International Immunology, Vol. 9, No. 10, pp. 1423–1430
© 1997 Oxford University Press
Altered effector responses of H-Y transgenic
CD8F cells
Ivica Arsov and Stanislav Vukmanović
Michael Heidelberger Division of Immunology, Department of Pathology, and Kaplan Comprehensive
Cancer Center, NYU Medical Center, 550 First Avenue, New York, NY 10016, USA
Keywords: CD8, effector, H-Y, transgene
Abstract
The primary role of CD8F T cells is to destroy virus-infected or tumor cells expressing cognate
antigens in the form of peptide–MHC class I complexes. This destruction is primarily achieved by
the actions of lytic mediators and/or lymphokines. In this report, we show that mature, H-Y/Dbspecific CD8F T cells from H-Y TCR transgenic mice were unable to efficiently release lytic
mediators after antigenic stimulation. However, anti-TCR antibody induced granule exocytosis and
target cell lysis, arguing against signaling and/or cytolytic machinery defects in CD8F cells, and
demonstrating that male antigen induced differentiation of ‘naive’ into effector CD8F cells.
Stimulation of H-Y-specific effector CD8F T cells with male stimulators, although insufficient to
induce lytic granule release, was sufficient for H-Y-specific IFN-γ production. Unexpectedly, this
effector-phase IFN-γ production was dependent on B7-2 engagement. We hypothesize that altered
effector functions in H-Y-specific CD8F cells are due to the low affinity of TCR–antigen–MHC
interaction and/or the elevated threshold of CD8F T cell activation.
Introduction
The ultimate result of CD81 T cell activation is the secretion
of different cytokines and directional release of lytic granule
contents containing pore-forming proteins. The vast majority
of CD81 T cell lines and clones generated in vitro lyse target
cells expressing cognate antigens in the form of short peptides
bound to MHC class I molecules. The major mechanism of
target cell lysis is mediated by perforin and granzymes (1,2),
which are stored in lytic granules of cytotoxic T cells and
released after antigen encounter (3,4). A minor contribution
to target cell lysis is mediated by the death-transducing
molecule, Fas, expressed on the surface of target cells (1,2,5).
The engagement of Fas with the ligand expressed by activated
CD81 T cells leads to target cell apoptosis. A normal effector
response of CD81 T cells which encounter ligand is the lysis
of target cells accompanied by secretion of IFN-γ, as well as
other cytokines, such as IL-3, tumor necrosis factor-α and
granulocyte macrophage colony stimulating factor.
The development of TCR transgenic mouse technology was
instrumental for the analysis of various aspects of CD81 T cell
biology, including the effector function of CD81 T cells. One of
the first TCR transgenic mice used the TCR recognizing male
antigen (6,7). Spleen cells derived from male mice efficiently
delete immature, H-Y TCR-expressing T cells and induce proliferation of mature CD81 T cells from female H-Y TCR trans-
genic mice (8,9). However, when CD81 T cells were injected
into male nude mice, they failed to induce graft versus host
disease, despite their vigorous proliferation in vivo (10). Further,
female H-Y TCR transgenic animals only infrequently reject
male skin grafts (11). Defective effector functions in vivo were
ascribed to the lack of CD41 T cell help (11), which was shown to
be important for non-transgenic H-Y-specific responses in vivo
(12–14). In this study we show that CD81 T cells from female
H-Y TCR transgenic mice are unable to lyse male cells in vitro,
but efficiently lyse irrelevant targets coated with anti-TCR antibodies in a perforin/granzyme-dependent manner. The release
of granular content could not be observed after stimulation with
male cells, while the same cells readily induced production of
IFN-γ. Interestingly, IFN-γ production was completely dependent on the co-stimulatory molecule B7-2. Collectively, these
results suggest that an altered effector function of H-Y-specific
transgenic CD81 cells, rather than the lack of CD41 help, may
be the basis for defective in vivo responses.
Methods
Animals
Female H-Y TCR transgenic animals were kindly provided by
Dr Janko Nikolić-Žugić (Sloan Kettering Institute, New York,
Correspondence to: S. Vukmanović
Transmitting editor: M. J. Bevan
Received 17 April 1997, accepted 12 June 1997
1424 Select responses of H-Y transgenic CD81 T cells
NY) and used at 6–8 weeks of age. C57BL/6 and C57BL/6
lpr/lpr mice were purchased from the Jackson Laboratory
(Bar Harbor, ME), and used at 3–5 weeks of age.
