0022-1 767/93/1506-2211$02.00/0
The Journal of Immunology
Copyright 0 1993 by The American Association of Immunologists
Vol 150, 2211-2221, No. 6, March 15,1993
Printed in U.S.A.
Use of Bispecific Heteroconjugated Antibodies
(Anti-T Cell Antigen Receptor x Anti-MHC
Class II) To Study Activation of T Cells with a
Full Length or Truncated Antigen
Receptor (-Chain
Samuel Wu,’* Yili Yang,* Scheherazade Sadegh-Nasseri,+ and Jonathan D. Ashwel12*
*Laboratory of Immune Cell Biology, Biological Response Modifiers Program, National Cancer Institute, and the
+LymphocyteBiology Section, Laboratory of Immunology, National Institutes of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, M D 20892
ABSTRACT.
Ligand-induced activation of T cells involves recognition of monovalent peptide Ag complexed with
a
cell surface MHC-encoded molecule. Incontrast, antibody-induced activation of
T cells typically requires external
cross-linking of the TCR. To examine the mechanisms that underlie the ability of these different stimuli to signal,
we have created bispecific chimeric antibody molecules (BA) that mimic Ag in several important aspects. AntiTCR-a,
-p, or anti-CD3-E Fab fragments were covalently
coupled toan anti-MHC class I1 Fab fragment. These BA
elicited IL-2 production or proliferation from Ag-specific T cell hybridoma cells or splenic T cells, respectively, in
the presence, but not the absence, of accessory cells expressing the appropriate M H C class II molecule. This
response wasprevented by soluble blocking antibodies against the TCR or MHCclass I I . When “presented” by MHC
class Il-bearing accessory cells, anti-TCR X anti-MHC class II BA, like cellsurface Ag, elicited IL-2 production from
T cell transfectants expressing full length TCR 5-chain but not fromotherwise identical cells expressing truncated
(;when immobilized ona plastic surface these EAwere potentstimulators that induced equal amounts of IL-2 from
the same cells. Purified Ag/MHC complexes immobilized on plastic were able to induce IL-2 production from T
cells expressing the full length, but notthe truncated, form of5.We hypothesizethat TCR-mediated T cell activation
requires stable aggregation of the TCR. In this model, activation by mobile cell surface Ag/MHC or BA occurs in
two steps, occupancy-induced TCR clustering followed by stable aggregation facilitated by the presence of a full
length (-chain. Immobilized high affinity anti-TCR antibodies, but not low affinity Ag/MHC complexes, directly
promote stable receptor aggregates, and thus would notrequire afull length [-chain. lournalofImmunology, 1993,
150: 2211.
A
ntireceptor antibodies have been widely used to
simulate physiologic ligand binding. In particular, anti-TCR antibodies have been used to stimulate T cells in studies of the events that follow activation
(1). The underlying assumption is that these antibodies
elicit responses similar, if not identical, to responses initiated by the natural ligand, in this case the combination of
Agandan
MHC-encoded glycoprotein. BothAg/MHC
complexes andanti-TCR antibodies induce phosphatidylinositol hydrolysis, increases in intracellular Ca2+,
protein kinase C activation, tyrosine kinase activation,
Received for publication July 6, 1992. Accepted for publication December 7 ,
1992.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2211
’ Howard Hughes Scholar.
’Addresscorrespondenceand
reprint requests to Dr. Jonathan D. Ashwell,
Room 18-40, Building 10, National Institutes of Health, Bethesda, M D 20892.
221 2
EFFECT OF TCR-5 TRUNCATION ON T CELL ACTIVATION
and lymphokine secretion (2, 3). The parameters that influence the ability of anti-TCR antibodies to stimulate T
cellshave
been extensively studied (4-11). Although
some studies have demonstrated T cell activation in the
absence of Ab3 cross-linking (12), soluble anti-TCR
Ab
have generallybeen found to beineffectiveat inducing
full T cell activation. External cross-linking (via FcR on
accessory cells or secondary Ab, for example), or physicalimmobilization of thestimulatoryAb,
is necessary
and sufficient to activate T cell hybridomas (8). The requirements are less clear for resting normal T cells; some
laboratories have reported that immobilized Ab alone are
capable of stimulatingrestingTcells
to proliferate,although others have found that an accessory cell-derived
costimulatory signal is required (13-16). In any case, in
the great majority of cases Ab multivalency seems to be
necessary for initiating TCR-dependent activation. Similar results have been found in the unusual case of haptenspecific T cells; making the fluorescein molecule multivalent by coupling it to a large polymeric backbone such
as polyacrylamide ordextranallows
the solublecompound to activate fluorescein-specific T cell clones in the
absence of MHC-encoded molecules (17, 18).
Based on the need for multivalent binding, TCR clustering or aggregation is generally thought tobe a required
intermediate stage for coupling the TCR to
intracellular
signaling pathways. However, it has also been proposed
that conformational changesin theTCR areresponsible for
TCR-mediated cellular activation (19, 20). One key difference between Ab and physiologic ligand is that activation by the latter presumably involves TCR recognition of
a monovalentAglMHC complex.Although AglMHC complexes are presented as an array on the surface of the APC,
theirmonovalentnatureprecludestrue
externalcrosslinking of TCR; local aggregation of ligand and occupied
TCR, however, does occur (21, 22). An unanswered question is, do antireceptor Ab stimulate T cells in a manner
analogous to AglMHC, or do they initiate signaling in a
fundamentally different way?
