By
From the * Centre d'Immunologie Institut National de la Santé et de la Recherche Médicale, Centre
National de la Recherche Scientifique de Marseille-Luminy, Case 906, 13288 Marseille, Cedex 09, France; and Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland
To study the role of CD8 in T cell function, we derived a CD8
/
(CD8
/
) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma
was transfected either with CD8
alone or together with CD8
. All three hybridomas released
interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though
CD8
/
cells did so more efficiently than CD8
/
and especially CD8
/
cells. More strikingly, only CD8
/
cells were able to recognize a weak agonist peptide derivative variant.
This recognition was abolished by Fab
fragments of the anti-Kd
3 monoclonal antibody SF11.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on
CD8
/
cells was ~5- and 20-fold more avid than on CD8
/a and CD8
/
cells, respectively. SF1-1.1.1 Fab
or Kd mutation D227K reduced the TCR photoaffinity labeling on
CD8
/
cells to approximately the same low levels observed on CD8
/
cells. These results
indicate that CD8
/
is a more efficient coreceptor than CD8
/
, because it more avidly
strengthens TCR-ligand binding.
MHC class I-restricted TCR Less is known about CD8 We have previously developed a system that allows assessment of TCR-ligand interactions by TCR photoaffinity labeling (16, 17). To this end, the Plasmodium berghei
cirumsporozoite peptide PbCS 262-260 (SYIPSAEKI) was
modified by replacing PbCS S-252 with photoreactive
iodo-4-azidosalicylic acid (IASA) and conjugating K-259
with 4-azidobenzoic acid (ABA). From mice immunized
with this derivative, Kd-restricted CTL clones were derived
that recognized this conjugate as well as the one lacking the
IASA group, but not the derivative lacking the ABA group
(17, 18). Selective photoactivation of IASA permitted crosslinking of the peptide derivative to Kd molecules (16). Incubation of soluble, monomeric Kd-peptide derivative complexes with cloned CTL and photoactivation of the ABA
group resulted in TCR photoaffinity labeling, which was
proportional to TCR-ligand binding (16). This labeling was not dependent on integrins but dependent on CD8
participation in TCR-ligand binding.
We have previously prepared a T cell hybridoma by fusing cloned T1 CTL with a TCR In this study we tested N9 CTL-derived hybridomas that
expressed either no CD8 (CD8 Peptide Derivatives and Photoaffinity Labeling Procedures.
All synthetic and analytic procedures were performed essentially as
described (16, 21). In brief, IASA-YIPSAEK(ABA)I and IASAYIPSAEK(BA)I were obtained by chloramine T-mediated iodination of ASA-Y(PO3H2)IPSAEK(ABA)I and ASA-Y(PO3H2)
IPSAEK(BA)I, respectively. Before deprotection and cleavage
from the resin, the peptides were NH2-terminally acylated with
4-azidosalicyloy-N-hyrdroxysuccinimidyl ester. After iodination
with 125I (~2,000 Ci/mMol) or nonradioactive iodine, the peptides were dephosphorylated with alkaline phosphatase and purified by reverse phase HPLC on a C-18 column. Kd and TCR
photoaffinity labeling experiments were performed as described
(16, 17, 19). In brief, purified soluble Kd was incubated with
freshly radiolabeled peptide derivatives at ambient temperature
for 2 h, followed by UV irradiation at Cell Lines.
The T cell hybridoma N9.1 (CD8 mAbs and Flow Cytometry.
The following mAbs were used:
H57-259 (anti-TCR C IL-2 Release Assay.
L-Kd or L-Kd D227K cells (5 × 106 cells/
ml) were incubated in DMEM supplemented with 0.7% FCS and
10 mM Hepes in 10 ml polypropylene tubes (Falcon Plastics, Oxnard, CA) with graded concentrations (10 To assess the antigen recognition of the hybridomas under study, they were incubated with L cells expressing covalent Kd-IASA-YIPSAEK(ABA)I complexes and the released IL-2 was measured. The L-Kd cells were sensitized
by incubation with the indicated concentrations of IASAYIPSAEK(ABA)I and IASA-YIPSAEK(BA)I, respectively,
followed by UV irradiation at
/
+ T cells express heterodimeric CD8 consisting of a disulfide linked
and
chain, whereas other cell types, such as NK cells or extrathymic intraepithelial T cells, express homodimeric CD8
(1, 2). Whereas CD8
can be surface expressed as CD8
/
homodimers, CD8
is expressed only as CD8
/
heterodimers (3). X-ray crystallography showed that CD8
is
folded in an Ig-like manner (2, 4). For CD8
/
, a major
CD8 binding site on MHC class I molecules is the acidic loop 222-229, located in the center of the
3 domain (5, 6). Moreover, the cytoplasmic tail of CD8
has been shown
to associate with the T cell-specific tyrosine kinase p56lck
(7, 8) and becomes phosphorylated on serine residues upon cell activation (8, 9).
