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Blood, 15 April 2002, Vol. 99, No. 8, pp. 2880-2889
IMMUNOBIOLOGY
B7-CTLA4 interaction promotes cognate destruction of tumor cells
by cytotoxic T lymphocytes in vivo
Xue-Feng Bai,
Jinqing Liu,
Kenneth F. May Jr,
Yong Guo,
Pan Zheng, and
Yang Liu
From the Department of Pathology and the Comprehensive
Cancer Center, Ohio State University Medical Center, Columbus.
 |
Abstract |
Costimulatory molecules B7-1 and B7-2 (hereby collectively called
B7) interact with CD28 and CTLA4 on T cells and promote antitumor
immunity. The function of B7-CTLA4 interaction in antitumor CTL
response remains controversial. Here we used CD28 / and
CD28+/ or CD28+/+ transgenic mice that
express the T-cell receptor specific for an unmutated tumor
antigen, P1A, and for tumor cells expressing a CTLA4-specific B7 mutant
to evaluate the function of CD28-B7 and CTLA4-B7 interactions in
induction and effector phases of antitumor immunity. We report that
B7-CD28 and B7-CTLA4 interactions promote tumor rejection. However,
this is achieved by distinct mechanisms. B7-CD28 interaction enhances
T-cell clonal expansion, though a role for this interaction in the
effector phase cannot be ruled out. In contrast, B7-CTLA4 interaction
enhances the CTL-mediated destruction of tumors, but not T-cell clonal expansion.
(Blood. 2002;99:2880-2889)
© 2002 by The American Society of Hematology.
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Introduction |
The identification of B7-11,2 and
B7-21-6 and their receptors, CD28 and
CTLA4,2,7,8 as prototypic costimulatory molecules has led
to several novel approaches in tumor immunotherapy, including the
expression of B7 on tumors9-12 and the use of antireceptor antibodies.12,13 Elucidation of the function of B7
receptors on T cells would facilitate the development of additional
therapeutic strategies targeted at this pathway. Despite extensive
analysis on the function of B7-CD28/CTLA4 interaction in the last
decade, it is still unresolved whether CD28 and CTLA4 have opposite
functions in immune regulation.
The prevailing notion that CTLA4 is a negative regulator during
T-cell activation was based on 3 lines of circumstantial evidence, namely, the effect of anti-CTLA4 monoclonal antibodies
(mAbs),14,15 the fatal lymphoproliferative diseases in
CTLA4-deficient mice,16-18 and the fact that CTLA4
associates with a tyrosine phosphatase, SHP-2.19,20 Recent
studies, however, have raised questions about this interpretation. For
example, because CTLA4 is expressed in developing thymocytes and
because of the effect of anti-CTLA4 mAb on anti-CD3-induced cell death
and deletion of myelin-basic protein-specific T
cells,21,22 one potential explanation of lymphoproliferative disease is an alteration of T-cell repertoire in
CTLA4-deficient mice.23 Consistent with this, recent
studies indicate that the lymphoproliferative diseases can be cured by eliminating potential autoreactive T-cell
repertoires.24,25 In addition, the effect of anti-CTLA4
mAbs depends on the condition of T-cell receptor (TCR) engagement
rather than the valence of the antibodies, and it is independent of the
cytoplasmic domain of CTLA4 that is involved in association with
SHP-2.26,27 Moreover, with one exception,28
most groups have failed to observe any inhibitory effect of B7 when
CD28/ T cells are used.29-31 Direct
comparison between CTLA4/ and CTLA4+/+ T
cells in vivo revealed no advantage of CTLA4/ T cells
during immune responses.32
Using B7-transfected cell lines and tumors, we have reported that B7
can promote the activation of CD28/ T cells in vitro
and promote tumor rejection in vivo.33,34 The cellular
basis for enhanced tumor rejection has not been clearly elucidated in
vivo. To address the function of B7 receptors during antitumor CTL
response, we developed an in vivo model involving the adoptive transfer
of tumor-specific naive T cells into RAG-2/ syngeneic
mice. We analyzed clonal expansion and effector function of
CD28+/ and CD28/ T cells in the
tumor-bearing RAG-2/ mice. Our results demonstrate that
B7-CD28 and B7-CTLA4 interactions play different roles in the antitumor
immune response. At the inductive phase, B7 on either tumor or host
antigen-presenting cells (APCs) interacts with CD28 on T cells
to promote clonal expansion. However, at the effector phase, CTLA4 on T
cells interacts with B7 on tumor cells to promote the cognate
destruction of tumor cells in vivo, though a role for CD28 in this
process cannot be ruled out.