Cell lines
P815 and A20 cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine,
1 mM 2-mercaptoethanol and antibiotics (RP10). The H-Y
TCR transgenic CD81 T cell line was derived from the
spleen of a H-Y TCR transgenic female mouse. The line was
maintained by weekly re-stimulations with irradiated spleen
cells from C57BL/6 male mice in RP10 supplemented with
5% rat concanavalin A supernatant. T cell-depleted, lipopolysaccharide (LPS) dextran sulfate-induced blasts were generated according to a previously described procedure (15).
LPS blasts used as targets for 51Cr and serine esterase
release assays were generated by 48 h incubation of spleen
cells in RP10 containing 10 mg/ml of LPS, serotype 0111:B4
(Sigma, St Louis, MO).
mAb
The following mAb were purified from hybridoma supernatants: anti-mouse CD3 ε (145–2C11), anti-mouse TCR αβ
(H57-597), anti-mouse TCR Vβ8.2 (F23.1), anti-transgenic TCR
α chain (T3.70 ) and anti-mouse CD4 (GK1.5). All hybridomas
were kind gifts from Dr Janko Nikolić-Žugić. Anti-mouse B7-1
and B7-2 (3A12 and GL1 respectively) antibodies were gifts
from Dr Yang Liu (New York University Medical Center, NY).
Flow cytometry
T cells were stained with saturating concentrations of T3.70
or F23.1 hybridoma supernatant, followed by FITC-conjugated
goat anti-mouse antibody (Southern Biotechnology Associates, Birmingham, AL) and analyzed using a FACScan flow
cytometer (Becton Dickinson, Mountain View, CA).
Cytotoxic T lymphocyte (CTL) assay
Targets were incubated with 51Cr-labeled sodium chromate
in RP10 media for 1 h at 37°C. Targets were then washed 3
times with PBS and 53103 or 13104 cells transferred to a
well of a round-bottomed 96-well plate. Effector cells were
added at varying numbers to the total volume of 200 µl. Plates
were incubated for 4 h at 37°C. At the end of this interval,
plates were centrifuged for 2 min at 1200 r.p.m., and supernatants (100 µl/well) were harvested and counted in a γ-counter.
The percent specific lysis was calculated as follows:
1003[(experimental release – spontaneous release)/(maximal
release – spontaneous release)]. Spontaneous and maximal
release were determined in the presence of medium or 1%
Triton X-100 respectively.
Serine esterase assay and ELISA for IFN-γ
For the serine esterase release assay, 105 effector cells were
incubated for 4 or 10 h, as indicated in figure legends, with
stimulator cells in a well of a round-bottomed 96-well plate at
37°C. Where indicated, serine esterase release was induced
with immobilized 2C11 antibody. For that purpose, 50 µl of
5 µg/ml of 2C11 was added to each well, incubated for 30 min
at room temperature and then washed with RP10 before
effectors were added. Total culture volumes in all cases were
100 µl/well. Aliquots of 50 µl of 4 h culture supernatants were
harvested and mixed with 950 ml of BLT substrate (0.20 mM
N-α-benzyloxycarbonyl-L-lysine thiobenzyl ester, 0.22 mM
5,59-dithio-bis(2-nitrobenzoic acid) and 0.01% Triton X-100 in
PBS). Samples were incubated for 20–40 min in a 37°C water
bath, after which they were placed on ice and the reaction
stopped with 10 µl of 0.1 M phenylmethylsulfonyl fluoride.
Absorbance of samples was measured at 412 nm and the
percent specific release was calculated following the formula
used for the CTL lysis assay. For the IFN-γ assay, cultures
were set up in an identical manner as for the serine esterase
release. IFN-γ was measured from 48 h culture supernatants
using an ELISA kit purchased from PharMingen (San Diego,
CA), according to the manufacturer’s recommendations.
Results
H-Y TCR transgenic CD81 T cells lyse anti-TCR-coated but
not male target cells
To examine the effector response of transgenic CD81 T cells,
spleen cells from female H-Y TCR transgenic mice were
stimulated in vitro with irradiated B6 male splenocytes with or
without exogenous IL-2 (to bypass the need for CD41 help).