. It has only ~30% sequence
homology with CD8
, and its hinge region is 13 amino
acids shorter than the one of CD8
(1, 2). CD8
broadens
the range of antigen recognition, e.g., CD8
/
expressing
T cell hybridomas were able to recognize ligand variants,
but CD8
/
expressing ones did not (10, 11). CD8
also
plays a decisive role in thymic differentiation and maturation, since CD8
"knock out" mice have dramatically reduced numbers of mature CD8+ cells (12, 13). Transgenic
mice expressing "tailless" CD8
also exhibited a reduced
number of mature CD8+ cells, indicating that the cytoplasmatic portion of CD8
has a functional significance (14).
The recent finding that CD8
considerably increases
CD8-p56lck association suggests that the tail of CD8
directly or indirectly interacts with this enzyme (15).
variant of the BW thymoma (19). This hybridoma expressed T1 TCR but no
CD8, because of a fusion mediated silencing of the CD8
promoter and the inability of CD8
to be surface expressed in the absence of CD8
(3, 20). As assessed by TCR photoaffinity labeling, TCR-ligand binding on this hybridoma
was >95% weaker than on T1 CTL (19). CD8
transfection of the hybridoma resulted in high level expression of
CD8
/
and weak CD8
/
expression (CD8
being
contributed by the endogenous gene). This transfection only
partially restored TCR-ligand binding, suggesting that
CD8
/
(19) may strengthen TCR-ligand binding more
efficienty than CD8
/
.
/
), only CD8
(CD8
/
),
or CD8
/
for antigen recognition (IL-2 release) and TCR-
ligand binding (TCR photoaffinity labeling). The results
indicate that CD8
/
cells more efficiently recognized antigen, especially a weak antagonist variant, because CD8
/
avidly strengthened TCR-ligand binding.
350 nm and FPLC gel
filtration. For TCR photoaffinity labeling, 8 × 106 cells/ml were
resuspended in DMEM supplemented with 2% FCS and 20 mM
Hepes and incubated in 1 ml aliquots with 1.2 × 107 cpm of Kd-
peptide derivative complex for 1 h at 26°C. After UV irradiation at 312 ± 40 nm, the cells were washed and lysed on ice in PBS supplemented with NP-40, Hepes, and protease inhibitors. Immunoprecipitation was performed with anti-TCR mAb H57297, and the immunoprecipitates were analyzed by SDS-PAGE
(10% linear, reducing conditions). The dried gels were evaluated
by autoradiography and densitometry. TCR photoaffinity labeling experiments were performed in triplicate and repeated at least
twice.
/
) was obtained by fusing cloned N9 CTL (18) with the BW5147 TCR
(BW
) thymoma as described (19). N9.1 cells were transfected
with CD8
cDNA inserted in the pH pH
APr-1-neo expression
vector (8) and selected in the presence of G418 (2.5 mg/ml). Various stable CD8
transfectants were tested by flow cytometry for
expression of CD8
, and one clone was found that was CD8
.
This clone was transfected with a CD8
BamHI genomic fragment DNA inserted in the pSV2-his expression vector (pCA257.10). A representative clone (WB1.2) was selected in the presence of histidinol (3 mM). All transfections were performed using protoplast
fusion (22). Murine fibroblast L cells were transfected with Kd or
KdD227K cDNA as described (23).
), 53-6-72 (anti-CD8
), H35-17 (antiCD8
), SF1-1.1.1. (anti-Kd
3), 20-8-4S (anti-Kd
1), and antiCD3 (145.2C11). For most experiments, single staining was performed with FITC- or PE-conjugated anti-CD8
and anti-Kd,
PE-conjugated anti-CD8
, and anti-TCR (mAb). Samples were
analyzed on a FACScan® (Becton Dickinson & Co., Mountain
View, CA) equipped with LYSIS II software.
7-10
14 M, in 10-fold
dilutions) of peptide derivative at 26°C for 2 h. After UV irradiation at
350 nm (16), cells were washed three times, resuspended
in DMEM supplemented with 5% FCS and 10 mM Hepes at 106
cells/ml, and plated in 100-µl aliquots into flat bottom 96-well microtiter plates (Falcon Plastics). The T cell hydridomas, resuspended in the same medium and at the same cell density, were
added in 100-µl aliquots. Alternatively, hydridomas were incubated either with anti-CD3 mAb (145.2C11, absorbed on plates)
or PMA (2.5 ng/ml) and ionomycin (0.5 µg/ml). After 24 h of
incubation at 37°C, supernatants (100 µl) were transferred into
fresh microtiter plates and incubated for 36-48 h with CTLL indicator cells (4 × 103 cells/100 µl/well). 1 µCi of [3H]thymidine
(Amersham Corp., Arlington Heights, IL), was added per well,
and after incubation for an additional 12 h, the cells were harvested and the incorporated [3H]thymidine was measured by
counting (Inotech Harvester and Trace 96
counter). Each experiment was performed in triplicate and was repeated at least
twice.
