triton X-100 as detergent and then precipitated using Protein Aagarose beads prebound to the designated antibody. Protein separation and immunoblotting were carried out using standard techniques using appropriate antibodies, and visualized with horseradish peroxidase-bound Protein A and chemiluminescence.
For PTEN analysis, 107 cells were harvested, washed and lysed in NP-40 lysis buffer, pH 7.5, which included the following: 50 mM HEPES, 100 mM NaCl, 0.5% NP-40, 5 mM EDTA, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 10 mM ß-glycerophosphate, 1 mM Na3VO4, 10% glycerol and 1 mM dithiothreitol (all from Sigma-Aldrich, St Louis, MO), and Complete protease inhibitor cocktail (cat. no. 1 697 498; Boehringer-Mannheim/Roche, Nutley, NJ). After incubating in 75 µl lysis buffer on ice for 10 min, the lysates were centrifuged at 10,000 g at 4°C for 10 min. The resulting supernatants were collected and detergent-soluble protein concentrations measured using the BCA protein assay kit (Pierce, Rockford, IL). Thirty micrograms of proteins per lane was separated by reducing SDSPAGE and resolved proteins were transferred to PVDF (Imobilon; Millipore, Bedford MA) membranes by standard electroblotting using 25 mM Tris/40% methanol/192 mM glycine/0.01% SDS buffer, pH 8.0. Non-specific proteins were blocked (1 h at room temperature) with 2% BSA in Tris-buffered saline/Tween 20 (TBST). TBST included: 20 mM TrisHCl, pH 7.5, 100 mM NaCl and 0.1% Tween 20. Filters were then probed overnight at 4°C in BSA/TBST with mouse monoclonal anti-PTEN antibody (PTEN-A2B1, 1:400 dilution) from Santa Cruz Biotechnology (Santa Cruz, CA; cat. no. SC-7974) or anti-PTEN rabbit polyclonal from Cascade (Woburn, MA). The filters were then washed using TBST and then blocked again with fresh 2% BSA/TBST for at least 1 h. Finally, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG as required (Santa Cruz; 1:2000 dilution) for at least 1 h. Finally, the membranes were washed using TBST, followed by TBS. Specific proteins were detected using a peroxidase-based chemiluminescence system (Boehringer-Mannheim/Roche; cat. no. 1 500 694) per kit specifications.
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Results |
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For the 58 line, cell-surface expression of wild-type and mutant 2B4 ß chains was analyzed by flow cytometry using a mAb specific for Vß3 (KJ25) and results are shown in Fig. 1
(A). All four single residue ß chain mutants (YA265, YL265, YA275 and YL275) were expressed at similar levels on the surface, only slightly reduced relative to wild-type. Expression levels were stable and did not vary following different independent infections. The polyclonality of infected populations ensures that clone-to-clone variations will not affect the results (see 21). Flow cytometric analysis of TCR
expression using TCR
-specific antibody A2B4 or CD3
using CD3-specific antibody 500A2 showed similar expression profiles as that of TCR ß (data not shown). Taken together, these data suggest that a single Y to A or Y to L mutation at either of the conserved Y residues in the transmembrane domain allows surface expression of the TCR.
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TCR complex surface expression and function of T cell hybridomas with two Y mutations (YA265/275 or YL265/275) in TCRß chain transmembrane domain
Previous work by Rodriguez-Tarduchy and others showed that mutation of the C-terminal Y to L in human TCR ß chain affected surface expression and apoptosis but not cytokine production, once surface expression levels were normalized (22). However, our mutants with Y to L at either residue 275 or 265 did not show significant differences in surface expression and cytokine production, and only marginal differences in apoptosis (Fig. 1). Although we cannot explain why mouse TCR mutants expressed in hybridomas differ from human TCR mutants expressed in Jurkat cells, we nevertheless hypothesized that the two transmembrane Y might in some way compensate for each other in the 58
hybridoma line. To test this, we mutated both the residues to either A or L and expressed these double-mutant ß chains in T cell hybridomas as outlined above. Both double mutations (YA 265/275 or YL 265/275) were found to dramatically reduce TCR surface expression (by 1 log or more, Fig. 2A
). The Y to L mutation diminished expression to a greater extent than did the Y to A mutations. Thus, Y to A or Y to L mutation in both the conserved Y residues of the transmembrane domain is detrimental for surface expression of the TCR molecules.
