Different composition of the human and the mouse gammadelta T cell receptor explains different phenotypes of CD3gamma and CD3delta immunodeficiencies.

Abstract

The gammadelta T cell receptor for antigen (TCR) comprises the clonotypic TCRgammadelta, the CD3 (CD3gammaepsilon and/or CD3deltaepsilon), and the zetazeta dimers. gammadelta T cells do not develop in CD3gamma-deficient mice, whereas human patients lacking CD3gamma have abundant peripheral blood gammadelta T cells expressing high gammadelta TCR levels. In an attempt to identify the molecular basis for these discordant phenotypes, we determined the stoichiometries of mouse and human gammadelta TCRs using blue native polyacrylamide gel electrophoresis and anti-TCR-specific antibodies. The gammadelta TCR isolated in digitonin from primary and cultured human gammadelta T cells includes CD3delta, with a TCRgammadeltaCD3epsilon(2)deltagammazeta(2) stoichiometry. In CD3gamma-deficient patients, this may allow substitution of CD3gamma by the CD3delta chain and thereby support gammadelta T cell development. In contrast, the mouse gammadelta TCR does not incorporate CD3delta and has a TCRgammadeltaCD3epsilon(2)gamma(2)zeta(2) stoichiometry. CD3gamma-deficient mice exhibit a block in gammadelta T cell development. A human, but not a mouse, CD3delta transgene rescues gammadelta T cell development in mice lacking both mouse CD3delta and CD3gamma chains. This suggests important structural and/or functional differences between human and mouse CD3delta chains during gammadelta T cell development. Collectively, our results indicate that the different gammadelta T cell phenotypes between CD3gamma-deficient humans and mice can be explained by differences in their gammadelta TCR composition.

Full text

Page 1

The Journal of Experimental Medicine
2537
BRIEF DEFINITIVE REPORT
JEM © The Rockefeller University Press $30.00
Vol. 204, No. 11, October 29, 2007 2537-2544 www.jem.org/cgi/doi/10.1084/jem.20070782
The ? ? TCR is a multimeric complex consist-
ing of a clonotypic TCR ? ? heterodimer, the
CD3 ? ? and/or CD3 ? ? dimer, and the ? ? dimer.
Because ? ? TCR signaling regulates the com-
mitment of double-negative (CD4 ? CD8 ? ) cells
to the ? ? T cell lineage and is required for their
subsequent diff erentiation into mature ? ? T cells,
the development of ? ? T cells depends on the ex-
pression of the ? ? TCR. Indeed, neither CD3 ? -
nor CD3 ? -defi cient mice have ? ? T cells ( 1 – 3 ).
Although the overall structure and function of
the ? ? TCRs in mice and humans are quite
similar, ablation of the highly related CD3 ? and
CD3 ? subunits has markedly diff erent eff ects
in these two species. Hence, ? ? T cell develop-
ment is severely impaired in CD3 ? -defi cient
mice but not in their human counterparts ( 3, 4 ).
Conversely, CD3 ? defi ciency results in a block
in human, but not mouse, ? ? T cell develop-
ment ( 5, 6 ).
In contrast to the ? ? TCR, the ? ? TCR has
been studied extensively and its minimal stoichi-
ometry is proposed to be TCR ? ? CD3 ? 2 ? ? ? 2
in mice and humans ( 7, 8 ). Unlike the mouse
? ? TCR, mouse ? ? TCR does not incorporate
CD3 ? even if this subunit is expressed intra-
cellularly ( 9, 10 ), explaining why ? ? T cells
develop normally in CD3 ? -defi cient mice ( 6 ).
Interestingly, the composition of the mouse ? ?
TCR complex changes in activated cells: a dif-
ferentially glycosylated form of CD3 ? becomes
CORRESPONDENCE
Wolfgang Schamel:
schamel@immunbio.mpg.de
G.M. Siegers and M. Swamy contributed equally to this article.
The online version of this article contains supplemental material.
Diff erent composition of the human
and the mouse ? ? T cell receptor
explains diff erent phenotypes of CD3 ?
and CD3 ? immunodefi ciencies
Gabrielle M. Siegers, 1 Mahima Swamy, 1 Edgar Fern á ndez-Malav é , 2,4
Susana Minguet, 1 Sylvia Rathmann, 3 Alberto C. Guardo, 4
Ver ó nica P é rez-Flores, 4 Jose R. Regueiro, 4 Balbino Alarc ó n, 2 Paul Fisch, 3
and Wolfgang W.A. Schamel 1
1 Max-Planck-Institute of Immunobiology and University of Freiburg, 79108 Freiburg, Germany
2 Centro de Biolog í a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientifi cas, Universidad Aut ó noma
de Madrid, 28049 Madrid, Spain
3 Department of Pathology, University of Freiburg Medical Center, 79110 Freiburg, Germany
4 Inmunolog í a, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain
The ? ? T cell receptor for antigen (TCR) comprises the clonotypic TCR ? ? , the CD3 (CD3 ? ?
and/or CD3 ? ? ), and the ? ? dimers. ? ? T cells do not develop in CD3 ? -defi cient mice,
whereas human patients lacking CD3 ? have abundant peripheral blood ? ? T cells express-
ing high ? ? TCR levels. In an attempt to identify the molecular basis for these discordant
phenotypes, we determined the stoichiometries of mouse and human ? ? TCRs using blue
native polyacrylamide gel electrophoresis and anti-TCR – specifi c antibodies. The ? ? TCR
isolated in digitonin from primary and cultured human ? ? T cells includes CD3 ? , with a
TCR ? ? CD3 ? 2 ? ? ? 2 stoichiometry. In CD3 ? -defi cient patients, this may allow substitution of
CD3 ? by the CD3 ? chain and thereby support ? ? T cell development. In contrast, the
mouse ? ? TCR does not incorporate CD3 ? and has a TCR ? ? CD3 ? 2 ? 2 ? 2 stoichiometry.
CD3 ? -defi cient mice exhibit a block in ? ? T cell development. A human, but not a mouse,
CD3 ? transgene rescues ? ? T cell development in mice lacking both mouse CD3 ? and CD3 ?
chains. This suggests important structural and/or functional differences between human
and mouse CD3 ? chains during ? ? T cell development. Collectively, our results indicate that
the different ? ? T cell phenotypes between CD3 ? -defi cient humans and mice can be
explained by differences in their ? ? TCR composition.