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Materials and methods |
Experimental animals
Transgenic mice expressing the TCR specific for tumor
antigen P1A35-43:Ld complex have been
described.35 TCR transgenes were back-crossed with
BALB/cByJ for at least 6 generations before they were used for this
study. BALB/c mice with a targeted mutation of CD28 were purchased from
Jackson Laboratories (Bar Harbor, ME). BALB/c mice with a targeted
mutation of RAG-2 were purchased from Taconic (Germantown, NY).
CD28+/ and CD28/ P1CTL mice were derived
from breeding between CD28/ mice and
CD28+/ P1CTL+ transgenic mice the F1 of
CD28/ BALB/c and BALB/c P1CTL. Cell line plasmocytoma
J558 transfected either with vector alone (J558-Neo), wild-type B7-1
(J558-B7), or B7-1 with a mutation from W to A at position 88 (J558-B7W) has been described.34
Antibodies and fusion protein
Anti-B7-1 (3A12 and 10.16A)6,36 and anti-B7-2
(GL-1) mAbs4 were purified from the hybridoma supernatants
using a protein G affinity column. For in vitro analysis, the
antibodies were biotinylated. Biotinylated or phycoerythrin
(PE)-conjugated anti-V 8, anti-CD28, and anti-CTLA4 mAbs were
purchased from PharMingen (San Diego, CA). Fusion proteins CD28
immunoglobulin and CTLA4 immunoglobulin, composed of the immunoglobulin
(Ig)G1 Fc portion and extracellular domains of CD28 or CTLA4, were
produced using the pIg vector (Clontech, San Diego, CA) according to
the manufacturer's instructions. Murine B7 immunoglobulin, which
consists of the extracellular domain of murine B7-1 and the Fc portion
of the murine IgG2a, was produced according to a described
procedure.37 Ascites of anti-CD28 mAb 37N38
and anti-CTLA4 mAb 4F1015 were produced using hybridomas
kindly provided by Drs James P. Allison (University of California,
Berkeley) and Jeffery A. Bluestone (University of Chicago, IL), respectively.
Flow cytometry
Cell surface expression of B7-1 was detected with mAb 10.16A.
Unlabeled anti-B7-1 was detected using goat-antihamster
IgG-fluorescein isothiocyanate (FITC) (Caltag, Mountain View, CA).
Biotinylated anti-B7-1 and anti-B7-2 were detected using
PE-streptavidin.
Spleen cells from P1CTL transgenic mice were used directly without in
vitro stimulation or were stimulated with P1A peptide (AA35-43, 0.1 µg/mL) for 3 days before analysis. In addition, single-cell
suspension was prepared from J558-B7 tumors surgically isolated from
RAG-2/ BALB/c mice at 4 days after adoptive transfer of
CD28+/+, CD28+/, or CD28/
P1CTL. CD8+ T cells were marked with FITC-labeled anti-CD8
mAbs. Cell surface expression of CTLA4 was measured using PE-conjugated
anti-CTLA4 mAb 4F10 (PharMingen). To detect the expression of
intracellular CTLA4, T cells were treated with perm/wash buffer
(PharMingen) before the addition of PE-labeled anti-CTLA4 mAbs. For
blocking studies, intact or permeabilized cells were preincubated with a 1:10 dilution of anti-CD8, anti-CD28, or anti-CTLA4 mAb for 1 hour at
4°C. Biotinylated anti-B7 immunoglobulin or heat-stable antigen
(HSA)-immunoglobulin fusion protein39 was added at a final
concentration of 25 µg/mL and was incubated for 2 hours. After 4 washes with perm/wash buffer, the cell-bound fusion protein was
detected using PE-conjugated streptavidin. Fluorescence was analyzed
using a Coulter XL analyzer (Beckman Coulter, CA). List mode data were
analyzed using the Flowjo 3.4 (Tree Star, La Jolla, CA).