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To determine whether the differences in function could be attributed entirely to differences in surface expression or whether additional functional defects could be noted, we used FACS to isolate sub-populations of hybridomas with matched expression levels. The YA265/275 mutant population was used because surface expression was less affected than the YL265/275. Even so, the mutant's expression was too low to allow us to isolate a population that matched wild-type levels even after three rounds of sorting. Therefore, we sorted the wild-type population for lowered levels of expression, to match with the YA265/275 population which was sorted for high-level expression. Despite matching surface expression, we found that the YA265/275 population produced significantly less IL-2 in response to peptide stimulation than did the wild-type cells (Fig. 2B, IV). Thus, the YA265/275 mutant combination affects signaling efficiency to a greater degree than predicted by surface expression alone, with the caveat being that under conditions of low expression even the wild-type receptor performs poorly.
Loss in function is specific to the conserved Y residues
It could be argued that any two mutations in such a short stretch of amino acids may alter the secondary structure of the mutant polypeptides and lead to the loss of function. To address this possibility, we chose another amino acid residue, threonine (T) at position 273, which is not as highly conserved in the transmembrane domain, as a candidate for double-mutation combination. We generated two other double-mutation combinations, i.e. TA273/YA275 and TA273/YL275, combining mutations at the T at position 273 and the Y at 275. Interestingly, both of these double mutants were expressed at the cell surface comparable to wild-type (Fig. 2A). The functional analysis of these mutants was consistent with their high surface expression (Fig. 2B
). Both TA273/YA275 and TA273/YL275 showed high levels of IL-2 production, in response to MCC (Fig. 2B
, I) or plate-bound antibody (Fig. 2B
, III), as well as apoptosis (Fig. 2B
, II).
Specific disruption of association to the TCR complex in mutants at position 265
We next evaluated the assembly of the TCR multimeric complex. We immunoprecipitated the TCR complexes using the pan-ß specific antibody (H-57). After the immunoprecipitation, the proteins were separated by gel electrophoresis, blotted, and probed with TCR-specific antibody H-28, CD3
-specific antibody HMT 3-1 and
-specific antibody H 146-C10. As shown in Fig. 3
, all the single mutants showed similar levels of TCR
association with TCRß. However, the two functionally deficient double mutants YA265/275 and YL265/275 showed very severe reductions of TCR
, CD3
and, especially,
association with TCR ß. We further examined
in TCR complexes immunoprecipitated from YA265/275 and wild-type populations which were sorted to match for surface expression. We found a significant reduction (5-fold) in
associated with the YA265/275 mutant as opposed to wild-type receptor (data not shown). Surprisingly, however, the presence of
in the TCR complex was also diminished when TCR ß was mutated at the single position 265 alone (Fig. 3
). These data suggest that the Y at position 265 is involved in the association of
to the TCR complex. This lack of apparent
association in the single mutants nevertheless allowed surface expression and signaling in the 58
hybridoma context (Fig. 1
).