Page 2

2538
STOICHIOMETRY OF THE ? ? TCR | Siegers et al.
http://www.jem.org/cgi/content/full/jem.20070782/DC1).
This was done for all experiments in which ? ? T cell clones
were used. In the fi rst experiment, we lysed human ? ? as well
as ? ? T cell clones and immunopurifi ed the TCRs with anti- ?
incorporated into the receptor ( 9 ) and ? can be substituted by
the FcR ? chain ( 10 ).
Contradictory fi ndings have been reported concerning
human ? ? TCR stoichiometry. Primary human ? ? T cells
were found to incorporate little or no CD3 ? into their surface
? ? TCRs ( 10 ). In contrast, human ? ? T cell clones and lines
were found to possess both CD3 ? ? and CD3 ? ? dimers ( 11, 12 ).
In light of the reported activation-induced changes in mouse
? ? TCR composition, it is possible that although CD3 ? is not
incorporated into TCRs of naive human ? ? T cells, this chain
becomes part of the receptor on ? ? T cell clones that have un-
dergone activation and expansion.
In this study, we use blue native PAGE (BN-PAGE) and
specifi c anti-CD3 antibodies to determine the stoichiometries
of human and mouse ? ? TCRs. These data are complemented
by studies on the human CD3 ? (hCD3 ? ) defi ciency pheno-
type, as well as those of CD3 ? ? -defi cient mice supplemented
with mouse or hCD3 ? transgenes. In conclusion, we show that
there are diff erences in the stoichiometries and, thus, subunit
requirements for the assembly of mouse and human ? ? TCRs.
RESULTS AND DISCUSSION
? ? T cells with high levels of ? ? TCR are present
in CD3 ? -defi cient patients
In CD3 ? knockout (CD3 ? ? / ? ) mice, ? ? T cell development
is blocked ( 3 ); however, this is not the case in CD3 ? -defi cient
humans. We have studied four CD3 ? -defi cient patients ( 13, 14 ),
including one ?20 yr old, and consistently found that ? ?
T cells are present in their peripheral blood ( Fig. 1 A ). As is
the case with ? ? T cells, the number of ? ? T cells in these
patients was at or just below the lower limit (P5) of healthy
CD3 ? -suffi cient controls. In the absence of CD3 ? , CD3 ex-
pression by ? ? T cells is reduced to ? 20% of that of healthy
controls ( 4 ). However, when we analyzed ? ? T cells from
these patients by fl ow cytometry using anti-CD3 antibodies,
we found that the amount of ? ? TCR per T cell was only re-
duced to 30 – 55% of healthy individuals, depending on the
antibody used ( Fig. 1, B and C ). These data show that hCD3 ?
can compensate, at least partially, for the lack of hCD3 ? in as-
sembly and surface transport of the human ? ? TCR. In fact,
in the absence of CD3 ? , these processes appear to occur more
effi ciently in ? ? T cells than in ? ? T cells. As a consequence,
? ? T cells can develop in CD3 ? -defi cient patients, indicating
that hCD3 ? can functionally replace hCD3 ? to promote ? ?
T cell development. In conclusion, the human ? ? TCR can
assemble and signal for selection effi ciently without hCD3 ? .
The human ? ? TCR includes CD3 ?
The diff erent subunit requirements for ? ? T cell development
in mice and humans could refl ect distinct ? ? TCR subunit
composition in these species. To clarify the composition of the
human ? ? TCR, we used established human ? ? T cell clones
as well as primary ? ? T cells. Because our ? ? T cell clones
contained ? 5% residual irradiated feeder cells expressing the
? ? TCR, we depleted ? ? TCRs after cell lysis by immuno-
purifi cation with anti-TCR ? antibodies (Fig. S1, available at
Figure 1. CD3 ? -defi cient patients show abundant peripheral blood
? ? T cells with high levels of ? ? TCR. (A) Presence of ? ? T cells in
hCD3 ? defi ciency. Peripheral blood cell counts from four CD3 ? -defi cient
individuals are plotted as a function of age in comparison with the
normal distribution (dashed line). Three were homozygous for a p.K69X
mutation (triangles), and one was compound heterozygous for p.M1V and
p.N28V/H29X (squares). CD3 ? -defi cient patients (circles) are included for
comparison. Filled symbols identify SCID patients, who died before 1 yr of
age. (B) CD3 expression is higher on CD3 ? -defi cient ? ? than ? ? T cells.
Flow cytometry histograms of anti-CD3 (SK7) – stained CD3 ? -defi cient
T cells (dashed lines) are compared with healthy controls (continuous lines)
either in ? ? (CD8 and CD4; top and middle) or ? ? (double negative; bottom)
T cells. Numbers indicate TCR expression (mean fl uorescence intensity)
on cells from CD3 ? -defi cient patients as a percentage of that on cells
from healthy donors. The vertical dashed line indicates the background
fl uorescence using an irrelevant antibody. (C) Quantifi cation of the CD3
expression on the indicated cell types from CD3 ? -defi cient patients as
a percentage of that on the same cell types from healthy donors
(percentage of CD3 expression). Data are expressed as the percent mean
fl uorescence intensity ? SEM from three different patients using the
anti-CD3 antibodies SK7 (left) or UCHT1 (right). Similar results were ob-
tained using other anti-CD3 antibodies, as well as other gating criteria
(not depicted). *, P ? 0.05 compared with ? ? T cells.