Adoptive transfer of purified transgenic T cells
Pools of spleen and lymph node cells from the P1CTL-transgenic
mice were incubated with a cocktail of mAbs (anti-CD4 mAb GK1.5, anti-FcR mAb 2.4G2, and anti-CD11c mAb N418). After the removal of
unbound mAbs, the cells were incubated with anti-immunoglobulin-coated magnetic beads. Antibody-coated cells were removed with a magnet. Unbound cells consisted of more than 90% CD8 T cells with no
detectable CD4 T cells. All CD8 T cells expressed the transgenic
receptor as revealed by staining with anti-V8 mAb. Purified T cells
were adoptively transferred by intravenous injection into
RAG-2/ mice with established tumors or had received
tumor cells on the day of adoptive transfer. In some experiments, the
CD8 T cells were labeled with carboxylfluoresceindiacetate succinimidyl
ester (CFSE) before adoptive transfer, as
described.40
Tumorigenicity assay 5 × 106 J558 cells were injected in
the flanks as described.11 Tumor size and incidence
were determined by physical examination.
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Results |
Inside P1CTL or on the cell surface, CTLA4 is the only detectable
non-CD28 receptor for B7-1 and is induced by CD28-independent
mechanisms
Two assumptions must be verified before one can use
CD28/ T cells to study the function of B7-CTLA4
interaction. First, CTLA4 expression must be autonomous from that of
CD28. Second, in addition to CD28 and CTLA4, activated T cells must
express no other B7 receptor. CTLA4 is inducible during T-cell
activation. It is known that anti-CD28 can enhance the expression of
CTLA441; however, it is unclear whether CD28 is required
for CTLA4 expression. We analyzed the expression of CTLA4 among resting
and activated P1CTL by flow cytometry. Profiles of CTLA4 expression on
gated CD8 T cells are presented in Figure
1 and Figure
2.
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| Figure 1.
Expression and identity of a non-CD28 receptor
for B7-1 on the cell surface of P1CTL.
(A) CD28-independent expression of CTLA4 on activated T cells. Naive
(i-ii), in vitro-activated (iii-iv), and ex vivo-activated (v-vi)
P1CTL were stained with either PE-conjugated anti-CTLA4 mAb (solid
lines) or isotype control (dotted lines). Data shown were gated CD8 T
cells, marked by FITC-conjugated anti-CD8 mAb. (B) Blocking of B7-1
immunoglobulin binding to activated CD28/ P1CTL by
anti-CTLA4, but not anti-CD8 mAbs. CD28/ P1CTL were
stimulated for 3 days in vitro with P1A peptide and were stained with
biotinylated B7-1 immunoglobulin or control HSA immunoglobulin followed
by PE-conjugated streptavidin. To verify the involvement of CTLA4, half
the cells were pretreated with either anti-CD8 or anti-CTLA4 mAb (100 µg/mL) for 30 minutes before the addition of biotinylated fusion
proteins. Data shown are normalized histograms using Flowjo software
(version 3.4). Essentially identical numbers of cells were analyzed to
produce the overlaid histograms. The number of gated CD8 T cells
analyzed were naive, 10 000 events; in vitro activated, 5000 events;
ex vivo activated CD28/ T cells, 4000 events; ex vivo-
activated WT CD8 T cells, 5000 events. These experiments were repeated
twice with similar results.
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| Figure 2.
CD28-independent expression of CTLA4 and absence of B7 receptors other
than CD28 and CTLA4 in permeabilized P1CTL T cells.
(A) CD28-independent expression of intracellular CTLA4 in activated
P1CTL. (B) CD28-independent expression of intracellular CTLA4 in
tumor-infiltrating P1CTL. The genotype of CD28 locus (+/
or /) and the antibodies (CTLA4 or ctrl for isotype
control) used for panels A and B are shown in the legends in panel B. (C, D) Blocking of B7- immunoglobulin binding to activated T cells by
anti-CD28 or anti-CTLA4. Activated CD28+/ P1CTL were
incubated first with either anti-CD28 or anti-CTLA4 mAb ascites. They
were then incubated with biotinylated B7 immunoglobulin or a murine
IgG2a mAb isotype control (ctrl). The amount of biotinylated B7
immunoglobulin or ctrl was determined by PE-conjugated streptavidin.
