By
From the Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
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Abstract |
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A recent crystal structure of the N15 /
-T cell receptor (TCR) in complex with an Fab derived from the H57 C
-specific monoclonal antibody (mAb) shows the mAb fragment interacting with the elongated FG loop of the C
domain. This loop creates one side wall of a cavity within the TCR Ti-
/
constant region module (C
C
) while the CD and EF loops of
the C
domain form another wall. The cavity size is sufficient to accommodate a single nonglycosylated Ig domain such as the CD3
ectodomain. By using specific mAbs to mouse TCR-
(H57) and CD3
(2C11) subunits, we herein provide evidence that only one of the two CD3
chains within the TCR complex is located in close proximity to the TCR C
FG loop, in
support of the above notion. Moreover, analysis of T cells isolated from transgenic mice expressing both human and mouse CD3
genes shows that the heterologous human CD3
component can replace the mouse CD3
at this site. The location of one CD3
subunit within the rigid constant domain module has implications for the mechanism of signal transduction
throughout T cell development.
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Introduction |
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Each TCR consists of a clonotypic TCR heterodimer
(Ti-/
or Ti-
/
subunits) in complex with the invariant CD3 chains (
,
,
, and
). The disulfide-linked
heterodimer represents the peptide-MHC ligand binding
unit, thereby determining the ligand specificity of an individual T cell. In contrast, the CD3 polypeptides which are
in noncovalent association with a given Ti heterodimer, mediate TCR-base signal transduction (for review see references 1). Although CD3-
and -
are present in only
one copy each (6), it appears that two copies of CD3
and
exist per TCR complex (9). The signaling function of the CD3 components involves a conserved motif,
termed an immunoreceptor tyrosine-based activation motif
(ITAM) present in one to three copies in the cytoplasmic domain of each CD3 subunit (12, 13). The various CD3
subunits exhibit different interactions with intracellular signaling factors and induce distinct patterns of cellular protein
tyrosine phosphorylation upon activation (14). How
peptide-MHC ligand binding to a Ti-
/
or Ti-
/
heterodimer subsequently initiates signaling via the CD3 molecules is currently unknown.
Aside from their role in signal transduction, the CD3
subunits are also required for cell surface expression of the
TCR heterodimers on mature T lymphocytes (20, 21), as
well as for pre-T cell receptor function on immature
CD4CD8
double negative (DN)1 thymocytes (22, 23).
Thus, without CD3
or -
subunit expression there is a
marked decrease or absence of TCR molecules on the cell
surface as shown by in vitro analysis (20, 21, 24). In addition, in genetically engineered mouse strains in which these
CD3 components are deleted by homologous recombination, there is a developmental blockade of thymocytes at
the DN stage (25). The CD3
subunit, in contrast to
CD3
and -
chains, is required for TCR expression only
at a later stage of thymic development. The absence of
CD3
in a knockout mouse specifically blocks the thymic
selection processes mediating the transition from double positive (DP) (CD4+CD8+) to single positive (CD4+CD8
or CD4
CD8+) thymocytes (30).
Although the overall stoichiometry of the TCR complex is commonly given as TCR-/
-CD3
/
/
2/
2, there
is no direct structural evidence to support this subunit
composition. Recently, a three-dimensional structure of
the N15 vesicular stomatitis virus-specific/H-2Kb-restricted
/
-TCR (31) in complex with an Fab fragment from the
H57 anti-mouse C
-specific mAb (32) provided a clue
with which to infer new details about the association between the TCR-
/
heterodimer and CD3
(33). We
identified a cavity within the TCR-
/
C module formed
by the C
FG loop, partially exposed C
domain strands,
and conserved glycans from both C
and C
domains that
can accommodate a single Ig-like domain. Based on size
and charge considerations, we suggested that the cavity
probably represents the CD3
binding site. To determine
whether there is a physical proximity between the C
FG
loop and the CD3
chain, we performed a set of competition assays between the H57 and the CD3
-specific 2C11
mAbs (34). The results of these experiments provide evidence that one of the two CD3
subunits in a TCR complex is physically adjacent to the TCR-
constant region
FG loop.