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Expression and function of mutants in primary splenic T cells
Given the discrepancies in responses between the hybridomas, it becomes crucial to determine which cell line, 28 or 58
, most closely models the responses of non-immortalized, primary T cells. To test this, we introduced the Y mutant receptors into Con A-stimulated primary T cells from 2B4 TCR transgenic mice using retroviral vectors. We found that mutation of the N-terminal Y to L or A reduced TCR surface expression to just above background, while expression of the YA265/275 or YL265/275 doubly mutated receptors could not be detected by flow cytometry of primary T cells (data not shown). However, the YF265/275 double mutant was expressed reasonably well (Fig. 6A
). Although the mean of expression of YF265/275 mutants was lower than wild-type (mean fluorescence intensity for mutant was 520, compared to 1270 for wild-type), the range of expression of cells in the population was variable in both populations and largely overlapped (Fig. 6A
) Despite this, the YF265/275 mutant receptor was very severely deficient in IL-2 production in response to MCC peptide (Fig. 6B
). Both wild-type and mutant populations responded equivalently to stimulation by polyclonal activators such as Con A and anti-CD3
(data not shown). Therefore, we conclude that the conserved transmembrane Y residues are important for both TCR complex assembly and signaling efficiency in the physiological context of primary T cells stimulated in vitro, and furthermore that the
28 hybridoma line best mimics the functional responses of primary T cells.
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Discussion |
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How might the transmembrane Y contribute to TCR surface expression? We think it is unlikely that the mutations of transmembrane Y destabilize the TCRß protein since they are embedded in the membrane and since A, L or F replacements do not decrease the hydrophobicity of the domain. Furthermore, replacements of a less highly conserved but nearby T were without consequence. It is more likely that the Y residues are involved in important inter-subunit proteinprotein contacts that are required for transport of the complex to the cell surface. Generally, in mature T cells the TCR complex must be complete, and especially including , for its release from the ER and transport to the surface. Nevertheless, we found that YA or YL265 mutant TCR complexes expressed in 58
hybridomas lacked the
dimer as detected by immunoprecipitation, despite being efficiently expressed on the surface. Thus, in the context of the 58
hybridoma,
association is not strictly required for surface expression. This was also the case for the Y to L mutation of the C-terminal transmembrane Y in the human TCRß chain expressed on Jurkat cells (22).
The likely structural model for the TCRß transmembrane domain is an helix based on crystallographic studies of rhodopsin (29) and glycophorin (30) transmembrane domains. In the
-helical model, the Y residues at 265 and 275 are located on the same face of the molecule (20), which is consistent with our observations that these residues may have overlapping function. We also speculate that the human and murine receptors may differ in the relative usage of N- versus C-terminal transmembrane Y for
association to explain some of the differences between our results and those of Rodrigo-Tarduchy (22).
It is unlikely that the transmembrane Y are important for intracellular retention of -less complexes, as they are in the BCR (3134). Mutations of BCR transmembrane polar residues were shown to block the endoplasmic reticulum retention of the BCR in the absence of Ig
and Igß (31), resulting in BCR surface expression levels higher than wild-type. In the case of the TCR ß chain mutations described here and previously (22), surface expression is on average lower than wild-type. In addition, Rodrigo-Tarducy showed that the human TCR ß chain mutation increases the amount of immature (endo-H sensitive, ER form) TCR
ß heterodimer in the cells (22), suggesting that the mutant has a reduced, rather than enhanced ability to exit the ER. Therefore, it is more likely that the TCRß transmembrane residues are required for interactions directly or indirectly with members of the TCR complex.
Mutations within the transmembrane domain have been shown to affect
dimerization and assembly with the TCR complex (3537). However, since pairwise interactions between
and TCRß or any other individual member of the TCR complex have been difficult to demonstrate, any mutation in TCRß could alter
associations indirectly. This is not to suggest that transmembrane domains are the only regions involved in TCR assembly. For example, others have shown that deletion or mutation of conserved residues in the
chain extracellular domain alter its association with the TCR complex, resulting in reduced surface expression and signaling (37,38).
Importantly, our mutational analysis also reveals the involvement of transmembrane Y in signaling in addition to surface expression. This was first described in our previous work (21) using the 28 line and conservative Y to F mutations, in which surface expression was only moderately reduced while signaling (IL-2 production, apoptosis) was almost eliminated. Finally, and most importantly, the Y to F double 265/275 mutation shows the same phenotype in primary T cells as it did in the
28 line, i.e. moderate reductions in surface expression while IL-2 production in response to receptor stimulation was abolished.