Page 3

JEM VOL. 204, October 29, 2007
2539
BRIEF DEFINITIVE REPORT
glycosylation is intrinsic to the ? ? TCR and not caused by
diff erent cellular environments of ? ? and ? ? T cells. Incorpor-
ation of CD3 ? into the ? ? TCRs of human clones and cell
lines was confi rmed by anti-CD3 ? immunoprecipitation and
subsequent anti- ? Western blotting (Fig. S1 and not depicted).
This is in line with earlier reports using ? ? T cell clones and
lines ( 11, 12 ). When primary human ? ? T cells were used,
CD3 ? could not be detected ( 10 ); however, the composition
of the mouse ? ? TCR changed upon cultivation of primary
? ? T cells such that ? was replaced by FcR ? ( 10 ). Likewise,
the TCR of primary human ? ? T cells might not contain CD3 ?
but may incorporate it during cultivation.
To determine whether CD3 ? is present in the ? ? TCR of
primary human T cells, we lysed PBMCs from a healthy do-
nor and purifi ed ? ? and ? ? TCRs with anti-TCR ? and anti-
TCR ? ? antibodies, respectively. Purifi ed proteins were left
untreated or deglycosylated and separated by SDS-PAGE
( Fig. 2 C ). Jurkat cells were used as a control. Indeed, the ? ?
antibodies. After nonreducing SDS-PAGE, purifi ed proteins
were detected using anti-CD3 ? and anti- ? antibodies ( Fig. 2 A ).
The ? ? TCR of the ? ? T cell line Jurkat and the ? ? clones
? ? B6 and ? ? PA (lanes 2 – 4), as well as the ? ? TCR of clones
? ? 19 and ? ? 46 (lanes 5 and 6), all contained CD3 ? . The re-
duced electrophoretic mobility of CD3 ? associated with the ? ?
TCR could be caused by more complex glycosylation ( 11 ).
To test this, we treated purifi ed TCRs with N-glycosidase F,
which cleaves N-linked carbohydrate moieties. Indeed, the
de glycosylated forms of ? ? TCR – and ? ? TCR – associated
CD3 ? are the same size ( Fig. 2 B , lanes 2, 4, and 6). This clearly
indicates that the ? ? TCR expressed on human ? ? clones
contains CD3 ? .
To identify the cause of this diff erential glycosylation, we
used an ? ? TCR – defi cient variant of Jurkat stably expressing
transfected TCRV ? 9 and V ? 2 chains, named J ? 9 ? 2 ( 15 ). The
CD3 ? of J ? 9 ? 2 had a similar mobility to CD3 ? in the ? ?
clones ( Fig. 2 B , lanes 5 and 7). Therefore, the complex CD3 ?
Figure 2. The human ? ? TCR includes CD3 ? . (A) Human ? ? T cell clones incorporate CD3 ? into the ? ? TCR. Anti- ? immunopurifi ed TCRs from Jurkat,
? ? ( ? ? B6 and ? ? PA), and ? ? ( ? ? 19 and ? ? 46) T cell clones were separated on nonreducing SDS-PAGE and analyzed via Western blotting using anti-
CD3 ? and anti- ? antibodies. In the control (C), which was loaded on another gel, anti- ? antibodies and protein G – coupled sepharose were incubated in
lysis buffer alone. (B) ? ? TCR – and ? ? TCR – associated CD3 ? chains are differentially glycosylated. Anti- ? immunopurifi ed TCRs from ? ? ( ? ? B6 and
? ? PA) and ? ? ( ? ? 19) T cell clones, as well as the Jurkat variant J ? 9 ? 2, were left untreated ( ? ) or subject to N-glycosidase F treatment (?) and analyzed
as in A. Glycosylated (CD3 ? ) and deglycosylated (dg-CD3 ? ) CD3 ? chains are indicated by arrowheads. (C) The ? ? TCR on primary human ? ? T cells con-
tains CD3 ? . TCRs from Jurkat and human PBMCs were immunopurifi ed using anti-TCR ? and anti-TCR ? ? antibodies and subjected to deglycosylation and
analysis as in B.