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Materials and Methods |
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Transgenic Mice.
Transgenic mice expressing the human CD3Molecular Modeling of the N15 TCR-H57 Fab Complex.
The molecular modeling was produced using the N15 TCR and H57 Fab complex crystal structure 3D coordinates (PDB code 1NFD; 33). The plot of the protein structure was created using the programs MOLSCRIPT (36) and RASTER3D (37).Flow Cytometric Analysis.
The following mAbs were used: R-PE-labeled anti-mouse CD3 ![]() |
Results |
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The three-dimensional structure of a
complex between the N15 /
-TCR and the anti-TCR-
chain mAb H57 Fab fragment resolved crystallographically
to 2.8Å has revealed several interesting features about TCR
structure relative to antibody structure. As shown in Fig. 1,
there is an obvious difference between the ligand binding
surface of the V domain modules of the two receptors
(V
V
versus VHVL), with a relatively flat antigen binding
surface in the case of the TCR, versus a concave surface in
the case of the Fab. The flatness of the N15 antigen binding surface and the concavity of the H57 Fab surface are
complementary to the surface of their respective ligands, Kb
and C
. Perhaps the most striking difference between these
immunoreceptors is the symmetry of the Fab molecule
compared with asymmetry of the TCR molecule. This
TCR asymmetry results from the unusual arrangement of
the C
and C
domains relative to one another. Moreover, as shown in Fig. 1, the H57 Fab binds to the 12-residue C
FG loop conserved among TCR C
domains
from multiple species (39). This loop architecture is
uniquely rigidified by a hydrophobic minicore and an internal hydrogen bonding network. In addition, we have
noted that this loop creates one side wall of a cavity, while
the CD and EF loops of the C
domain form the other
side. The floor of the cave, which measures ~25Å in
depth, ~20Å in height, and ~25Å in width, is presumably
formed by the plasma membrane on the T cell surface. The
murine CD3
subunit, which is predicted to have an Ig
fold (40), is nonglycosylated unlike CD3
and CD3
, and
could readily fit into a cavity of this size.
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To investigate our hypothesis that a CD3 subunit might occupy
this cavity within the C module and hence lie proximal to
the C
FG loop, a set of competition assays between the H57
and 2C11 mAbs were performed using direct immunofluorescence analysis by FACS®. 2C11 was previously shown
to specifically bind to the mouse CD3
subunit (34). As
shown in Fig. 2 A, unlabeled competitor 2C11 or H57 was
added before the addition of FITC-2C11, FITC-H57, or
FITC-labeled anti-CD4, and log fluorescence was determined by quantitative FACS® analysis. As expected, unlabeled 2C11 blocks the subsequent binding of FITC-2C11
and, likewise, unlabeled H57 blocks the binding of FITC-H57. In contrast, neither unlabeled 2C11 nor H57 alters
the binding of the unrelated FITC-H129.19. More importantly, note that under these conditions the anti-CD3
mAb completely blocks the binding of FITC-H57 mAb,
whereas H57 only partially reduces the mFI of FITC-2C11
binding to CD3
on mouse splenic T cells. A second anti-CD3
mAb, 500A2, also completely blocked FITC-H57 binding and, as with FITC-2C11, the binding of PE-500A2 was reduced by 50% when T cells were preincubated with unlabeled H57 (data not shown). Moreover,
similar results were obtained by using 2C11 Fab or H57
Fab fragments for inhibition analysis. That the findings are
not secondary to "general" steric blockade of the TCR by
any mAb or Fab fragment binding to the complex is evident from the inability of V
-specific antibodies to block
FITC-H57 binding on TCR transgenic T lymphocytes
(data not shown).