Data in panels A, C, and D were repeated at least 5 times, and those in
panel B were repeated twice.
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Naive CD28+/+ and CD28/ P1CTL had no
detectable cell-surface CTLA4, as expected (Figure 1Ai-ii). To test
whether CTLA4 can be induced on activated T cells, we activated
CD28+/+ and CD28/ transgenic T cells with
their specific antigen, P1A, for 3 days in vitro. Regardless of CD28
expression, the transgenic T cells had low, but detectable, cell
surface CTLA4 (Figure 1Aiii-iv). To determine whether CTLA4 expression
is autonomous of CD28 during an in vivo immune response, we adoptively
transferred CD28+/+ and CD28/ P1CTL into
RAG-2-deficient syngeneic mice that bore J558-B7+ tumors.
Three days later, the expression of CTLA4 in the tumor-infiltrating T
cells was analyzed. As shown in Figure 1Av-vi, substantial populations of tumor-infiltrating P1CTL isolated from J558-B7 tumors expressed CTLA4. Levels of CTLA4 were comparable between CD28+/+ and
CD28/ P1CTL. Thus, on P1CTL, CTLA4 is expressed by
CD28-independent mechanisms.
Because the overwhelming proportion of steady-state CTLA4 resides
within the cells and translocates to the site of TCR
engagement,42 we included 0.1% saponin in the staining
buffer to measure cell surface and intracellular CTLA4 molecules
simultaneously. Again, naive T cells did not express any detectable
intracellular CTLA4 (data not shown). As shown in Figure 2A,
CD28+/ P1CTL expressed a high level of CTLA4, as
expected. Interestingly, similar levels of CTLA4 were detected in
CD28/ T cells. Thus, in the presence of high doses of
antigen, CTLA4 expression does not depend on CD28. Although the ex vivo
P1CTL expressed lower CTLA4 than that activated by a high dose of
peptide in vitro, significant levels of CTLA4 were observed among the CD28+/ and CD28/ P1CTL. A comparison of
the CTLA4 levels between the CD28+/ and
CD28/ T cells indicated that, to some extent, CD28-B7
interaction can enhance intracellular CTLA4 expression, as has been
reported.41,43
CD28 and CTLA4 are 2 known receptors for B7-1.2,7
Recently, genetic evidence was reported that implied the existence of
additional B7 receptor(s), though no data to date support the direct
binding of B7 to CD28/ CTLA4/ T
cells.44 To test whether CD28 and CTLA4 are the only
receptors for B7-1 on P1CTL, we activated CD28+/ and
CD28/ P1CTL cells in vitro with the specific peptide
and tested their binding to B7-1 immunoglobulin. As shown in Figures 1B
and 2C-D, activated T cells bound significantly to the biotinylated
B7-1 immunoglobulin, but not to control fusion protein or mouse IgG. Thus, biotinylated B7-1 immunoglobulin, but not control biotinylated HSA immunoglobulin, bound to intact CD28/ T cells. This
binding was almost completely blocked by anti-CTLA4 mAbs. The
specificity of the blocking is confirmed because a control anti-CD8 mAb
had only a small effect. These results indicated that most B7-1 binding
to CD28/ T cells was mediated by CTLA4.
Because most CTLA4 on P1CTL resided intracellularly (Figure 2A-B), we
also tested B7-1 binding after permeabilization. Either anti-CD28 or
anti-CTLA4 mAbs partially blocked B7-1 immunoglobulin binding to
CD28+/ T cells. Importantly, a combination of the 2 mAbs
completely blocked B7-1 immunoglobulin binding to activated T cells.
These results confirmed that CD28 and CTLA4 account for all B7-1
receptors in activated P1CTL. Thus, one can use CD28/
P1CTL to study the function of CTLA4.
B7 on host antigen-presenting cells, but not on tumors, are
responsible for T-cell clonal expansion: roles for CD28 and
CTLA4
We first compared CD28+/ and CD28/
P1CTL for their proliferation and cytokine production in response to
the P1A antigen in vitro. As shown in Figure
3A, CD28+/ and
CD28/ T cells proliferated vigorously to P1A antigen
stimulation, though CD28+/ T cells required 100-fold less
P1A antigen to achieve maximal proliferation. In the presence of
anti-B7-1 and anti-B7-2 mAbs, CD28+/ and
CD28/ T cells responded equally well to P1A peptide.