One tool to probe the mechanisms underlying TCR signaling has been provided by T cell hybridomas bearing
partial or mutated TCR. Variants of 2B4.11, a pigeon cytochrome c-specific T cell hybridoma, that lackTCR-5 and
-q chains havebeen characterized (23). One such cell, MA
5.8, was found to produce high levels of IL-2 in response
to plate-bound anti-CD3 or anti-TCR-ap Ab but not to
Ag/MHC, although this result couldnot be unambiguously
interpreted because these mutant cells expressed only 5%
as much cell surface TCR as the
wild type. However, when
cell surface TCR expression was restored to normal by
transfection of another 5- 2B4.11 derivative with either a
full length 5 cDNA or a cDNA that encoded a truncated
form of 5 (lacking the C-terminaland intracellular 57 amino
acidresidues)(24),a
similardichotomy wasobserved:
whereas both transfectants, FL (full length 6) and CT108
(truncated l),
responded well to immobilized anti-TCR-ap
or anti-CD3 Ab,
only the full length5 transfectant produced
IL-2 in response to pigeon cytochrome c.
To address how Ab and Ag-stimulation differ, we have
attempted to create Abmolecules that partially mimic Ag.
To this end, Fab fragments of various anti-TCR chain Ab
were ligated to the Fab fragment of an anti-MHC class I1
Ab. These bispecific heteroconjugated Ab constructs resemble Ag inthat they interact in a monovalent fashion with
the TCR and the MHC class I1 molecule and require the
presence of APC bearing the properMHC class11. We have
used these BA and an A g M H C unresponsive T cell hybridoma expressing atruncated 5-chain toexplore the
mechanism by which TCR occupancy or perturbation initiates T cell activation.
’
Abbreviations used in this paper:Ab, antibody; BA, bl-specific Ab: SPDP,
N-succin1midyi-3-(2-pyridyldithiol)propionate; AdMHC, Ag bound by MHCencoded molecules; FceRI,
Fc
receptor for Ig E.
Materials and Methods
Animals
BIO.A mice were the kind gift of Dr. Ronald Schwartz
(NationalInstitute of Allergy and InfectiousDiseases,
NIH).
Production and purification of Ab
The Ab used to make BA are listed in Table I. A2B4, H57,
and 2C 11 were preparedfrom tissue culture supernatant and
purified over a protein A-Sepharose affinity column. 103.6.2 andY-3P (anti-MHC classI1 I-Ab (25)) were prepared
from ascites by ammonium sulfate precipitation and passagesover an Ultrogel AcA34column
andaprotein
A-Sepharose affinity column. Purified Ab were dialyzed
into borate buffer (0.17 M boric acid, 0.12 M NaCl, 0.2%
sodium azide, pH 8.5) and concentrated by immersible-CX
filters (Millipore Corp., Bedford, MA).
Culture medium
Cells were maintained and assayed in RPMI 1640 (Biofluids Inc., Rockville, MD) supplemented with 10% heat
inactivated FCS, 4 mM glutamine,100 U/ml penicillin, 150
pg/ml gentamicin,and 5 X
M 2-ME(complete medium).
2B4.11, a cytochrome c-specific T cell hybridoma, was producedby polyethyleneglycol-mediated fusionof lymph
nodeT cells from Ag-primed BIO.A mice to an AKRderived thymoma, BW5 147 (26-28). Although originally
C D ~ +the
, 2~4.11
and its variants are all now C D ~ - ,
assessed
as
by flow
cytometry.
2 M.2,
a
lq-deficient
221 3
Journal of Immunology
rified from detergent solubilized LPS-stimulated C B N J B
2B4.11 variant, was transfected with full length or cytocell blasts using the 1 4 - 4 4 mAb for affinity chromatogplasmic truncated TCR (-chain cDNA constructs to yield
raphy as described (34). Purified Eak:Epkwas stored in 30
FL and CT108, respectively(24,29).
CT108 lacks the
mM octylglucoside in Tris (100 mM), NaCl (140 mM),
intracytoplasmic residues 108 to 164. L cells transfected
with MHC classI1 genes of various allelic origins are listed sodium azide (0.02%) pH 8.3 at 4°C. Emk:Epkmolecules
in Table I, and were the kind gift of Dr. Ronald Germain
were "loaded" with peptide as described (34). Briefly, pu(NationalInstitute of Allergy and InfectiousDiseases,
rified Emr:Eprmolecules (at a concentrationof 1 mM) were
NIH).
adjusted to a pH of 4.5 in the presence of 100 pM pigeon
fragment 81-104, and after a 60-min incubation at 37°C
the
reaction mixture was neutralized.
Bispecific Ab (BA) preparation
Fab fragments were prepared by digesting the Ab at 37°C
with a 1:20 or 1:100 (w/w) ratio of papain/Ab in a buffer
of 0.1 M sodium phosphate, 0.1 M NaC1, 1 mM EDTA, 20
mM cysteine, pH 8.0 (30). The reaction was terminated by
adding iodoacetamide to a final concentration of 30 mM.