350 nm, which produced
covalent cell-associated Kd-peptide derivative complexes.
As shown for a representative experiment in Fig. 1, all
three hybridomas produced IL-2 upon incubation with L
cells expressing Kd-IASA-YIPSAEK(ABA)I, but CD8
/
cells more efficiently, especially at low degrees of sensitization. Because all three hybridomas expressed comparable
levels of TCR and LFA1 (Table 1) and responded similarly
upon stimulation with anti-CD3 mAb or PMA and ionomycine (Fig. 1, insets), these results indicate that CD8
/
,
but less CD8
/
, increased the efficiency of antigen recognition.
Fig. 1.
CD8/
cells more efficiently recognize IASA-YIPSAEK
(ABA)I and IASA-YIPSAEK(BA)I than CD8
/
or CD8
/
cells. The
IL-2 released by CD8
/
(A), CD8
/
(B), and CD8
/
(C) cells was
measured as [3H]thymidine uptake by CTLL indicator cells after incubation with L-Kd cells sensitized with IASA-YIPSAEK(ABA)I (
) or
IASA-YIPSAEK(BA)I (
). In insets, the IL-2 responses are shown as observed after incubation with anti-CD3 mAb or PMA and ionomycin.
[View Larger Version of this Image (17K GIF file)]
More strikingly, the peptide derivative variant IASAYIPSAEK(BA)I, which lacks the azido function of the
ABA group, was efficiently recognized only by CD8/
cells (Fig. 1). Half-maximal IL-2 release was observed at
>10-fold higher degree of sensitization than the wild-type
conjugate. Substitution of the ABA group with BA reduced the efficiency of recognition by N9 CTL by ~50-fold (18).
As assessed by TCR photoaffinity labeling (see below), Kd
complexes with IASA-YIPSAEK(BA)I bound to the N9
TCR approximately seven times less efficiently than those
containing IASA-YIPSAEK(ABA)I (see Fig. 3 D). The observation that this weak agonist was significantly recognized
only by CD8
/
cells is in accordance with reports indicating that CD8
broadens the range of antigen recognition by CD8+ T cells (10, 11) and in addition provides a
quantitative correlation between antigen recognition and
TCR-ligand binding.
To find out why CD8/
cells efficiently recognize
IASA-YIPSAEK(BA)I, we performed the previous experiment (Fig. 1 C) in the presence of Fab
fragments of the
anti-Kd
3 mAb SF1-1.1.1. This reagent has been shown
to impair participation of CD8 in TCR-ligand binding
while leaving CD8 mediated adhesion unaffected (19). As
shown in Fig. 2 A, this reagent abolished the efficient recognition of IASA-YIPSAEK(BA)I. Similarly, CD8
/
cells
failed to recognize significantly this conjugate on L cells expressing mutant Kd D227K, though they well recognized
the wild type conjugate (Fig. 2 B). This Kd mutation has
been shown to dramatically impair CD8-MHC class I interactions (5, 6, 19). The finding that CD8
/
cells under
conditions that prevent CD8 participation in TCR-ligand binding failed, as CD8
/
cells, to significantly recognize
the weak agonist IASA-YIPSAEK(BA)I suggests that
CD8
/
more avidly strengthens TCR-ligand binding
than CD8
/
.
The utilized system allowed to test this possibility, because it permits direct assessment of CD8 participation in
TCR-ligand binding by TCR photoaffinity labeling with soluble covalent Kd-peptide derivative complexes (16, 17, 19).
TCR photoaffinity labeling with Kd-125IASA-YIPSAEK
(ABA)I on CD8/
cells was ~5 and 20 times more efficient than on CD8
/
cells and CD8
/
cells, respectively
(Fig. 3, A and B). The background, e.g., nonspecific labeling, was 1-2%, as observed in the presence of the
-Kd
1
mAb 20-8-4S, which blocks specific TCR-ligand binding
(16, 19). Because CD8
/
cells expressed more CD8
than N9 CTL or CD8
/
cells expressed CD8
/
(Table 1),
these results demonstrate that heterodimeric CD8
/
indeed more avidly strengthens TCR-ligand binding than
homodimeric CD8
/
. This is in accordance with preceeding experiments, in which various CD8
and CD8
plus CD8
transfectants of CD8
/
N9.1 cells or subclones
of CD8
/
WB1.2 were likewise tested (unpublished results).