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To quantify the mFI reduction in FITC-2C11
resulting from unlabeled H57 preincubation in individual
CD4 and CD8 T cell subsets, we performed the analogous
competition experiments by three-color analysis, in addition using PE-labeled anti-mouse CD4 and Red613-labeled anti-mouse CD8. We collected data comparable to that
shown in Fig. 2 A but on individual splenic CD4+ and CD8+
T lymphocytes. As shown in Fig. 2 B (left) for CD4+ T
cells, although unlabeled 2C11 completely blocked the
binding of FITC-2C11 to the mouse T cells, the unlabeled
H57 mAb reduced the binding of the FITC-2C11 by
~50% (mFI, from 402 to 183). On the other hand, unlabeled 2C11 almost completely blocked the binding of FITC-H57 (mFI, from 398 to 45; Fig. 2 B, right). Identical results
were obtained when CD8+ T cells subset were examined.
Three conclusions may be drawn from these data. First,
one CD3 subunit is close to the H57 mAb binding site on
the C
FG loop. Second, given that there are two CD3
components per TCR (10, 11) and the H57 mAb can only block 50% of the FITC-2C11 binding, the second CD3
subunit must exist at a distance from the C
FG loop.
Third, this postulated TCR subunit arrangement is found
in both CD4+ and CD8+ T cell subpopulations.
To test whether replacement of the murine CD3 chain with the human
CD3
subunit might alter the binding affinity of the H57 mAb to the C
FG epitope, we used a well-characterized
transgenic mouse strain (tg 600) engineered to express the
human CD3
component (reference 35; here referred to as
hCD3
tg). Splenocytes from littermates that do not express
the human CD3
(WT), as well as a heterozygous hCD3
tg
mouse, were stained with directly labeled FITC-2C11
mAb at concentrations ranging from 5 × 10
6-10
10 M. As shown in Fig. 3 A, the hCD3
tg mouse expressed only
~50% of the normal cell surface level of mouse CD3
.
However, 2C11 binding affinity to the mouse CD3
subunit is not altered by the presence of the human CD3
component (Kd ~10
8 M). The capacity of the unlabeled
2C11 mAb to block binding of FITC-H57 to heterozygous hCD3
tg T cells was also examined. As shown in Fig.
3 B, the same amount of unlabeled 2C11 that blocks FITC-H57 binding to the WT mouse T cells only partially
blocks the FITC-H57 binding to hCD3
tg T cells. This
suggests that the human CD3
subunit can replace the
mouse CD3
subunit in agreement with the finding of others (10, 41), and that it can occupy the cavity within the
mouse TCR C
C
module. Fig. 3 B also demonstrates that the incubation of the mouse splenocytes with the
2C11 mAb does not block the binding of H57 to the
TCR-
chain by downmodulation of the TCR complex
from the T cell surface (42, 43). Thus, unlabeled 2C11 preincubation does not change the binding of the anti V
8
mAb, FITC-F23.1, to T cells.
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In contrast to the results comparing mouse CD3 in
WT and hCD3
tg mice where the level of 2C11 expression is ~50% on the latter (Fig. 3 A), H57 reactivity is
equivalent in both (Fig. 4).This implies that the affinity of
H57 mAb to the C
FG loop is not altered by the presence
of the human CD3
component. Had human CD3
altered the affinity of H57, then the curves of mFI at different molar concentrations of FITC-H57 would have shown
a difference in the two mouse strains. Unlabeled 2C11
competitor mAb shifts the binding curve by ~2 logs, at
concentrations of FITC-H57 below 10
6 M. At the highest concentration of FITC-H57 (10
6 M), the labeled antibody binds similarly in the presence or absence of unlabeled competitor 2C11 mAb. These finding are consistent with the notion that the epitopes defined by H57 and
2C11 mAbs are distinct but nevertheless partially overlapping.
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Discussion |
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The crystal structure of the complex between the N15
TCR and an Fab fragment of the anti-C mAb H57 led us
to the hypothesis that one CD3
may physically associate
with the H57 mAb epitope on C
(33). In this paper we
demonstrate that one of the two CD3
subunits of the
TCR lies adjacent to the C
FG loop.