The digested product was dialyzed against borate buffer,
passed over protein A-Sepharose, and then applied to an
Ultrogel AcA 34 column to remove Fc fragments and trace
contaminants of intact IgG. Analysis by SDS-PAGE indicated that all of the material migrated at 50 kDa. Fab fragments were cross-linked using the heterobifunctional reagent, SPSP, as described(3 1,32). Fab
fragments (one antiTCR,one 10-3.6.2) wereeachdialyzedagainst
0.1 M
potassium phosphate, 0.1 M NaCl, pH 7.5 (coupling buffer), and incubated separately for 0.5 h at room temperature
with twofold molar excess of SPDP in ethanol. The 103.6.2 Fab fragment was redialyzed against 0.1 M sodium
acetate, 0.1 M NaC1, pH 4.5; dithiothreitol was added to a
final concentration of 0.02 M. The reduced 10-3.6.2 Fab
fragmentwasimmediatelypassedthroughaPharmacia
PDlO column equilibrated with coupling buffer, and then
added to the nonreduced anti-TCR Fab fragment. Reduction of the SPDP-anti-MHC class I1 Fab fragment before
adding the SPDP-anti TCR Fab or SPDP-anti CD3 Fab
fragment minimizes the production
of anti-TCR X antiTCR, anti-CD3-E X anti-CD3-~, oranti-MHC class I1 X
anti-MHC class I1 Fab homodimers. After an 18-h incubation at 37"C, 1 mg of iodoacetamide was added and the
protein eluted from an Ultrogel ACA34 column (IBF Biotechnics, Savage, MD). Appropriate fractions were pooled
and rechromatographed on a Pharmacia FPLC Superose 6
column, and the dimeric bispecific anti-TCR X anti-MHC
class I1 fraction was taken for the experiments. In some
experiments, FPLC separation was performed twice to ensure purity of the Fab X Fab heteroconjugates. There were
no differences in the functional characteristics of the heteroconjugate fractions that were purified once or twice by
FPLC, indicating that one passage was sufficient to yield
a pure preparation.
Preparation of MHC class Il/pigeon fragment 81-1 04
complexes
Pigeon cytochrome c fragment 81-104 (pigeon fragment
81-104) was prepared as described (33). Emk:Epkwas pu-
Stimulation of T cell hybridomas
For most experiments, T hybridoma cells were cultured in
duplicateortriplicate
in 96-well,flat-bottomedmicrotiter plates (no. 3596, Costar, Cambridge, MA)in a volume
of 200 pl of completemediumatadensity
of 5 X lo4
cells/well. In some assays, the accessory cells RT, DCEK,
or FT were added at a concentration of 5 X lo4 cells/well.
In other experiments, the T cells were stimulated with Ab
that was adherent to plastic microtiter
wells (Costar no.
3596). This was accomplishedby incubating the plates with
the indicated purified intact Ab or BA at varying concentrations in 50 pl of PBS. After 2 h atroom temperature, the
wells were washed three times with complete medium and
used. In some experiments, T cells were stimulated
with
purified Emk:Epk/pigeonfragment8 1-104 complex. In
theseexperiments,varyingconcentrations
of preformed
Emk:Epk/peptide
complexes were directly placed in culture
wells (Immunolon 4, Dynatech Laboratories, Inc., Chantilly, VA) to allow for binding to the plastic surface. The
detergent concentration was diluted by adding 200 pl PBS,
pH 7.2, followed by several washeswith complete medium,
and 5 X lo4 Thybridomacells were added.Regardless
of the means of stimulation,afteranovernightculture
aliquots of supernatant were removed and IL-2 content determined.
IL-2 assay
After approximately 20 h of coculture with the indicated
stimuli, aliquots of supernatant were removed and frozen
to ensure that no viable cells were included. IL-2 content
was determined by incubating twofold serial dilutions of
supernatant with an IL-Zdependent T cell, CTLL (4 X IO3
cells/well). After 16 to 18 h, 1 pCi of ['HITdR was added
to each well for the final 6 to 8 h of culture. The incorporation of ['HITdR was determined by liquid scintillation
counting,andthe results areexpressedasunits
of IL-2
activity. One U is defined as the dilution of supernatant
causing CTLL cells to incorporate half-maximal amounts
of ['Hlthymidine (maximal levels were establishedin each
EFFECT OF TCR-6 TRUNCATION O N T CELL ACTIVATION
221 4
Table I
Antibodies and cells used
loool
Antibodies
(Abbreviation)
H57-597 (H57)
A2B4-2 (A2B4)
145-2C11 (2C11)
10-3.6.2
Transfected
accessory cells
Specificity
Reference
TCR-P chain
TCR-a
27
chain
CD3-E
MCC class II (LAk)
51
52
53
MHC Class I I
+ A2B4 X 10-3.6.2
Expression
RT7.3H3B (RT)
54
FT7.1C6 (FT)
55
DCEK.HC (DCEK)
I-Ak
I-Ab
LEk
* H57
X
10-3.6.2
+ 2Cll x 10-3.6.2
R. Cermain
and
J. Miller,
unwblished
0
experiment using MLA 144 supernatant as a source of
IL-2).