Page 4

2540
STOICHIOMETRY OF THE ? ? TCR | Siegers et al.
each antibody molecule bound to the complex produces a
discrete change in electrophoretic mobility. At nonsaturating
antibody concentrations, a partial shift, indicating ? ? TCR
bound to only one antibody (TCR?Ab) and cross-linked
products, in which one antibody bound to two TCRs (marked
with X), were observed (lanes 2 and 3). These data show that
the human ? ? TCR incorporates two CD3 dimers.
To verify the specifi city of anti-CD3 ? and anti-CD3 ?
antibodies, we expressed individual mouse and human TCR
subunits in Drosophila S2 cells and performed subsequent
immunopurifi cations, verifying antibody specifi city for HMT3.2
(anti-hCD3 ? ) and APA1/2 (anti-hCD3 ? ; Fig. S2 A, available
at http://www.jem.org/cgi/content/full/jem.20070782/DC1).
Using these antibodies in the NAMOS assay revealed that one
copy each of CD3 ? and CD3 ? are present in the human ? ?
TCR ( Fig. 3 B , lanes 5 – 8). This is in agreement with the fact
that CD3 ? pairs with either CD3 ? or CD3 ? ( 18 ). An anti-
TCR ? ? antibody produced only one shift, whereas anti- ?
produced two shifts (lanes 9 – 14). Although ? is a homodimer,
the antibody could not bind twice to most ? ? TCRs (lane 12).
This is caused by steric hindrance, because ? is very small
(16 kD) ( 8 ). The same band patterns were observed for several
other human ? ? T cell clones analyzed (unpublished data). In
conclusion, the digitonin-solubilized human ? ? TCR has a
stoichiometry of TCR ? ? CD3 ? 2 ? ? ? 2 .
The mouse ? ? TCR has a stoichiometry of TCR ? ? CD3 ? 2 ? 2 ? 2
The mouse ? ? TCR was reported to lack CD3 ? ( 10 ). We
aimed to study mouse ? ? TCR stoichiometry using our re-
agents and methods. Splenocytes from TCR ? ? / ? mice ( 19 )
carrying transgenes for the TCRV ? 1.1 and TCRV ? 6 chains
(TCR ? ? / ? ? 1 ? 6tg) ( 20 ) served as a source of primary ? ? T
cells. Initially, we compared the ? ? TCR from wild-type Bl/6
mice with the ? ? TCR from TCR ? ? / ? ? 1 ? 6tg mice by anti- ?
immunopurifi cation and anti-CD3 ? Western blotting ( Fig. 4 A ).
As expected, the ? ? TCR did not contain CD3 ? (lanes 3 and 4).
BN-PAGE showed that the digitonin-solubilized ? ? TCR
TCR and ? ? TCR roughly contained equal amounts of CD3 ?
(lanes 4 and 8, when normalized to ? ). The CD3 ? double
band from ? ? T cells exhibited a slower electrophoretic mo-
bility than that from ? ? T cells (lanes 3 and 7). In addition,
shorter ? chains, which probably represent diff erential mRNA
splicing, were incorporated into the ? ? TCR (lanes 7 and 8).
In an earlier study, CD3 ? was not found associated with the
? ? TCR from primary human T cells ( 10 ). It is likely that,
because of its diff erent glycosylation, the ? ? TCR – associated
CD3 ? had similar mobility to CD3 ? and, therefore, could not
be resolved when biotinylated proteins were detected by SDS-
PAGE and streptavidin Western blotting ( 10 ). In conclusion,
our data show that the human ? ? TCR contains CD3 ? in
cultured as well as in primary ? ? T cells.
The human ? ? TCR has a stoichiometry of TCR ? ? CD3 ? 2 ? ? ? 2
BN-PAGE is a method used to study the native structures of
multiprotein complexes ( 16 ). In our experiments, we used
this technique to analyze the size of the human ? ? TCR
compared with that of the ? ? TCR. After digitonin lysis of
Jurkat and the human ? ? T cell line Peer, as well as ? ? T cell
clones ? ? 19 and ? ? 46, TCRs were purifi ed, separated by
BN-PAGE, and detected by immunoblotting with an anti- ?
antibody ( Fig. 3 A ). The ? ? TCR, with a stoichiometry
of TCR ? ? CD3 ? 2 ? ? ? 2 ( 7, 8 ), had the same size as the ? ?
TCR, suggesting a similar stoichiometry for the ? ? TCR.
Similar results were obtained from nonpurifi ed TCRs (un-
published data).
To determine the stoichiometry of the human ? ? TCR,
we made use of the native antibody mobility shift (NAMOS)
assay that we previously developed to determine the ? ? TCR
stoichiometry ( 8, 17 ). The digitonin-extracted ? ? TCR was
incubated with diff erent amounts of an anti-CD3 ? antibody,
UCHT1, and then subjected to BN-PAGE ( Fig. 3 B , lanes 2 – 4).
At the highest antibody concentration, the ? ? TCR shifted
twice (lane 4, arrowhead labeled TCR?2Ab). This indicates
that the ? ? TCR has two binding sites for UCHT1, because
Figure 3. The human ? ? TCR has a stoichiometry of TCR ? ? CD3 ? 2 ? ? ? 2 . (A) The digitonin-solubilized ? ? TCR is the same size as the ? ? TCR. TCRs
from Jurkat, Peer, and the ? ? T cell clones ? ? 19 and ? ? 46 were purifi ed, separated by BN-PAGE, and analyzed via Western blotting using the anti- ? anti-
body. (B) Digitonin-extracted TCRs from ? ? T cell clone ? ? 19 were incubated with the indicated amounts of antibodies against hCD3 ? (UCHT1), hCD3 ?
(HMT3.2), hCD3 ? (APA1/2), ? (G3), and hTCR ? ? (5A6.E9), separated by BN-PAGE and analyzed as in A. The number of shifts correlates with the number of
antibody binding sites in the TCR complex, as indicated by arrowheads. The marker protein is ferritin in its 24-meric (f1, 440 kD) and 48-meric (f2) forms.