The cells were then tested for their ability to lyse B6 male
LPS dextran sulfate-induced blasts. Irrespective of whether
IL-2 was absent (data not shown) or present (Fig. 1A), CD81
transgenic cells failed to lyse LPS dextran sulfate-induced
male blasts. Identical results were obtained using LPS- or
concanavalin A-induced blasts (data not shown).
The frequency of CD81 T cells expressing the transgenic
TCR α chain in the peripheral T cells from H-Y TCR transgenic
female mice can be quite low (16) and it could be argued
that the frequency of H-Y-specific effector cells was too low
to be detected at the effector to target ratios used. To avoid
this, we re-stimulated transgenic cells several times with male
stimulator cells and obtained a transgenic CD81 line. Virtually
all cells in this line expressed the transgenic α chain (Fig. 2).
When tested for the ability to lyse male blasts, the results
were similar to those observed after primary stimulation—no
lysis of male cells was observed (Fig. 1B). In contrast, both
male and female LPS-induced-blasts (Fig. 1B) or irrelevant
FcR-expressing targets (P815 mastocytoma) (Fig. 1C) were
efficiently lysed in a redirected cytotoxic assay using 2C11,
T3.70 or F23.1 antibodies. The lysis of both male and female
targets can be induced with anti-CD3 antibody with nearly
equal efficiencies, suggesting no H-Y antigen contribution to
the lysis. It therefore appears that CD81 T cells from the
transgenic mouse have both signaling and cytolytic machinery
capable of inducing lysis. Furthermore, stimulation with male
antigen can induce differentiation of transgenic CD81 T cells
into effector cells with cytolytic potential, but cytolytic activity
can only be triggered by anti-TCR antibody and not by
male antigen.
Redirected lysis of targets is mediated by perforin/granzyme
release
The cytolytic activity of CD81 cells in a redirected assay
could be due to the release of preformed lytic granule contents
or to the induction of FasL expression and activation of
Select responses of H-Y transgenic CD81 T cells 1425
Fig. 1. TCR-mediated lytic function of H-Y TCR transgenic CD81 T cells. H-Y TCR effectors were tested for their ability to kill male B6 LPS
dextran sulfate blasts (A), B6 LPS blasts using 2C11 antibody (B) or P815 cells using different anti-TCR antibodies (C). The final concentration
of antibodies was 1 µg/ml.
Fas-dependent apoptosis in target cells. To examine the
mechanism of anti-CD3 antibody-mediated lysis of P815 cells
by the H-Y-specific CD81 T cell line, we first carried out
the serine esterase release assay. Granule exocytosis was
observed upon stimulation with anti-TCR antibodies (Fig. 3)
and correlated well with the actual lysis observed in the 51Crrelease assay. No granule release or lysis was observed when
both male or female C57Bl/6 cells were used as targets unless
anti-CD3 antibody was included. These results indicated that
redirected lysis of P815 and B6 cells seen in our system
might be a consequence of degranulation triggered by 2C11
antibody. To confirm that granules stored in this CD81 T
cell line are cytolytic, a redirected assay using 2C11 was
performed (i) in the presence of 1 mM EGTA, that abrogates
perforin-mediated killing without affecting Fas-mediated killing
(5), and (ii) using Fas mutant B6 lpr/lpr (17) blasts as targets.
As shown in Fig. 4(A), EGTA completely abolished 2C11induced lysis of P815 cells. Moreover, B6 lpr/lpr blasts were
lysed, albeit less efficiently than normal B6 blasts (Fig. 4B).
Based on the results presented so far we conclude that
the lytic machinery and the TCR signaling leading to the
granule exocytosis are intact in the H-Y TCR transgenic CD81
T cells.
Identical stimulation with male stimulators results in IFN-γ
secretion, but not in serine esterase release
The above described absence of H-Y-specific lysis could in
theory be explained by a distinct possibility that the transgenic
TCR recognizes an H-Y epitope with restricted expression.
For example, in a hypothetical example even 100% lysis of a
subpopulation that represents 5% of LPS-induced blasts
would still result in overall 5% lysis that can be hard to detect.