T cell proliferation assay
Freshly prepared spleen cells were cultured in triplicate in
96 well, flat-bottomed microtiter plates in a volume of 200
pl of complete medium at a density of lo5 cells/well with
indicated concentrations of BAS.After 48 hr the cells were
pulsed with 1 pCiof [3H]TdR, incubated overnight, and
harvested. The incorporation of r3H]TdR was determined
by liquid scintillation counting, and theresults expressed as
the averaged cpm.
Results
BA stimulation of T hybridoma cells
BA were prepared from Fab fragments of the Ab listed in
Table I, and experiments were performed to determine if
these bispecific heterochimeric Ab molecules could stimulate T cells. The Ag-specific 2B4.11 T cell hybridoma was
incubated with varying concentrations of three BA, each
specific for the I-Ak-encoded MHC class I1 molecule and
a different TCR chain. Inthe
presence of an I-Aktransfected L cell, all BA preparations, regardless of their
TCR chain specificity, induced2B4.11 cells to produce
IL-2 in a dose-dependent fashion (Fig. 1). Several approaches were usedto determine if BAcorecognition of the
TCR and MHC class I1 was important in this process (Fig.
2). When titered into microtiter wells containing AakApk
(I-Ak)-bearingL cells, the A2B4X 10-3.6.2 BA stimulated
IL-2 production. In contrast, in the absence of I-Ak-bearing
accessory cells, or in the presence of an L cell bearing the
inappropriate MHC class I1 molecule (I-Ab),the BA did not
induce IL-2 from the T cell hybridoma. Inasmuch as A2B4
does no&itself activate 2B4.11 T cells under these culture
conditions (27), it was possible to test whether the intact
Ab could prevent T cell activation by the BA by blocking
the binding of the BA to the TCR. The addition of soluble
A2B4 mAb, or soluble 10-3.6.2 mAb, which block BA
corecognition of the TCR and MHC class 11, respectively,
100
10
1
BA (W)
FIGURE 1. The 284.1 7 T cell hybridoma is activated by BA
in the presence of an MHC class11-bearing accessory cell.
Five X 1O4 284.11 T cells were incubated with 5 X l o 4 RT
cells plus the indicated concentrations of anti-TCR-a X antiMHC class II (A284 X 10-3.6.2; O),anti-TCR-fl X anti-antiMHC class II (H57 X 10-3.6.2; O),or anti-CD3-E X antiM H C class I I ( 2 C l l X 10-3.6.2; A). After anovernight
culture, supernatants were removed and assayed for IL-2
content.
prevented IL-2 production. Addition of equal amounts of an
irrelevant anti-MHC class I1 Ab, Y-3P (anti-I-Ab), had no
effect. Similar results were also obtained with the other BA
and 2B4.11cells, and mere aggregation of 2B4.11 andclass
11-bearing L cells with anti-H-2Kk Ab did not cause IL-2
production (data not shown). These results attest to the
“MHC-restricted” nature of the stimulus and the requirement for binding both the TCR and MHC class 11.
BA activate normal T cells
To determine if normal T cells can respond to BA, BIO.A
spleen cells, whose B cells and macrophages bear the LAkencoded class I1 molecule, were incubated with varying
concentrations of H57 X 10-3.6.2 (anti-TCR-P X anti-1Ak) and 2Cll X 10-3.6.2 (anti-CD3-E X anti-I-Ak). Both
BA preparations induced excellent T cell proliferative responses in a dose-dependent manner (Fig. 3). Under these
conditions, intact H57 and 2C11 cannot beused as potential blocking reagents, because they will activate the T
cells as a result of external cross-linking by FcR present on
splenic accessory cells (8). To circumvent this problem,
H57 or 2Cll Fab fragments were used in an
attempt to
block T cell proliferation. The splenic T cells’ response to
the BA was blockedby these soluble Fab fragments, as well
as by anti-MHC class I1Ab (Fig. 3). An irrelevant antiMHC class I1 Ab hadno effect on the proliferative response
to BA. Moreover, spleen cells from BIO.A (3R) mice,
which express I-Ab-encodedMHC class I1 (not recognized
221 5
Journal of Immunology
FIGURE 2. Blocking Ab against the TCR
or the appropriate MHC class I1 prevent BA
activation. Five X l o 4 284.11 cells were
cultured with the indicated concentrations
of anti-TCR-a X anti-MHC class II for 24 h
u n d e r the following conditions: (0)alone
(no accessory cells), (0)
RT (LAk) accessory cells, (A)FT (LAh) accessory cells, (A)
RT + soluble A2B4 (1 m M final), (a)RT +
soluble 10-3.6.2 (1 mM final), and (0)
RT
+ Y-3P (anti-l-Ab; 1 mM final). IL-2 was
measured as in Figure 1.
10 =
Y
.a3
+e
N
-
d
h
1:
L
-,-
.01
.1
1
Medium
RT
m
RT + soluble AZB4
RT + soluble 10-3.6.2
RT + soluble Y-3P
10
100
[A2B4 x 10-3.6.21(pM)
by 10-3.6.2), did not proliferate in response to the same BA
(data not shown). Together, these data prove that BA can
indeed stimulateT cells, andthat this stimulationis “MHCrestricted.”