Page 5

JEM VOL. 204, October 29, 2007
2541
BRIEF DEFINITIVE REPORT
not depicted), indicating that hCD3 ? can functionally replace
mCD3 ? in the mouse ? ? TCR, whereas mCD3 ? cannot.
Sequence- and structure-wise, hCD3 ? is more related to
mCD3 ? than to mCD3 ? (Fig. S3, available at http://www.jem
.org/cgi/content/ full/jem.20070782/DC1) ( 24, 25 ). The
property of hCD3 ? to replace mCD3 ? is probably indepen-
dent of the signal-trans ducing immunoreceptor tyrosine-
based activation motif sequence of the cytoplasmic tail, because
? ? T cell development is unaff ected in mice lacking the CD3 ?
immunoreceptor tyrosine-based activation motif ( 26 ). Thus, the
functional diff erences between hCD3 ? and mCD3 ? might map
to the extracellular region, as the transmembrane regions are
highly conserved between the diff erent CD3 chains (Fig. S3).
Indeed, the ectodomains are critical for TCR assembly, suggest-
ing that hCD3 ? can assemble within mouse TCR ? ? to form a
functional ? ? TCR, whereas mCD3 ? cannot. This conclusion
is in line with the fi nding that mouse TCR ? can assemble with
hCD3 ? ? ( 15 ), in contrast to mCD3 ? ? .
In our mice, ? ? TCR expression was reduced to 60%,
whereas that of ? ? TCR was 80 – 100% of wild-type TCR levels.
As in hCD3 ? -defi cient patients ( Fig. 1 ), in CD3 ? ? ? / ? -
hCD3 ? tg mice, the ? ? TCR expression level was less af-
fected by the absence of CD3 ? than that of the ? ? TCR
( Fig. 5, E and F ) when compared with wild-type mice.
The stoichiometries of human and mouse ? ? TCRs cor-
relate well with the phenotypes for human and mCD3 de-
ficiencies. Mice lacking CD3 ? exhibit normal ? ? T cell
development ( 6 ), consistent with the fi nding that mCD3 ? is
not part of the mouse ? ? TCR ( 9, 10 ). In contrast, CD3 ? ? / ?
mice do not contain ? ? T cells ( 3 ), because CD3 ? is an oblig-
atory subunit of the mouse ? ? TCR. In humans, both CD3 ?
and CD3 ? are part of the ? ? TCR ( Figs. 2 and 3 ; and Fig. S1).
CD3 ? -defi cient ? ? TCRs are still able to support ? ? T cell
development in humans ( Fig.1 ), likely because hCD3 ? can
partially substitute for hCD3 ? . Remarkably, hCD3 ? is also
has a similar mobility to the ? ? TCR for which the stoi-
chiometry has been determined to be TCR ? ? CD3 ? 2 ? ? ? 2
( Fig. 4 B , lanes 1,4, 6, and 8) ( 8 ). To ascertain mouse ? ? TCR
stoichiometry, the NAMOS assay was applied using antibodies
that had been controlled for specifi city (Fig. S2 B). Anti-CD3 ? ,
as well as anti-CD3 ? , antibodies produced two shifts, indicating
that the mouse ? ? TCR contains two CD3 ? ? dimers ( Fig. 4 B ,
lanes 6 – 11). Because CD3 ? always pairs with either CD3 ?
or CD ? and because the mobility of the ? ? TCR in BN-
PAGE was the same as that of the ? ? TCR, we concluded that
the digitonin-solubilized mouse ? ? TCR has a stoichiometry
of TCR ? ? CD3 ? 2 ? 2 ? 2 . This stoichiometry is in agreement
with the conserved charge distributions in the transmembrane
segments of the ? ? and ? ? TCR subunits and with the 1:2
ratio of TCR ? ? /CD3 ? in primary mouse ? ? T cells ( 21 ).
Mouse TCR ? can bind to mouse CD3 ? ? (mCD3 ? ? ) but
not to mCD3 ? ? ( 9, 10, 21 ), whereas human TCR ? binds
both hCD3 ? ? and hCD3 ? ? ( 15 ). These results are in agree-
ment with the diff erent stoichiometries determined in our
experiments for human and mouse ? ? TCRs.
Human, but not mouse, CD3 ? can restore ? ? T cell
development in CD3 ? /CD3 ? double-defi cient
(CD3 ? ? ? / ? ) mice
We asked whether the diff erent subunit requirements for
mouse versus human ? ? TCR formation were caused by
sequence diff erences in their respective CD3 ? subunits. As
expected, our CD3 ? ? / ? and CD3 ? ? ? / ? mice both lack ? ?
T cells ( Fig. 5 B and not depicted) ( 3, 22 ). This was not caused
by limiting amounts of CD3 ? , because a mCD3 ? transgene
(mCD3 ? tg) could not rescue ? ? T cell development ( Fig. 5 C ).
In contrast, the CD3 ? ? ? / ? mouse strain carrying an hCD3 ?
transgene (CD3 ? ? ? / ? hCD3 ? tg) ( 23 ) has as many ? ? T cells as
wild-type mice ( Fig. 5, A and D ). These cells could be detected
in the thymus, spleen, lymph nodes, and blood ( Fig. 5 and
Figure 4. The mouse ? ? TCR has a stoichiometry of TCR ? ? CD3 ? 2 ? 2 ? 2 . (A) The ? ? TCR on primary mouse ? ? T cells does not contain CD3 ? . TCRs
from splenocytes from wild-type (Bl/6) and TCR ? ? / ? ? 1 ? 6tg mice were immunopurifi ed with an anti- ? antiserum. They were left untreated or were degly-
cosylated, separated via SDS-PAGE and analyzed by Western blotting as in Fig. 2 (B and C) . (B) The mouse ? ? TCR has two CD3 ? ? dimers. Splenocytes
from wild-type (Bl/6) and TCR ? ? / ? ? 1 ? 6tg mice were lysed in digitonin, and purifi ed TCRs were incubated with the indicated amounts of antibodies
against CD3 ? (145-2C11) or CD3 ? (17A2), separated by BN-PAGE, and analyzed by Western blotting as in Fig. 3 . Lanes 1, 4, 6, and 8 show TCRs alone. The
number of shifts correlates with the number of antibody binding sites in the TCR complex, as indicated by arrowheads. The marker protein is ferritin in its
24-meric (f1, 440 kD) and 48-meric (f2) forms.