To bypass this problem we took advantage of the fact
that anti-CD3-induced granzyme release by the H-Y-specific
transgenic CD81 cell line can be detected. One of the CD81
effector functions that could readily be induced by male
stimulator cells is the IFN-γ secretion (15). We therefore used
the same stimulator cells in an attempt to compare cytokine
production and the release of lytic mediators by transgenic
CD81 T cells. Our preparations of male stimulator cells
induced very efficient production of IFN-γ by H-Y-specific
CD81 T cells, which was comparable to the induction of
IFN-γ by anti-CD3 antibody (Fig. 5A). Strikingly, the same
preparation of male stimulator cells failed to induce serine
esterase release (Fig. 5B). Antigen-specific serine esterase
release was not visible even after longer incubations [a 10 h
assay is shown in Fig. 5(C)—longer assays revealed compar-
1426 Select responses of H-Y transgenic CD81 T cells
Fig. 3. Serine esterase release by H-Y TCR transgenic cells is
efficiently induced by 2C11, but not by B6 male LPS blasts. Effector
cells (13105) were incubated with 53104 stimulator cells and
serine esterase release was determined after 4 h. Where indicated,
5 µg/ml of 2C11 antibody was added. The results are expressed as
means of triplicate cultures 6 SD.
Fig. 2. Expression of H-Y transgenic TCR by the CD81 T cell line
derived from female H-Y TCR transgenic mice. T3.70 and F23.1
antibodies were used to detect H-Y TCR α or β chains respectively,
followed by FITC-conjugated anti-mouse Ig.
able increase in both female or male stimulator-induced serine
esterase release]. Finally, increasing the total number of
antigen-presenting cells (APC) in the culture should increase
the number of potentially rare antigen-expressing stimulators.
Although a 4-fold increase in the numbers of APC induced
.100-fold increase in male-specific IFN-γ release, significant
male-specific serine esterase release was not observed (Table
1). Even 53104 stimulators induce visible IFN-γ secretion, yet
8-fold more stimulator cells does not cause significant malespecific serine esterase release (in a 10 h assay). Thus, the
stimulator cell preparation that contained sufficient numbers of
antigen-expressing cells, as determined by IFN-γ production,
failed to induce serine esterase release.
Male antigen-induced IFN-γ secretion is co-stimulation
dependent
The possibility of a very restricted tissue expression of the
H-Y antigen is suggested by findings that preparations of
stimulator cells enriched for dendritic cells are the best
stimulators for transgenic CD81 T cells (9). This could be due
to selective antigen expression by dendritic cells, and/or
due to a constitutive expression of the B7-1 co-stimulatory
molecule by dendritic cells (18) and a possible unusual
requirement for co-stimulation by transgenic CD81 T cells.
To test the possible involvement of the B7 family of costimulatory molecules in the H-Y-specific IFN-γ response, we
blocked the B7-1 and B7-2 interaction with their ligands by
adding specific antibodies. The H-Y-induced IFN-γ production
was highly dependent on the expression of the B7-2 molecule
on the stimulator cells, since it was abolished by anti-B7-2
antibody, but not by anti-B7-1 antibody (Fig. 6). The absence
of significant inhibition by anti-B7-1 antibody probably reflects
different kinetics of B7-1 and B7-2 expression after LPS
activation (19). It therefore appears that IFN-γ secretion by
H-Y transgenic CD81 T cells is dependent on co-stimulation.
Although these findings do not specifically rule out the possibility of selective antigen expression, they demonstrate costimulation requirements for IFN-γ secretion of H-Y-specific
effector cells. Given that IFN-γ secretion is usually co-stimulation independent in effector CD81 cells (20) and to a certain
degree even in naive T cells (21), this finding demonstrates
another altered effector function of transgenic CD81 T cells.
Discussion
We show in this report that H-Y-specific transgenic CD81 T
cells exhibit altered effector function in vitro when stimulated
with male antigen. Male cells fail to induce granule exocytosis
under conditions that readily induce IFN-γ secretion. In addition, antigen-induced IFN-γ secretion showed a requirement
for co-stimulation, an unusual finding for effector CD81 T cells
(20). The altered effector function of H-Y-specific cells is not
due to a defect in TCR signaling or granule exocytosis
machinery as lytic effector function can be triggered by antiTCR antibodies. The anti-TCR-induced lysis is largely perforinmediated since redirected lysis can be blocked by Ca21
chelation and Fas-deficient B6 lpr/lpr blasts are sensitive to
anti-CD3-mediated lysis. It seems, therefore, that male antigen
is effective in priming of CD81 T cells, but that the same
antigen fails to trigger serine esterase release.