Bivalent Fab heteroconjugates are responsible for
stimulatory activity of BA
It is possible that contaminatingheterotrimers of anti-TCR
X anti-TCR Fab X 10-3.6.2 Fab or higher order multimers
accounted forthe stimulatory capacity of BA. To determine
whether Fab multimers were involved in the T cell activation, an FPLC elutionprofile of the BA Ab preparations
was generated and each fraction analyzed for its ability to
stimulate 2B4.11 cells. One such elution profile, in which
intact IgG (- 150 kDa)and Fab fragments(-50 kDa) were
used to calibrate the column, is shown (Fig. 4). The major
protein peak, eluting with a retention volume of -72 ml
(solid line), corresponded to that predicted for Fab X Fab
dimeric heteroconjugates.A second smaller andtrailing
peak represented unreacted Fab fragments. No other specieswasdetected; notably, no materialwasfoundthat
eluted with a higher apparent M , than the Fab X Fab BA.
Concurrent bioassay demonstrated that the biologic activity
in thesamplewas
found in the Fab X Fabdimeric
heteroconjugate fraction. Similar elution and activity profiles were obtained with all three types of BA preparation.
In addition, for many preparations the BA sequentially underwent two,rather than one, separation by FPLC. In those
cases even theunreacted Fab peak was eliminated after the
first FPLC run, and no unexpected high M , material was
detectable. For those preparations as well, all of the biologic activity was found in the single (Fab X Fab) protein
peak (data not shown). Therefore, BA formed by the conjugation of two monomeric Fab fragments were respon-
sible for the T cell activating properties of the BA preparations.
BA stimulation is dependent on the mode of
presentation and an intact TCR c-chain
A (-negative variant of 2B4.11 has been transfected with
either a full length (FL) ora mutated (cDNA that encodes
a ( protein truncated at residue
108 (CT108; missing the
COOH-terminal 57 amino acid residues) (24). As shown
in Figure 5 , FL cells produced large amounts of IL-2 when
stimulated with Ag in the presence of I-Ek-transfected L
cells. As previously reported (24), CT108 cells did not respond in this experiment,even at the highest concentration of Ag tested. In other experiments, CT108 did beginto secrete 1L-2 whentheconcentration
of pigeon
fragment 81-104 was increased to 100-300 pM (data not
shown). This same dichotomy was evident when the BA
were analyzed. Although 2C11 X 10-3.6.2, A2B4 X 103.6.2,and H57 X 10-3.6.2 all inducedIL-2production
by FL cells, they were ineffective at inducing a response
from CT108 cells. There was a shift of 100-fold in the
BA dose-response curves between the two cells. The celldependent difference between the potency of the BA was
not due to an intrinsicproperty of the BA themselves;
when immobilized on a plastic surface, these BA induced
both FL and CT108 cells to produce IL-2 (Fig. 6). Importantly, the dose-responsecurvesto
plastic-adherent BA
wereidentical,rulingout
a simple inherentquantitative
difference in the potency of these BA to stimulate FL vs
CT108cells.Thus, themanner in whichthe BA is encountered by the TCR determines the cellular response;
when “presented” by MHCclass 11-bearing accessory
cells the BA resemble cell-boundAg/MHC, and when
immobilized they resemble Ab.
c
-
EFFECT OF TCR-6 TRUNCATION O N T CELL ACTIVATION
221 6
160000 1
a.
A
J
140000
-
120000
-
100000
-
80000
1
v
-@-
Medium
-0" H57Fab
* 10-3.6.2
* Y3P
6ooooi
40000
20000
1
0
160000
140000
P
1
120000
100000
Eu
80000
1
6oooolI / // --
4-
40000
r
Medium
2CllFab
4- 10-3.6.2
Y3P
20000
0
.01
.1
1
10
ficiently close proximity. To test this, one would have to
compare the ability of an immobilized low affinity ligand
to activate FL and CT108 cells. Because we do not have
very low affinity anti-TCR Ab, we asked whether an immobilized form of the natural TCR ligand, pigeon fragment
81-104 plus Eak:Epk,could stimulate FL and/or CT108
cells. The affinity (Kd) of Eak:Epk/cytochromec complexes
for soluble TCR from two Ag-specific T cell hybridomas
similar to 2B4.11has recently been estimated tobe
to
M, and the Kd of the 2B4.11 TCR itself wasfound to
be 5.3 X
M (35). These values are many orders of
magnitude lower than the Kd of monovalent Fab' of KJ25
anti-TCR Vp (2.4 X
M, (35)) orA2B4-2 (3.6 X
M, (R. H. Schwartz, personal communication)). Purified
Eak:Epkwas loaded with pigeon fragment 81-104 as described in Materials and Methods. Varying dilutions of the
Ag/MHC complex were adhered to plastic microtiter wells
andused to stimulate FLand CT108 cells (Fig. 7a).