The overall shape of the TCR C domain module is remarkably asymmetric (Fig. 1). The C domain bends more
acutely towards the V
domain compared with the angle
formed between the C
and V
domains. C
also has an
unusually long and well-structured FG loop (the H57 mAb
epitope) projecting down from the V
-C
interface in all
TCRs studied to date (33, 44, 45). Although half of the C
domain's ABED sheet is surface exposed, it does not make
contact with the C
domain. As described in detail by
Wang et al. (33), this asymmetry creates a cave-like structure or cavity below the
chain sufficient in size to accommodate a single Ig domain. The partially exposed
ABED
sheet of the C
domain forms an extensive ceiling. The CD and EF loops of the C
domain, along with
the glycans attached to C
N185, C
N121, and C
N186 form one side wall, and the FG loop of C
and the glycan
emanating from C
N236 form the other side wall of the
cavity. The glycans project outward and do not obstruct
the cavity. The floor of the cave is presumably formed by
the plasma membrane on the T cell surface (33). It is noteworthy that the accessible surface at the
/
-TCR cavity
contains multiple basic residues.
The murine CD3 subunit consists of 87 residues in the
extracellular segment, and has Ig-like characteristics suggesting that this segment can readily fold into a small Ig domain (40). CD3
is the only CD3 subunit with a nonglycosylated Ig-like ectodomain in the TCR complex of man
and mouse (40). Moreover, the CD3
subunit has twice as
many acidic residues as basic ones and is therefore negatively charged (pI = 4.5, whereas the predicted pIs of the
CD3
and CD3
extracellular domains are more basic, being 8.76 and 5.82, respectively). The charge complementation between the acidic residues of the CD3
subunit and
the basic cavity also argues in favor of the CD3
subunit
occupying this site. The initial hypothesis regarding the
proximity of CD3
and the C
FG loop was confirmed
here by a set of competition assays in which the 2C11 and
H57 mAbs, which bind to epitopes on CD3
and TCR-
,
respectively, were able to alter each other's binding capacity. We further showed that although there are two CD3
subunits per TCR-
subunit, only one of the CD3
components is in close proximity to the C
FG loop. Given
that the
/
-TCR includes CD3 components (46) and is
predicted to contain an equivalent insertion in the corresponding constant domain loop (47) analogous to the C
FG loop, we can reasonably predict that a cavity with a comparable arrangement for CD3
exists in
/
T cells.
If the TCR C module cavity associates with one CD3,
where is the second CD3
located? In vitro translation
studies have shown that when CD3
is translated alone it
tends to form disulfide-linked oligomers. However, using
more physiological TCR-
/
-CD3 expression conditions,
namely simultaneous cotranslation of all the TCR subunits, the cotranslation of CD3
or CD3
was sufficient to keep
CD3
in a monomeric state (48). Both CD3
and CD3
compete for binding to CD3
(49). In addition, it was
shown that the CD3
/
pair associates with TCR-
,
whereas the CD3
/
pair associates with TCR-
. This association takes place upon formation of an intrachain disulfide bridge between TCR-
and -
(10). Additional studies have shown that CD3
and TCR-
pair via their
ectodomains, whereas the association of the other CD3
components with TCR-
and -
is largely mediated by interactions within their transmembrane regions. The proximity of TCR-
and CD3
has been suggested by cross-linking experiments in humans (50) and mice (7). Based on
these findings, it is likely that the CD3
subunit that is physically associated with the C
FG loop is paired with
CD3
and that the second CD3
, which pairs with the extracellular domain of the CD3
subunit, associates via its
transmembrane domain with the TCR-
(51). The ability
of the CD3
/
dimer-specific mAb 7D6 (52) to partially
inhibit FITC-H57 binding to T cells is also consistent with
this view (data not shown).