The results obtained using in vitro assays are compatible
with the in vivo findings that H-Y TCR transgenic females only
Select responses of H-Y transgenic CD81 T cells 1427
Fig. 4. Redirected cytotoxicity by the H-Y-specific CD81 T cell line is at least partially mediated by perforin/granzyme release. Redirected
cytotoxicity assays were carried out in the presence of 1 mM EGTA and 1.5 mM Mg21 (A), or using B6 lpr/lpr blasts as targets (B). Final
concentration of 2C11 antibody was 1 µg/ml.
Fig. 5. H-Y TCR transgenic cells secrete IFN-γ, but fail to release serine esterases in response to B6 male LPS dextran sulfate-induced blasts.
Blasts were prepared by incubating T cell-depleted spleen cells with 25 µg/ml of each LPS and dextran sulfate for 12 h. Experiments were
performed using 13105 effector cells and 23105 stimulator cells per well. For IFN-γ ELISA (A), we used soluble 2C11 antibody (5 µg/ml) and
A20 cells as APC (13105/well). Immobilized 2C11 antibody was used for the 4 h serine esterase release assay (B). Plate-bound as well as
FcR cross-linked (using either A20 or P815 cells) anti-CD3 antibody were equally efficient in inducing IFN-γ secretion or serine esterase
release (data not shown).
1428 Select responses of H-Y transgenic CD81 T cells
Table 1. The effect of stimulator cell numbers on IFN-γ or
serine esterase release of H-Y TCR transgenic cells
No. of stimulator
cells/well (3105)
0.5
1
2
4
Specific serine
esterase release (%)
IFN-γ pg/ml)
Female
Male
Female
Male
ND
ND
ND
NT
40
220
5400
NT
12
13
17
27
7
19
27
32
Blasts were prepared by incubating T cell-depleted spleen cells
with 25 µg/ml of each LPS and dextran sulfate for 12 h. Then, 13105
effector cells were incubated with indicated numbers of stimulator
cells per well. Cultures were incubated for 48 h for IFN-γ ELISA or
10 h for the serine esterase release assay. NT, not tested; ND, not
detectable.
sporadically reject male skin grafts and pancreatic islets (11)
and fail to induce a graft versus host reaction (10). The
absence of a normal response could be explained by the
lack of CD41 T cell help, which was shown to be important
for H-Y-specific responses in vivo (12–14). While it remains
to be seen whether CD41 T cells from H-Y TCR transgenic
female animals are able to provide help in vivo, our results
argue against the CD41 help absence, as we have bypassed
this requirement by providing exogenous IL-2. In vitro antigen
stimulation of CD81 cells in the presence of exogenous IL-2
usually results in the generation of potent killer cells, but this
is obviously not the case with H-Y-specific transgenic CD81
cells. We therefore believe that the cause for altered CD81
effector functions lies in the specific interaction of the H-Yspecific TCR with the antigen–MHC complex at the effector
stage.
The absence of lysis of male targets by the H-Y TCR
transgenic T cells could in theory be due to low levels of
expression of the H-Y epitope or expression by only a small
number of male spleen cells. We cannot formally rule out the
possibility that the H-Y epitope recognized by this line is
presented by only a subset of spleen cells, e.g. dendritic
cells. The recently identified H-Y epitope presented by H-2Db
is ubiquitously expressed (22). However, this epitope failed
to induce IFN-γ secretion from transgenic cells even at very
high concentrations (data not shown), arguing that the epitope
recognized by the transgenic TCR might be different from
the one recognized by cytotoxic T cell lines isolated from
non-transgenic female B6 mice. Still, we feel that potential
selective expression of H-Y epitope is not the reason for
the altered H-Y-specific responses because: (i) the same
stimulator cells that induce IFN-γ secretion do not induce
serine esterase release, (ii) increasing the time of incubation
or the number of stimulator cells (conditions that should
enhance the chance of interaction with ‘rare’ cells) does not
induce male-specific serine esterase release and (iii) the
IFN-γ secretion is completely B7-2 dependent.