Whereas the IL-2 response of FL cells was detectable at 0.3
pg/well of Ag/MHC complex and reached a plateau at 0.9
pg/well, the CT108 cells did not respond at even 1.5 pg/
well. Neither cell produced IL-2 when incubated with the
same amounts of purified Emk:Epkthat had not been
loaded
with Ag peptide (data not shown). As previously shown, in
the same experiment both cells responded equivalently to
plastic-bound anti-TCR Ab (Fig. 7b).This result was reproduced in three independent experiments. Recalling that
immobilized monovalent (for the TCR) BA were equally
stimulatory for both FL andCT 108 cells, these data indicate
that in the absence of full length 5, ligand immobilization
is not sufficient to generate an IL-2 response; a high affinity
TCR-ligand interaction is also required.
2C11 x 10-3.6.2 (W)
FIGURE 3. Spleen T cells are activated by BA in an "MHCDiscussion
restricted manner. Whole spleen cell populations from
BIO.A mice were cultured with the indicated concentrations
How receptor occupancy initiates signal transduction has
of either ( a ) anti-TCR-/3 X anti-MHC class II (H57 X 10-3.
been extensively studied. In many instances, cellular acti6.2) or ( b )anti-CD3-E X anti-MHC class II (2C11 X 10-3.6.2)
vation involves ligand binding followed by receptor agBA. The wells had the following additions: a, (0)medium
gregation, which in turn leads to signal generation. In the
H57 Fab (100 p M final), (0)
10-3.6.2 (100 p M
alone, (0)
final), (A)Y-3P (100 p M final). b, (0)medium alone, (0) case of the receptor for IgE (FceRI), Ab that cross-link
2 C l l Fab (100 p M final), (0)
10-3.6.2 (100 p M final), (A) receptor-bound IgE, or multivalent ligands recognized by
Y-3P (100 p M final).
the FceRI-bound IgE, activate basophiVmast cells to de-
Stimulation with immobilized Ag/MHC
The possibility was considered that for cells with a truncated [, the dependence of BA-mediated stimulation on the
manner of presentation (i.e., cell surface or plasticadherent) might reflect a need for stable TCR aggregates,
whose formation in the absence of full length 5 might rely
solely on external forces. It would be expected that the
stability of such TCR aggregates would be influenced by
both the state of ligand mobilization and the affinity of the
ligand-TCR interaction, because at some point a low affinity ligand would be unable to maintain the TCR in suf-
granulate (36). Although binding of bivalent Ab or (Fab'),
fragments against IgE causes degranulation, monovalent
Fab fragments do not. There is evidence that the density of
receptor aggregates and/or their degree of stability may be
a determinant in cellular activation. In the case of Abcrosslinked FceRI, the smallest aggregate (the receptor-bound
IgE dimer) is capable of triggering histamine release, but
in some cells IgE trimers and higher order oligomers of IgE
induce degranulation more effectively (37). Receptor mobility hasalso been implicated in thisprocess; aggregations
of two receptor-IgE complexes are predominantly mobile,
although clusters of more than two rapidly become immobile after formation (38). In the case of the insulin receptor,
221 7
Journal of Immunology
0.301
0.25
t
i
d
0
Fraction Number
FIGURE 4. FPLC elution profile of BA and their T cell stimulatory activity. An A2B4 X 10-3.6.2 BA preparation was purified
by sequential passage over an ACA 34 column followed by FPLC separation. The FPLC ODzsoelution profile is shown as a solid
line. The arrows indicate the expected elution fraction of BA trimers, dimers, and uncoupled Fab fragments. Aliquots were taken
from each fraction and the ability to induce IL-2 production by 2B4.11 cells (2 x 1 04/well) in the presence ofRT accessory cells
(2 X 1 o4/well) was measured (dotted line).
diverge. Our experiments explore thisdifferencewitha
antireceptor Ab simulate insulin activity (39). Both antiinsulin receptor bivalent(Fab’)* and monovalent Fab frag- stimulus that has some of the properties of both Ag/MHC
and anti-TCR Ab. Using a T cell hybridoma or normal T
ments caninhibit the binding of insulin, but onlythe (Fab’),
cells, BA behaved like an “MHC-restricted’ TCR ligand:
has agonist properties. Furthermore, addition of anti-Fab
Ab to cellstreated with antiinsulin receptor Fab fragments IL-2 production and T cell proliferation required accessory
cells bearing the correct MHC-encoded class I1 molecule,
restores the agonist property. For growth factor receptors
and blockade of either the TCR or MHC class
I1 prevented
such as those for epidermal
growth factor andplateletderived growth factor, Schlessinger and coworkers (40-43)T cell activation. In contrast to cells transfected with full
has proposed a model of “allosteric receptor oligomerizalength <,cells expressing truncated5 responded very poorly
to BA presented by MHC class 11-bearing accessory cells.
tion.” In this model, ligand binding stabilizes oligomeric
receptorcomplexes,whichpossessenhanced
ligandThus, theability of BA to stimulate normal and (-truncated
binding affinity and elevated protein tyrosine kinase acT cells was strikingly similar to that of AglMHC.
tivity. Such hetero- or homooligomers, involvingreceptors
FL and CT108 cells respondedidentically to plasticand perhaps accessory proteins, would bring about the acimmobilized BA. Therefore, although a full length (-chain
tivation of receptor tyrosine kinase activity by allosteric
was required for ‘‘occupancy’’-generated activation, it was
subunit interaction, resulting in intracellular signaling.