An interesting feature of the potential interaction of
CD3 within the TCR C domain module relates to certain
critical C
residues that are preserved in pre-T
(pT
), a
30-kD glycoprotein whose expression is restricted to early
CD4
CD8
DN T lineage cells (53). The pre-TCR, a
disulfide-linked heterodimer of a functionally rearranged
TCR-
chain and the pT
chain, noncovalently associated
with the CD3 components is expressed in DN immature thymocytes (56). During development, the DN to DP thymocyte transition is induced by an as yet unknown ligand
that binds to the pre-TCR, resulting in expression of mature type
/
TCR heterodimers on DP thymocytes (57-
59). Although there is only 12% identity between pT
and
C
in humans and mice (60), the C
residues involving
important polar interactions with the C
domain are all
conserved in the pT
sequence. Given that conserved
structural elements account for close to half of the pT
-C
amino acid sequence identities, it is very obvious that the
heterodimer interface of pT
-C
will be extremely similar
to that of C
-C
. With this in mind and since genetic disruption of the CD3
gene results in T cell development
blockage at the DN stage, it is likely that the CD3
subunit
is accommodated in the pre-TCR module in a manner similar to its mode in the mature
/
-TCR.
One can imagine that certain mouse CD3 residues contribute to the contacts made between H57 and the TCR.
In this regard, the total buried molecular surface area at the
N15 TCR-H57 Fab interaction is 1460A2 (720A2 for N15
and 740A2 for H57) (33), consistent with the range of values observed for other intact protein antigen-Fab interactions (61). However, one unusual feature of the N15-
H57 interaction is the predominance of contacts made by
the light chain (430A2 buried surface) rather than the heavy
chain (310A2 buried surface) (33). This unusual characteristic led us to postulate that the mAb H57 heavy chain may
interact primarily with TCR C
but in addition with some
CD3
residues. In agreement with that notion is the large
rotational mobility (~20°) of the Fab relative to the TCR
C
FG loop observed in the two independent copies of the
TCR-Fab complexes in the asymmetric unit of the crystal
(33). Nevertheless, analysis of the hemizygous hCD3
tg mice revealed that although those T cells contain only half
the surface copy number of the mouse CD3
, H57 affinity
is unaltered. This result suggests that the assembly of the
TCR complex is not affected by the expression of the human CD3
, and that the extracellular domain of the human
CD3
subunit can fit into the mouse TCR C domain
module cavity (Fig. 3 B). Given that the extracellular domain of the mouse and human CD3
share 53% identity in
their amino acid sequence, it is possible that any putative
CD3
residues that interact with the H57 heavy chain are
conserved among mouse and human CD3
.
Although the precise functional role of the FG loop in
C is far from clear, it is likely to have an important impact
on signaling and/or TCR assembly and structure. The
unique FG loop is conserved among sequences of mouse,
rat, human, and rabbit TCRs (39). Although absent in
TCRs sequences from some other species, it is replaced by
a potential glycosylated addition site whose glycan may
serve a similar structural function (33). Undoubtedly this loop influences the mobility and disposition of V
relative
to C
domains. Although the minihydrophobic patch of
this loop as resolved from the crystal structure fixes the
overall loop orientation relative to the V
and C
domains, local movements are permitted (33). How, if at all,
peptide-MHC interactions might transfer information to
the CD3 signaling subunits by affecting the FG C
loop
remains to be tested experimentally.
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Footnotes |
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Address correspondence to Ellis L. Reinherz, Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA 02115. Phone: 617-632-3412; Fax: 617-632-3351; E-mail: ellis_reinherz{at}dfci.harvard.edu
Received for publication 22 December 1997 and in revised form 5 March 1998.
We thank Drs. Linda K. Clayton and Raute Sunder-Plassmann for helpful comments on the manuscript and
Dr. Cox Terhorst for generous provision of the human CD3 transgenic mice.
This work was supported by National Institute of Health grants AI-19807 (to E.L. Reinherz) and AI-39098 (to H.-C. Chang).
Abbreviations used in this paper
DN, double negative;
DP, double positive;
mFI, mean fluorescence intensity;
pT, pre-T
;
tg, transgenic;
WT, wild-type.
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