Page 6

2542
STOICHIOMETRY OF THE ? ? TCR | Siegers et al.
? ? TCR stoichiometry is TCR ? ? CD3 ? 2 ? 2 ? 2 , as proposed by
Hayes and Love ( 21 ). Clarifi cation of both mouse and human
? ? TCR stoichiometries fi nally explains the diff erent pheno-
types observed in CD3-defi cient humans and mice. We show
that, in contrast to mCD3 ? , an hCD3 ? transgene is able to
rescue ? ? T cell development in mice lacking both mCD3 ?
and mCD3 ? . This indicates important structural and func-
tional diff erences between hCD3 ? and mCD3 ? chains, as
already suggested from the analysis of ? ? T cells ( 23 ). Indeed,
the phenotype of CD3 ? ? ? / ? hCD3 ? tg mice ( Fig. 5 ) resem-
bles that of CD3 ? -defi cient humans ( 4, 23 ), as opposed to
that of CD3 ? ? / ? mice ( 3 ). This is true for ? ? ( 23 ) as well as
for ? ? T cells ( Fig. 5 ). Thus, this humanized CD3 ? -defi cient
mouse strain may be a valuable tool to further study the im-
pact of CD3 ? -defi ciency in ? ? as well as ? ? T cell patho-
physiology in humans.
MATERIALS AND METHODS
Cells, mice, and antibodies. Human ? ? and ? ? T cell clones were generated
as previously described ( 28 ). TCR ? ? ? / ? Jurkat cells transfected with V ? 9 ? 2
(J ? 9 ? 2) were previously described ( 15 ). Human PBMCs were isolated from a
healthy donor using a Ficoll gradient. TCR ? ? / ? ( 19 ) and V ? 1.1V ? 6tg mice
(a gift of P. Pereira, Institut Pasteur, Paris, France) ( 20 ), both on a C57BL/6
able to rescue ? ? T cell development in CD3 ? ? ? / ? mice,
indicating its ability to substitute for mCD3 ? in the mouse ? ?
TCR as well. In contrast, CD3 ? -defi cient patients do not
develop ? ? T cells ( Fig. 1 A ) ( 5 ). Presumably, hCD3 ? cannot
substitute for hCD3 ? in human ? ? TCR formation and
function. Along this line, replacement of the mTCR ? connect-
ing peptide by the one of mTCR ? promotes the exclusion of
mCD3 ? from the complex ( 27 ), suggesting that the connect-
ing peptide of mTCR ? is involved in the association with
CD3 ? but does not permit the assembly of CD3 ? . A diff er-
ence in the connecting peptide sequences of human and mouse
TCR ? could be responsible for the diff erential involvement
in ? ? TCR assembly of CD3 ? in both species.
Conclusions
Using both conventional immunopurifi cation followed by
Western blotting and our novel NAMOS assay, we have
determined the human digitonin-solubilized ? ? TCR stoichi-
ometry to be TCR ? ? CD3 ? 2 ? ? ? 2 . The CD3 ? chain is diff er-
entially glycosylated depending on its association with the ? ?
or the ? ? TCR ( 11 ), likely accounting for contradictory
results previously reported ( 10, 12 ). We show that the mouse
Figure 5. hCD3 ? can substitute both mCD3 ? and mCD3 ? in ? ? T cell development. Thymocytes and splenocytes from wild-type (A), CD3 ? ? / ?
(B), CD3 ? ? ? / ? mCD3 ? tg (C), and CD3 ? ? ? / ? hCD3 ? tg (D) mice were surface stained with anti-CD3 (145-2C11) and anti-TCR ? ? (GL3) antibodies and analyzed
by fl ow cytometry. Percentages of cells in the marked regions and the total number of ? ? T cells (in millions) in the thymi are shown within and above the
dot plots, respectively. (E) CD3 expression is higher on CD3 ? -defi cient ? ? than ? ? T cells. Flow cytometry histograms of anti-CD3 (2C11) – stained
CD3 ? ? ? / ? hCD3 ? tg T cells (dashed lines) are compared with wild-type mice (continuous lines) either in ? ? (top) or ? ? (bottom) T cells from the thymus
(left) or spleen (right). (F) Quantifi cation of the CD3 expression on ? ? or ? ? T cells from CD3 ? ? ? / ? hCD3 ? tg mice as a percentage of that on the same cell
types from wild-type mice (percentage of CD3 expression). The CD3 high population was used for the ? ? TCR in thymocytes. Data are expressed as the
percent mean fl uorescence intensity ? SEM from two independent experiments. *, P ? 0.05 compared with ? ? T cells. mio, millions.