not needed for activation after TCR immobilization. A key
The mechanismby which the TCRtransduces signals is
observation is that although immobilized Ag/MHC is able
not known. There are at least two qualitatively different
to activate FL cells to produce IL-2, it is inefficient at acmechanisms by which TCR occupancy might lead to sigtivating CT108 cells.Assuming that thefundamental mechnaling. In one model, occupancy of single TCR induces
anism of activation is similar between Ab and Ag/MHC,
conformational changes in ap that initiate signal propahow do these dataconform to different models of the mechgation. In another scenario, TCR occupancy
results in local
anism of T cell activation? The results do not fit well with
clustering, which inturn initiates signalingevents. Because a simple conformational mechanism (TCR occupancy reof the complex natureof the TCR’s ligand, andits requisite
sulting in a change in a p conformation and then signaling),
association with the plasma membrane of a second cell,
because anti-TCR X anti-MHC classI1 BA as well as intact
much of our understanding in this area comes from work
anti-TCR Ab, regardless of which chain theybind (inin which anti-receptor Ab have been used to trigger the T
cluding the non-Ag-binding CD3-E chain),can trigger celcell. Although the ability of a T cell to be stimulated by
lular activation. The requirement for multivalent binding,
Ag/MHC or by Ab are usually inseparable, the
2B4.11
of Ag (17, 18) as well as Ab, also argues against this type
variant and its derivatives expressing truncated5 have proof conformational mechanism. The
inability of Ag/MHC or
vided a case in which the activities of these two stimuli
BA presented by MHC class 11-bearing accessory cells to
<-
221 8
EFFECT OF TCR-5 TRUNCATION ON T CELL ACTIVATION
/"
a. Antigen
100
lZ01
80
-F
L
CT108
.00001 .0001
.001
.01
.1
I
2C11 x 10-3.6.2(pglwell)
.1
1
10
pCC
'"1
81-104
(m)
100
100
lZO1
3
*a
3
b. 2Cll x 10-3.6.2
8ol
F
L
*- a 1 0 8
60
.00001 ,0001
.001
.01
.1
1
A2B4 x 10-3.6.2 (pglwell)
120
loo]
80
2
cr
loo-/ c. A2B4 x 10-3.6.2
80
0
.00001 .0001
J:
.001
.01 1
1
.1
H57 x 10-3.6.2 (pglwell)
O
L
.1
'"1
-
FIGURE 6. Stimulation of FL andCT108cells by BA immobilized on plastic. FL (0)
and CT108 (0)
cells were CUItured in microtiter wells that had been precoated with varying concentrations of the indicated BA.
"
1
10
100
d. H57 x 10-3.6.2
sol
60
.1
100 1
10
BA C
W)
FIGURE 5. Stimulation of FL andCT108cells
byAgor
soluble BA plus accessory cells. FL (0)
and CT108 (0)
cells
were cultured with (a) Ag plus I-Ek-bearing DCEK accessory
cells or (b-d)the indicated soluble BA plus I-Ak-bearing RT
accessory cells.
proximitywithimmobilizedBA,
not immobilizedAg/
MHC, is sufficient to stimulate cellsthat express truncated
<. These differences suggest that TCR clustering and the
formation of stable TCR aggregates are distinct phenomena. This can be understood if one considers the possibility
that TCR signaling is initiated in two steps. First, occupancy results in TCR clustering at the interfaceof the T cell
and the APC. Second, these clusters may be further modified in the presence of a full length TCR <-chain to form
a stable TCR aggregate. It is an intriguing speculation that
when in an occupancy-induced cluster, 1; might foster interactions between TCR themselves andor, perhaps, other
molecules such as cytoskeletal components, to promote
stable aggregation. In the case of Ab stimulation, TCR drawn
together at the celVplastic interface would be physically
prevented from moving in the membrane, thus stabilizing
TCR associations without requiring the participation of <.
This model would predict that in the absence of the tail
required for converting a TCR cluster into a stable aggregate, a relativelyhigh affinity TCR-ligand interaction must
be provided to maintain stable TCR associations. This pre-
<
stimulate IL-2 production by CT108 indicates that in the
absence of full length <,clustering alone is insufficient to
triggerfullactivation. Yet, drawingthe TCR intoclose
lournal of Immunology
loo
1
221 9
a. Adherent Ag/MHC
1
I
I
-0-
0.1
a108
I 0.01
1.51.20.0
51.00.750.50.25
Bound Ag/MHC (pg/Wefl)
loo
(44) or ubiquitination ( 4 9 , that in turn leads to stable aggregation. Thus, stable aggregate formation, the common
event in the initiation of TCR signaling, may be achieved
either by external immobilization or by conformationinduced and 5-dependent mechanisms. This type of conformational effect is distinct from a conformational change
in ap that serves as a proximal initiator of signal generation.
