By
From the Basel Institute for Immunology, CH-4005 Basel, Switzerland
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Abstract |
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The striking and unique structural feature of the T cell receptor (TCR) chain is the bulky solvent-exposed FG loop on the C
domain, the size of almost half an immunoglobulin domain.
The location and size of this loop suggested immediately that it could be a crucial structural link
between the invariant CD3 subunits and antigen-recognizing
/
chains during TCR signaling.
However, functional analysis does not support the above notion, since transgene coding for TCR
chain lacking the complete FG loop supports normal
/
T cell development and function.
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Introduction |
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The /
T lymphocytes use clonally distributed TCR
to recognize cell-bound antigens, usually in the form
of peptides embedded in MHC molecules. The
/
TCR
is an oligomeric complex containing variable, covalently
bound
and
chains responsible for antigen recognition
and four noncovalently associated monomorphic subunits, CD3
,
,
, and
chain. The invariant subunits are crucial
for efficient assembly of the TCR and, hence, for surface
expression (1). In addition, they couple extracellular ligand
binding into cytoplasmic signaling machinery and, therefore, form an essential and the most proximal component
of TCR signal transduction (2).
Although some of the sequential biogenesis steps of the
TCR complex are quite well-characterized, the final complex on the cell surface is surprisingly poorly defined: not
only is the overall topology of the complex unknown, but so
is even the basic stoichiometry of the TCR, the most commonly proposed structure being TCR2CD3
2 (1).
Recently resolved three-dimensional structures of ectodomains of TCR and
/
chains have now offered some
potential insights into the puzzle of the TCR complex topology (3). The most striking feature of the structure of
the C
domain is the large 14-amino acid long FG loop
that protrudes freely into the solvent on the external face of
the C
domain. It was soon proposed that this loop would
interact with CD3 and, therefore, be part of the relay team
in TCR signal transduction (3). Recent more detailed structural analyses and simple elegant antibody/epitope mapping
of the TCR have added further details and suggested that
the loop would form part of the interface between CD3
and the C
domain (6, 7).
Here we report our finding that the TCR chain lacking
the complete 14-amino acid FG loop is able to support normal T cell development and function in transgenic mice.
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Materials and Methods |
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TCR- Mutagenesis.
Transfection of Cell Lines.
Infectious retroviral stocks were generated by transfecting packaging cell lines GP+E-86 (9) with retroviral expression vectors LXSN (neomycin resistant) coding for wild-type or mutant TCRMice.
BALB/c and C56BL/6 mice were purchased from IFFA-Credo. The TCR-Flow Cytometry and Antibodies.
Immunofluorescence stainings were done as described previously (12). Flow cytometric analysis was performed with a FACSCaliburTM equipped with CellQuest software (Becton Dickinson). The reagents used were mAbs biotinylated 145-2C11 (anti-CD3T Cell Functional Assays.
For T cell proliferation, 2 × 105 spleen cells were cultured in triplicate with various concentrations of staphylococcal enterotoxin B (SEB) and SEC 2 superantigens in 200 µl of IMDM supplemented with 10% FCS in 96-well flat-bottomed plates. Proliferative responses were assessed after 48 h of culture. Cultures were pulsed 8 h before harvesting with 1 µCi [3H]TdR (40 Ci/nmol; Radiochemical Center, Amersham Pharmacia Biotech), and incorporation of [3H]TdR was measured by liquid scintillation spectrometry. Helper T cell responses were tested by immunizing mice (three per group) with 100 µg of NIP-OVA in CFA in the tail base. For control, mice received PBS in CFA (referred to as CFA only in Fig. 3). After 14 d, sera from immunized mice were pooled and tested for the presence of anti-NIP IgG by ELISA as described (15). Plates coated with 5 µg/ml of NIP-BSA and then blocked with PBS/1% BSA received dilutions of the sera. Binding of the anti-NIP IgGs was revealed by alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates). Allogeneic killer cells were generated as described previously (8). In brief, 107 responders (H-2b splenocytes from wild-type TCR-
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Results and Discussion |
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To test whether the deletion of the complete 14-amino acid FG loop in the C domain would be
deleterious for the TCR assembly and surface expression,
we transfected TCR
T cell thymoma 58 with retroviral
vectors coding for either a control or a mutant
chain together with a wild-type
chain (10). To our initial surprise, the TCR surface expression was only slightly lower
in the mutant case (Fig. 1 A). However, we must point out
that the observed 30-50% reduction in the surface expression represents a handicap in the TCR assembly which, although small, is real since we have used a very efficient retroviral transfection system that allows us to create bulk
transformants containing thousands of individual clones and
which, therefore, provides us with a reliable statistical average. Functional analyses of these transfectomas consistently
showed that the cells transfected with the mutant TCR
chain responded slightly less (about threefold) to antigenic
stimulation as exemplified here by the dose-response curves
to influenza hemagglutinin peptide HA 110-119 (Fig. 1 B) or SEC 3 superantigen (Fig. 1 C).