In most studies on CD81 T cell effector function cytotoxicity
is paralleled by cytokine secretion. However, several cases
of split CD81 T cell responses were observed (23–28).
Cytotoxic T cell responses in the absence of cytokine secretion
Fig. 6. H-Y-specific induction of IFN-γ can be blocked by antibody
against B7-2. Assay was performed using the same numbers of
effectors and stimulators as described in Fig. 5. All antibodies were
used at a concentration of 5 µg/ml. The results represent means of
triplicate cultures 6 SD.
observed in some of these models is perhaps simpler to
explain by weaker stimulation given that lower avidity TCR
engagement with antigen–MHC is necessary to trigger the
lytic response compared to cytokine secretion (26–28). In a
recent study, however, the response of a CD81 T cell clone
to self peptide was accompanied by IFN-γ production and
Fas-mediated killing, but the induction of serine esterases
was absent (29). We also observe IFN-γ production in the
absence of serine esterase release. This phenotype could
perhaps be explained by the fact that stimulator cells used
in our assays express high levels of the co-stimulatory molecule B7-2 which may provide compensation for the low-affinity
interaction of the transgenic TCR with the antigen. Indeed, we
were able to completely inhibit H-Y-induced IFN-γ production
using anti-B7-2 antibody (Fig. 6). This B7 dependence of
CD81 T cell effector functions was seen in some experimental
models in which the antigen was a self peptide (20,30–32).
It is nevertheless unclear why this strong co-stimulation cannot
support H-Y-induced serine esterase release. Perhaps the
signals transmitted through B7-1 and B7-2 ligands are not
the same as those involved in granule exocytosis, as suggested by studies on anergy induction in CD81 T cells
where cytokine production and not cytotoxic response was
abrogated in anergic CD81 T cells (23,33). Thus, a relatively
weaker stimulation of H-Y-specific CD81 T cells, partially
compensated by co-stimulatory interactions, can explain the
altered effector function of H-Y-specific transgenic CD81 T
cells. The reason for the weak interaction is still unclear and
could be due to low affinity of TCR–antigen–MHC interaction
and/or elevated threshold of CD81 T cell activation. Our
experiments do not discriminate between these possibilities
and are consistent with both. The affinity of transgenic H-Y
TCR has not been directly measured, but was suggested to
be lower than the affinity of other receptors on CD81 T cell
Select responses of H-Y transgenic CD81 T cells 1429
clones which exhibit normal cytotoxic response, including
other H-Y-specific clones (34–36). If the activation threshold
of H-Y-specific CD81 T cells is elevated an interesting question
of the possible cause arises. The existence of split CD81 T
cell responses to the self antigens (29,37,38) suggests the
possible involvement of tolerance to self, but the nature of
the tolerizing antigen is unknown. We could only speculate
based on the findings that HLA-B7-presented human H-Y
peptide differs from the one encoded by the homologous
gene on the X chromosome by only two amino acids and that
the female analog can also sensitize SMCY-specific CD81
cells (39). Thus it is possible that the female analog presented
by the same MHC class I molecule may be involved, at least
in some cases, in partially tolerizing H-Y-specific T cells
against the male antigen. This possibility can only be
addressed once the H-Y antigen for this particular TCR is
identified.
In summary, we found that H-Y antigen can efficiently prime
CD81 T cells from H-Y TCR transgenic female mice in
the presence of exogenous IL-2. These cells lysed FcRexpressing targets when triggered by anti-TCR antibodies.
Despite the fact that lytic mediators were efficiently generated,
these cells failed to release pre-stored mediators in response
to H-Y antigen, but readily produced IFN-γ. This split response
is reminiscent of a CD81 T cell response to self-peptide and
might be important for our understanding of how the response
of CD81 T cells to low avidity ligands is generated.
Acknowledgements
The authors thank Janko Nikolić-Žugić and Yang Liu for reagents,
and Yang Liu for reading the manuscript. This work was supported
by the Markey Charitable Trust Junior Investigator Award and NCI
core support grant 5P30 CA16087.
Abbreviations
APC
CTL
LPS
antigen-presenting cell
cytotoxic T lymphocyte
lipopolysaccharide
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