In a recent study, Malissen and coworkers described a
BW5147 transfectant that expressed TCR whose {-chains
have a large internal deletion in the cytoplasmic portion
(residues 66-157). These cells responded to stimulation
with anti-TCR Ab and AgMHC, although not to normally
stimulatory anti-Thy- 1 and anti-Ly-6Ab (46). Although the
BW5147 transfectants appear to differ in their phenotype
from CT108, the data obtained with the internal deletion
and CT108 truncation are in fact compatible. The BW5147
transfectants were selected for very high expression of cell
surface TCR, substantially higher than thelevels expressed
by the T cell hybridomas we have used (B. Malissen, personal communication). Inasmuch as Ag dose-response
curves were not done, one cannot quantitatively compare
the response of the BW5 147cells with a mutated 5 to those
with full length (-chains. It is noteworthy that 2B4.11 derivatives lacking any (-chain at all produce IL-2 when the
Ag concentration is sufficiently high (23), and CT108 cells
respond to Ag at very high concentrations (100-300 pM,
data not shown) as wellasto cell surface-bound BAat
concentrations 30- to 100-fold greater than those required
to activate FL cells (Fig. 5) (because of limits in theamount
of purified AgMHC that could be generated, we were unable to increase the amount of this ligand to equivalently
high points in the dose-response curve). Furthermore, we
have studied a CT108 subclone that expresses abnormally
high levels of cell surface TCR and, as expected, it is more
sensitive toAg/MHCthan the typical CT108 cells (responding to 30 pM pigeon fragment 81-104). Thus, all of
the data are consistent with the notion that loss of the internal portion of 5 severely impairs the ability of a cell to
respond to TCR occupancy, but does not completely prevent it if concentration of ligand (and thus the degree of
occupancy) is sufficiently high. This may indicate that clustering of TCR at high density is functionally equivalent to
formation of a stable aggregate.
Our findings allow us to build on a similar model that was
considered to deal with the initial observation that
5-deficient or (-truncated 2B4.11 cells responded well to
anti-TCR mAb but not to Ag/MHC (24). The fact that antiTCR X anti-MHC class I1 BA mimic AgMHC allows us
to exclude theories that would explain the differences between Ab and
AgMHC by invoking differences in the TCR
subregions or epitopes involved in ligand binding.
Interestingly, another study has used bispecific whole Ab
directed against the TCR and an MHC-encoded molecule
(in this case, class I) to study T cell activation (47). The
1
b. Adherent 2 C l l
.001
.01
F
.1
1
2 C l l (pg/well)
FIGURE 7. Stimulation of FL andCT108 cells bypurified
Ag/MHC complexes or anti-TCR rnAb immobilized on plastic. FL and CT108 cells were incubated in microtiter wells
coated with eitherEuk:Epk/pigeonfragment81-104complexes or 2C11 (anti-TCR).
diction is borne out in our studies using immobilized Agl
MHC complexes whose low affinity for the TCR might be
expected to be insufficient to maintain such TCR aggregates. In contrast, cells with a full length {-chain, which
would promote the conversion of clusters to stable aggregates, responded well to immobilized Ag/MHC. As 2B4.11
is CD4', itwill be of interest to determine if the introduction
of CD4 in its native or in an intracellularly truncated form
would, by virtue of enhancement of TCR affinity for ligand,
allow CT 108 cells to respond to Ag/MHC immobilized on
plastic.
Note that in this type of model, TCR conformation may
still play an essential role in signaling. All of the data are
compatible with the notion that initial aggregation leads to
conformational changes in the TCR, perhaps 5 itself because of covalent modification such as phosphorylation
EFFECT OF TCR-< TRUNCATION ON T CELL ACTIVATION
2220
system differed from that used here in that
the bispecific Ab
bound TCR and MHC class I molecules on the same cell.
However, the conclusion was that “immobilization” of the
TCR is animportant aspect in signaling, which isconsistent
with other attempts to trigger T cell activation by Abmediated cross-linking of the TCR to other T cell surface
molecules (48).
The formation of stable receptor clusters might be advantageous for a number of reasons, including prolonging
the time that individual TCR interact with one another (49).
Indeed, coupled with the hypothesis that 5 promotes stable
aggregation, this possibility accounts for the inability of a
low affinity ligand such as AgMHC to activate cells expressing a truncated c-chain. In addition to interactions between TCR, stable aggregation will bring associated molecules involved in signaling (e.g., kinases, phospholipases,
phosphatases, etc.) into closer proximity. Similar to our
observations with cells expressing a truncated it has recently been reported that T cells expressing either the brain
or the T cell form of the ~59~Y”
tyrosine kinase respond to
Ab, but only the latter respond to Ag (50). Taken together,
these data may imply that the Ab vs Agh4HC response
phenotype is dependent on a TCR-tyrosine kinase(s) interaction that is dictated by a full length 5-chain.Studies to
determine directly the effect of full length or truncated 5 on
TCR aggregate formation are currently being pursued.
c,
7.
8.
9.
IO.
11.
12.
13.
Acknowledgments
The authors thank Dr. David Segal for helpful advice on the preparation
of bispecific heterochimericAb, Dr. Ettore Appella foruse of the FPLC,
Dr. Ronald Germain for the gift of the MHC class 11-transfectedL cells,
andto Drs. Ronald Schwartz and Bernard Malissen for personal communications. We also thank Drs. R. Germain, Richard Klausner, Henry
Metzger, and Allan Weissman for critical review of the manuscript and
helpful discussions.
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