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To more rigorously assess the functional potential of the TCR containing the mutant chain in normal physiological settings in
vivo, we generated transgenic mice expressing either a
wild-type or a loop-deleted version of the TCR
chain.
The
transgenes, as in the above transfection studies, were
derived from 14.3d T cell hybridoma expressing the TCR
specific for influenza hemagglutinin peptide HA 110-119 in the context of I-Ed MHC class II molecules (16). In fact,
it was the very same
chain (V
8.2-J
2.1) whose three-dimensional structure was first solved, thus providing us
with the inspiration for the current study (3). Two characteristics of the transgenic lines used here were considered
essential for straightforward interpretation of the data. First,
the level of
/
TCR expression was identical in both
lines (Fig. 2). Presumably the small handicap of the mutant
chain in the TCR assembly could be compensated by
higher intracellular expression. Second, both transgenes
were bred to TCR-
/
background to avoid any contribution of endogenous
chains for the observed
/
T cell
behavior (11).
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/
T cell development proceeds undisturbed
and similarly in both TCR
chain transgenic lines as
shown by flow cytometric analysis of thymic and lymph
node cells (Fig. 2). Even the skewing into single positive
CD4 thymocytes, as noted earlier for our wild-type TCR-
transgenic mice (8), occurs to the same extent in both lines.
As predicted, mAb H57-597 (anti-C
[13]) does not bind
to mutant TCR
chain (Fig. 2 H [6]). Interestingly, mAb F23.1 (anti-V
8.1, 2, 3 [14]) binds equally well to both
chains, whereas mAb MR5-2 (anti-V
8.1, 2 [17]) fails to
react with the mutant, suggesting that the FG loop may
form part of the MR5-2 epitope (not shown). Since the
cellularity of thymi is normal in both cases, we assume that
pre-TCR-mediated T cell expansion occurs normally in
these mice.
Peripheral T cell responses were measured in several types of assays, and none of them, to our disappointment, showed any significant differences between mice of
the different transgenic lines. The in vitro responses to anti-TCR antibodies (not shown) and to SEC 2 and SEB superantigens were repeatedly similar in all mice tested (Fig. 3,
A and B). In addition, the in vivo CD4+ T cell responses
measured by T cell help for hapten-specific IgG production
were basically indistinguishable between control and mutant mice (Fig. 3 C). Finally, /
T cells from mutant TCR
chain transgenic mice made as vigorous cytotoxic T cell
responses against allogeneic targets as their control counterparts (Fig. 3 D). We also monitored the representation of
four different V
families by flow cytometry in peripheral
T cells in order to reveal any subtle in vivo biases, but none
were found (Table I). In addition, limited DNA sequence
analyses of V
2 and 8 families from single
/
T cells
revealed no obvious "mutant"-specific features (data not
shown).
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Thus far, we have found only a
quantitative role in the TCR assembly process for the large
solvent-exposed FG loop on the C domain. In transfectants, the TCR will assemble in the absence of the loop in
the
chain but slightly less efficiently compared with the
wild-type structure. Of course, the reduced surface expression leads to somewhat impaired function. However, we
were able to show in vivo that TCRs are functionally expressed at the same level with or without the FG loop, and
we did not find any qualitative or quantitative differences
in their activity. This finding seems to rule out the models
where the FG loop has an absolute role in TCR signaling.
However, the apparent absence of any effect in vivo could
also be due to the fact that some subtle compensatory mechanisms have been turned on in vivo (but not in cell
lines), e.g., TCR affinities could be modulated, or new carbohydrate structures on the C
domain could partially replace the FG loop functionally. Interestingly, all nonmammalian species studied to date, including birds, amphibians,
reptiles, and fish, do not have the FG loop on their C
domain (18); hence, our in vivo findings may not be that surprising.
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Footnotes |
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Address correspondence to Sylvie Degermann or Klaus Karjalainen, Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland. Phone: 41-61-605-1249; Fax: 41-61-605-1364; E-mail: degermann{at}bii.ch, or karjalainen{at}bii.ch
Received for publication 30 November 1998 and in revised form 21 January 1999.
The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland. ![]() |
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