Page 7

JEM VOL. 204, October 29, 2007
2543
BRIEF DEFINITIVE REPORT
G.M. Siegers, grant R05/01 from the Deutsche Jose Carreras Leuk ä mie-Stiftung to
P. Fisch, and grants SFB620 B6 and Z2 from the Deutsche Forschungsgemeinschaft to
W.W. Schamel and P. Fisch. The support of the Fundacion Ramon Areces to the
Centro de Biologia Molecular is acknowledged.
The authors have no confl icting fi nancial interests.
Submitted: 17 April 2007
Accepted: 17 August 2007
REFERENCES
1 . Malissen , M. , A. Gillet , L. Ardouin , G. Bouvier , J. Trucy , P. Ferrier , E.
Vivier , and B. Malissen . 1995 . Altered T cell development in mice with
a targeted mutation of the CD3 ? gene. EMBO J. 14 : 4641 – 4653 .
2 . DeJarnette , J.B. , C.L. Sommers , K. Huang , K.J. Woodside , R.
Emmons , K. Katz , E.W. Shores , and P.E. Love . 1998 . Specifi c require-
ment for CD3 ? in T cell development. Proc. Natl. Acad. Sci. USA. 95 :
14909 – 14914 .
3 . Haks , M.C. , P. Krimpenfort , J. Borst , and A.M. Kruisbeek . 1998 . The
CD3 ? chain is essential for development of both the TCR ? ? and
TCR ? ? lineages. EMBO J. 17 : 1871 – 1882 .
4 . Recio , M.J. , M.A. Moreno-Pelayo , S.S. Kilic , A.C. Guardo , O. Sanal ,
L.M. Allende , V. Perez-Flores , A. Mencia , S. Modamio-Hoybjor ,
E. Seoane , and J.R. Regueiro . 2007 . Diff erential biological role of
CD3 chains revealed by human immunodefi ciencies. J. Immunol. 178 :
2556 – 2564 .
5 . Dadi , H.K. , A.J. Simon , and C.M. Roifman . 2003 . Eff ect of CD3 ? de-
fi ciency on maturation of ? ? and ? ? T-cell lineages in severe combined
immunodefi cieny. N. Engl. J. Med. 349 : 1821 – 1828 .
6 . Dave , V.P. , Z. Cao , C. Browne , B. Alarcon , G. Fernandez-Miguel ,
J. Lafaille , A. de la Hera , S. Tonegawa , and D.J. Kappes . 1997 . CD3 ?
defi ciency arrests development of the ? ? but not the ? ? T cell lineage.
EMBO J. 16 : 1360 – 1370 .
7 . Alarcon , B. , M. Swamy , H.M. van Santen , and W.W. Schamel . 2006 .
T-cell antigen-receptor stoichiometry: pre-clustering for sensitivity.
EMBO Rep. 7 : 490 – 495 .
8 . Swamy , M. , S. Minguet , G.M. Siegers , B. Alarcon , and W.W. Schamel .
2007 . A native antibody-based mobility-shift technique (NAMOS-
assay) to determine the stoichiometry of multiprotein complexes. J. Immunol.
Methods . 324 : 74 – 83 .
9 . Hayes , S.M. , K. Laky , D. El-Khoury , D.J. Kappes , B.J. Fowlkes , and
P.E. Love . 2002 . Activation-induced modifi cation in the CD3 complex
of the ? ? T cell receptor. J. Exp. Med. 196 : 1355 – 1361 .
10 . Hayes , S.M. , and P.E. Love . 2002 . Distinct structure and signaling
potential of the gamma delta TCR complex. Immunity . 16 : 827 – 838 .
11 . Krangel , M.S. , B.E. Bierer , P. Devlin , M. Clabby , J.L. Strominger ,
J. McLean , and M.B. Brenner . 1987 . T3 glycoprotein is functional al-
though structurally distinct on human T-cell receptor ? T lymphocytes.
Proc. Natl. Acad. Sci. USA. 84 : 3817 – 3821 .
12 . Van Neerven , J. , J.E. Coligan , and F. Koning . 1990 . Structural com-
parison of ? ? and ? ? T cell receptor-CD3 complexes reveals identical
subunit interactions but distinct cross-linking patterns of T cell receptor
chains. Eur. J. Immunol. 20 : 2105 – 2111 .
13 . Arnaiz-Villena , A. , M. Timon , A. Corell , P. Perez-Aciego , J.M.
Martin-Villa , and J.R. Regueiro . 1992 . Brief report: primary immuno-
defi ciency caused by mutations in the gene encoding the CD3 ? subunit
of the T-lymphocyte receptor. N. Engl. J. Med. 327 : 529 – 533 .
14 . van Tol , M.J.D. , O. Sanal , R. Langlois van den Bergh , Y. van de Wal ,
M.T.L. Roos , A.I. Berkel , J.M. Vossen, and F. Koning . 1997 . CD3 ?
chain defi ciency leads to a cellular immunodefi ciency with mild clinical
presentation. The Immunologist . Suppl. 1 : 41 – 42 .
15 . Alibaud , L. , J. Arnaud , R. Llobera , and B. Rubin . 2001 . On the role of
CD3 ? chains in TCR ? ? /CD3 complexes during assembly and mem-
brane expression. Scand. J. Immunol. 54 : 155 – 162 .
16 . Swamy , M. , G.M. Siegers , S. Minguet , B. Wollscheid , and W.W.
Schamel . 2006 . Blue native polyacrylamide gel electrophoresis (BN-PAGE)
for the identifi cation and analysis of multiprotein complexes. Sci. STKE .
2006 : pl4 .
17 . Schamel , W.W. , I. Arechaga , R.M. Risueno , H.M. van Santen , P.
Cabezas , C. Risco , J.M. Valpuesta , and B. Alarcon . 2005 . Coexistence
background, were mated, generating the TCR ? ? / ? ? 1 ? 6tg strain. CD3 ? ? / ?
( 3 ), CD3 ? ? ? / ? mCD3 ? tg, and CD3 ? ? ? / ? hCD3 ? tg ( 23 ) mice were previously
described (D. Kappes [Fox Chase Cancer Center, Philadelphia, PA] and
C. Terhorst [Beth Israel Deaconess Medical Center, Boston, MA] provided
the mCD3 ? tg and CD3 ? ? ? / ? mice, respectively). Mice were killed between
6 and 12 wk of age, and lymphocytes were isolated from tissues indicated in
the fi gures using standard protocols. Animal research was approved by the
Regierungspr ä sidium-Freiburg (G.02/84) and the local animal care com-
mission. Antibodies are described in Supplemental materials and methods
(available at http://www.jem.org/cgi/content/full/jem.20070782/DC1).
Flow cytometry. Normal distributions for ? ? and ? ? T cell numbers were
obtained from the literature ( 29, 30 ). The normal ranges (which include 90%
of the data) were depicted in a logarithmic scale as median values (dashed
line) between the 5th and 95th percentiles (P5 and P95). In CD3-defi cient
patients, ? ? T cells were defi ned as CD4 ? and CD8 ? or CD8 bright , thus ex-
cluding most ? ? T cells ( ? 8%). ? ? T cells were defi ned as surface TCR ? ?
using the antibodies 11F2 or Immu510. In patients, ? ? T cell counts may be
underestimated because of the ? ? TCR expression defect.
Mouse cells were stained with PE-conjugated GL3, FITC-conjugated
H57-597, and biotinylated 145-2C11 antibodies. Streptavidin – PE-Cy5 was used
as a second-step reagent. Stained cells were analyzed in a fl ow cytometer (FACS-
Calibur) using CellQuest software (both purchased from Becton Dickinson).
Cell lysis, TCR purifi cation, and deglycosylation. Cells were lysed
using 1% digitonin or 0.5% Brij96V, and immunoprecipitations were per-
formed using the antibodies 448, Jovi1, and 5A6.E9, as previously described
( 17 ). TCRs bound to the beads were treated with 1 U N-glycosidase F
(Roche Diagnostics).
For TCR immunopurifi cations used in BN-PAGE, 10 7 cells were incu-
bated with 200 ? M pervanadate and lysed, and phosphorylated proteins were
purifi ed with 2 ? g 4G10 and 5 ? l protein G – coupled sepharose (GE Health-
care). Native elution was done in BN buff er including 50 mM phenylphos-
phate, the detergent indicated in the fi gures, and phosphatase to dephosphorylate
the TCR ( 16, 17 ). In experiments in which ? ? T cell clones were used, ? ?
TCRs were depleted by two sequential immunodepletions using Jovi1 and
? F1 bound to protein G – coupled sepharose (Fig. S1).
Gel electrophoresis and Western blotting. SDS- and BN-PAGE were
performed using standard protocols ( 16 ). Ferritin in its 24- and 48-meric forms
was used as the marker protein (f1 and f2, 440 and 880 kD, respectively).
In brief, for the NAMOS assay (unpublished data), antibodies were added to
10 ? l of eluted purifi ed TCR before separation by BN-PAGE (4 – 9%).
Western blotting was performed according to standard protocols using 448
(1:5,000), M20, and M20 ? (both 1:1,000) antisera.
Online supplemental material. Supplemental materials and methods pro-
vides the specifi cities and sources of the antibodies. Fig. S1 A shows fl ow
cytometric analysis of a human ? ? T cell clone stained for ? ? and ? ? TCR.
Fig. S1 B shows Western blotting of anti – ? ? TCR depletion of a human ? ?
T cell clone lysate. Fig. S1 C shows immunopurifi cation of the TCR from
a human ? ? T cell clone using an anti-CD3 ? antibody. Fig. S2 (A and B)
shows that anti – human and anti – mouse CD3 ? and CD3 ? antibodies are
specifi c for their respective chains by immunoprecipitation of individually
expressed CD3 chains in Drosophila S2 cells. In Fig. S3, a sequence alignment
of hCD3 ? and mCD3 ? is discussed. Online supplemental material is avail-
able at http://www.jem.org/cgi/content/full/jem.20070782/DC1.
We would like to thank M. Reth for his scientifi c support; G. Turchinovich and
S. Glatzel for technical assistance; P. Pereira, D. Kappes, and C. Terhorst for mice;
and S. Kilic, O. Sanal, and L. Allende for patients ’ samples.
This work was supported by an Emmy Noether Fellowship to W.W. Schamel
from the Deutsche Forschungsgemeinschaft (SCHA 976/1), Ministerio de Educacion
y Cultura (MEC) grant BMC2002-01431 to E. Fern á ndez-Malav é , MEC grant
BFU2005-01738/BMC to J.R. Regueiro, the European Union – funded grant EPI-
PEP-VAC to S. Minguet, a University of Freiburg Wiedereinstiegsstipendium to