The efficient TCR photoaffinity labeling on CD8/
cells
was reduced in the presence of SF1-1.1.1 Fab
to the same
low levels as observed on CD8
/
cells (Fig. 3, A and B).
The TCR photoaffinity labeling on CD8
/
was also reduced in the presence of this reagent, but for unknown reasons to a slightly lesser degree than on CD8
/
cells.
The lack of a significant inhibition of the TCR photoaffinity labeling on CD8
/
cells in the presence of SF1-1.1.1
Fab
showed that this reagent does not affect the TCR-
ligand interaction per se, but rather the CD8 participation
in TCR-ligand binding. Similarly, when TCR photoaffinity labeling on CD8
/
cells was performed with soluble Kd D227K-IASA-YIPSAEK(ABA)I, nearly five times weaker
labeling was observed than with the wild type ligand (Fig. 3
C). In contrast, on CD8
/
cells, both ligands exhibited essentially the same weak TCR photoaffinity labeling, confirming our previous finding that this Kd mutation does not
significantly affect the actual TCR-ligand binding, but
rather its dependence on CD8 (Fig. 3 B and reference 19).
The peptide derivative variant IASA-YIPSAEK(BA)I, lacking an orthogonal photoreactive group, can cross-link to Kd, but not to TCR; therefore, the binding of Kd-IASAYIPSAEK(BA)I to N9 TCR was assessed by its ability to inhibit the TCR photoaffinity labeling by Kd-IASA-YIPSAEK(ABA)I. As shown in Fig. 3 D, the TCR photoaffinity labeling on N9 CTL was inhibited in a linear fashion in the presence of graded amounts of Kd-125IASA-YIPSAEK(BA)I. By extrapolation, 50% of inhibition was observed at ~6.8fold molar excess of variant ligand.
Taken collectively, the results of the TCR photoaffinity
labeling experiments correlate well with those of the IL-2
release experiments. Most strikingly, SF1-1.1.1 Fab and Kd
mutation D227K inhibited the efficient recognition of the
conjugate variant IASA-YIPSAEK(BA)I by CD8
/
cells,
because they inhibited the TCR photoaffinity labeling
(Figs. 2 and 3). We thus conclude that CD8
/
cells more
efficiently recognize antigen than CD8
/
cells, because
CD8
/
more avidly strengthens TCR-ligand binding. Although our results do not rule out that other factors may
play a role as well (i.e., that CD8
/
has superior signaling
capabilities or more efficiently mediates CD8-dependent
adhesion), they clearly demonstrate that CD8
significantly
increases CD8 participation in TCR-ligand binding, and
this predictably is important for antigen recognition, especially of weak agonists.
It remains to be explained why CD8/
more efficiently increases TCR-ligand binding than CD8
/
. It is
conceivable that either CD8
/
more avidly binds MHC
class I molecules or that it more efficiently "couples" with
TCR/CD3, thus defining an orientation of CD8 relative
to the TCR that favors coordinate ligand binding. Although CD8
/
and CD8
/
both have been shown to
bind MHC class I molecules (5, 24), it is unknown whether
they do so with different affinities. In support of the second
possibility, we have previously observed that the dynamics
of TCR-ligand interactions on CTL clones are modulated
by CD8 in a time- and temperature-dependent manner
(16, 19). Moreover, several reports indicate that CD8 interacts with TCR/CD3 complex (25, 26), which in view
of our present data may imply that CD8
plays an important role in such interactions. Interestingly, CD8
has been
shown to significantly increase the association of CD8 with
the tyrosine kinase p56lck (15). In the CD4 system, p56lck is
known to be involved in coupling CD4 with TCR/CD3
(27). If the same were true for CD8, it would explain why
CD8
/
is a more efficient coreceptor than CD8
/
. The
system described here, by including kinetic experiments
and further genetic engineering of the hybridomas, should
now permit detailed analysis of the role of CD8
in T cell
function.
Address correspondence to I.F. Luescher, Ludwig Institute for Cancer Research, Ch. des Boveresses 155, 1066 Epalinges, Switzerland.
Received for publication 8 July 1996
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale/Centre National de la Recherche Scientifique, Ligue Nationale Contre le Cancer and Association pour le Recherche sur le Cancer.We thank Dr. C. Servis and C. Horvath for technical assistance, Drs. D.R. Littman and N. Killeen for L-Kd cells, Dr. F. Godeau for soluble KdD227K, Dr. J.-C. Cerottini for helpful discussions, and Anna Zoppi for preparing the manuscript.
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