How might the transmembrane Y contribute to signal transduction? In the case of the 28 hybridoma, we have previously shown that the TCR complex is intact and includes
in the YF265/275 double mutant. Nevertheless, since more drastic replacements do appear to disrupt
, especially in the 58
line, it is possible that the transmembrane Y are important in the signal transduction mechanism involving
. Triggering of the TCR appears to involve clustering and reorganization of TCR complexes and co-receptors together with adaptors and signaling molecules in detergent-insoluble lipid `rafts' (39). Reorganization of the receptor within the membrane bilayer is therefore an important component of signaling; membrane-imbedded residues such as the transmembrane Y may play an important role in this process. Future experiments are planned to investigate the partitioning of YF265/275 mutant receptors in the membrane during activation.
In this work we have also attempted to uncover the basis of differences we observed when TCRß mutants are expressed in different hybridomas and cell types. One obvious difference between 28 and 58
hybridomas is the presence or absence of endogenous TCRß expression. Recent experiments (40) have shown that in mice expressing two distinct transgenic Vß specificities, both Vßs can be co-immunoprecipitated. Co-assembly of two different Vß molecules per complex is also supported by fluorescence energy transfer experiments, which show that two TCRß molecules are within the same close range of each other as they are to CD3 components (40). The presence of more than one TCR ß chain per TCR complex could affect our experiments in which mutant receptors were expressed in cells (hybridomas
28 and
3A3, and primary T cells) which bear endogenous receptors (21). We found that when a wild-type D10 (Vß8) ß was co-expressed at high levels with the 2B4 wild-type or YF265/275 double mutant in 58
cells, signaling efficiency through the 2B4 receptor was greatly reduced. However, the co-expression of wild-type D10 TCRß did not affect the YF265/275 mutant phenotype any more than it did the 2B4 wild-type receptor. Therefore, it is unlikely that co-expression of wild-type receptor with mutant explains the signaling differences between
28 and 58
hybridomas. Instead, we speculate that immortalized hybridomas can differ from each other and from primary T cells and the Jurkat thymoma line in their TCR-mediated responses because fusion with the hybridoma partner can lead to clonal variance. Clonal variance can lead to differences in expression of co-stimulatory molecules, or downstream signal transducers, which could result in different signaling outcomes. Our own data support this notion since we have found that individual 58
and
28 hybridoma clones vary widely in their IL-2 production and apoptotic responses to peptides (S. K. and L. M. S., unpublished). In the experiments described here we have avoided problems with clonal variance by comparing only polyclonal populations derived from retroviral infection. However, our experiments in primary T cells help to define the consequence of TCRß transmembrane domain mutations in a physiologically relevant context. Future experiments will focus on the primary T cells and the
28 hybridomas which most closely mimic them.
Previous reports indicate that the apoptotic response is more dependent on signals emanating from than is cytokine production (5,41,42). However, we saw nearly normal apoptotic responses in YA265 mutant-bearing hybridomas that appear to lack
in immunoprecipitation assays. Our biochemical experiments cannot rule out the possibility that
may be weakly associated with the complex in YA265 mutants and is unable to survive detergent lysis, but still capable of transmitting signals relatively normally. Alternatively, the requirement for
in TCR signaling appears to be greatly dependent on TCR specificity. For example, mice deficient in CD3-
/
were reconstituted with
lacking its three ITAM sequences and this reconstitution allowed normal T cell development and function. However, crossing these mice to a H-Y-specific TCR transgene revealed dramatic disruptions in development and function (43), while the P14 TCR transgenes were relatively unaffected by the absence of
ITAMs (44). Therefore, future experiments will introduce transmembrane Y mutations into receptors with a range of affinities to test whether receptor affinity alters sensitivity to TCRß transmembrane mutations.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cell |
Con A concanavalin A |
MCC moth cytochrome c |
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Notes |
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Transmitting editor: C. Terhorst
Received 24 January 2000, accepted 2 November 2000.
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References |
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