Page 8

2544
STOICHIOMETRY OF THE ? ? TCR | Siegers et al.
of multivalent and monovalent TCRs explains high sensitivity and wide
range of response. J. Exp. Med. 202 : 493 – 503 .
18 . Koning , F. , W.L. Maloy , and J.E. Coligan . 1990 . The implications of
subunit interactions for the structure of the T cell receptor-CD3 complex.
Eur. J. Immunol. 20 : 299 – 305 .
19 . Mombaerts , P. , A.R. Clarke , M.L. Hooper , and S. Tonegawa . 1991 .
Creation of a large genomic deletion at the T-cell antigen receptor
? -subunit locus in mouse embryonic stem cells by gene targeting. Proc.
Natl. Acad. Sci. USA. 88 : 3084 – 3087 .
20 . Malissen , M. , P. Pereira , D.J. Gerber , B. Malissen , and J.P. DiSanto .
1997 . The common cytokine receptor ? chain controls survival of ? ?
T cells. J. Exp. Med. 186 : 1277 – 1285 .
21 . Hayes , S.M. , and P.E. Love . 2006 . Stoichiometry of the murine ? ?
T cell receptor. J. Exp. Med. 203 : 47 – 52 .
22 . Wang , B. , N. Wang , M. Salio , A. Sharpe , D. Allen , J. She , and C. Terhorst .
1998 . Essential and partially overlapping role of CD3 ? and CD3 ? for
development of ? ? and ? ? T lymphocytes. J. Exp. Med. 188 : 1375 – 1380 .
23 . Fernandez-Malave , E. , N. Wang , M. Pulgar , W. Schamel , B. Alarcon ,
and C. Terhorst . 2006 . Overlapping functions of human CD3 and mouse
CD3 in T cell development revealed in a humanized CD3-defi cient
mouse. Blood . 108 : 3420 – 3426 .
24 . Sun , Z.Y. , S.T. Kim , I.C. Kim , A. Fahmy , E.L. Reinherz , and G. Wagner .
2004 . Solution structure of the CD3 ? ? ectodomain and comparison
with CD3 ? ? as a basis for modeling T cell receptor topology and signaling.
Proc. Natl. Acad. Sci. USA. 101 : 16867 – 16872 .
25 . Arnett , K.L. , S.C. Harrison , and D.C. Wiley . 2004 . Crystal structure
of a human CD3 ? ? dimer in complex with a UCHT1 single-chain
antibody fragment. Proc. Natl. Acad. Sci. USA. 101 : 16268 – 16273 .
26 . Haks , M.C. , T.A. Cordaro , J.H. van den Brakel , J.B. Haanen , E.F. de
Vries , J. Borst , P. Krimpenfort , and A.M. Kruisbeek . 2001 . A redundant
role of the CD3 ? -immunoreceptor tyrosine-based activation motif in
mature T cell function. J. Immunol. 166 : 2576 – 2588 .
27 . Werlen , G. , B. Hausmann , and E. Palmer . 2000 . A motif in the alpha-
beta T-cell receptor controls positive selection by moldulating ERK
activity. Nature . 406 : 422 – 426 .
28 . Fisch , P. , M. Malkovsky , E. Braakman , E. Sturm , R.L. Bolhuis , A.
Prieve , J.A. Sosman , V.A. Lam , and P.M. Sondel . 1990 . ? ? T cell clones
and natural killer cell clones mediate distinct patterns of non – major histo-
compatibility complex – restricted cytolysis. J. Exp. Med. 171 : 1567 – 1579 .
29 . Comans-Bitter , W.M. , R. de Groot , R. van den Beemd , H.J. Neijens ,
W.C. Hop , K. Groeneveld , H. Hooijkaas , and J.J. van Dongen . 1997 .
Immunophenotyping of blood lymphocytes in childhood. Reference
values for lymphocyte subpopulations. J. Pediatr. 130 : 388 – 393 .
30 . Ikinciogullari , A. , T. Kendirli , F. Dogu , Y. Egin , I. Reisli , S. Cin ,
and E. Babacan . 2004 . Peripheral blood lymphocyte subsets in healthy
Turkish children. Turk. J. Pediatr. 46 : 125 – 130 .