Because anti-B7-1 and anti-B7-2 mAbs did not inhibit the
proliferation of CD28/ T cells, B7 expressed on the
APCs was insufficient to costimulate T-cell proliferation through a
pathway other than CD28. This was most likely attributed to the
relatively low level of B7 on the APCs given that B7-1 can costimulate
the clonal expansion of CD28/ CD4 T cells when it is
overexpressed on Chinese hamster ovary cells.33
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| Figure 3.
Functional characterization of the CD28+/
and CD28/ P1CTL.
(A) Proliferative response of transgenic spleen cells to varying
concentrations of the P1A peptide was measured by pulsing the culture
for 6 hours with 3H-TdR, starting at 42 hours of culture. A
mixture of anti-B7-1 and anti-B7-2 (1 µg/mL) mAbs was added at the
beginning of the culture. Data presented are means and SE of
triplicates of cpm. (B, C) Role of CD28 in production of cytokines,
IL-2 (B) and IFN- (C) in response to antigenic P1A peptide (0.1 µg/mL). Cytokines released into the supernatants at 48 hours after
antigenic stimulation were measured by sandwich ELISA. (D) Cytotoxicity
of activated CD28+/ and CD28/ P1CTL.
Spleen cells activated in vitro for 4 days were used as the effectors,
whereas the macrophage cell line P388D1, pulsed with (+P) or without
( P) P1A peptide (1 µg/mL) was used as the target. (E) Anti-B7 mAbs
inhibit IFN- production by CD28/ P1CTL, as detailed
in panel C, except that anti-B7-1 and anti-B7-2 mAbs (10 µg/mL)
were added into the culture. (F) Anti-B7 mAbs (added before the
addition of effector T cells and present during CTL assay only) did not
inhibit the cytolysis of P1A peptide-pulsed P388D1 target cells by
P1CTL. Data shown are representative of at least 2 independent
experiments.
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When stimulated by optimal amounts of P1A peptides,
CD28+/ T cells produced a large amount of interleukin-2
(IL-2). In contrast, no IL-2 was detectable from the culture of
CD28/ T cells, even when a sufficient dose of antigen
was used to induce maximal proliferation (Figure 3B). In comparison
with CD28+/ T cells, CD28/ T cells
produced a significant, though 3- to 5-fold lower, amount of interferon
(IFN)- (Figure 3C) and IL-4 (not shown). Moreover, regardless of
the CD28 genotype, activated P1CTL was cytotoxic to a macrophage cell
line pulsed with specific antigen. Cytotoxicity levels appeared to be
3- to 5-fold lower in CD28/ T cells (Figure 3D). Both
propertiesthe essential role in IL-2 production and the ability to
reduce the amounts of antigen required for proliferationare
consistent with known functions of CD28.31,45
Interestingly, anti-B7-1 plus anti-B7-2 significantly inhibited the
production of IFN- by CD28/ P1CTL (Figure 3E). These
results suggest that B7-CTLA4 interaction promotes cytokine production
by T cells. However, blocking with anti-B7 had no effect on the
cytotoxicity of CD28/ P1CTL (Figure 3F).
We labeled transgenic T cells with CFSE and injected them into
RAG-2/ mice that bore either J558-B7 or J558-Neo
tumors. T-cell division was analyzed at 4 days after adoptive transfer.
As shown in Figure 4, both
CD28+/ and CD28/ P1CTL proliferated
vigorously in tumor-bearing mice. Within the same time frame, little
proliferation was observed in mice that bore no tumors. Thus, the bulk
of the proliferation was tumor driven.
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| Figure 4.
Optimal in vivo clonal expansion of P1CTL requires CD28
on the T cells.
Purified CD28+/ (blue lines) or CD28/
(red lines) CD8+ P1CTL were labeled with CFSE and were
adoptively transferred into RAG-2/ mice that bore
either J558-B7 (C) or J558-Neo (B) tumors or into
RAG-2/ mice that received no tumor cells (A).
Mononuclear cells were harvested on day 4 after transfer. Division of
P1CTL was analyzed by flow cytometry. Data shown are CFSE intensity of
gated V8+ T cells, as measured by flow cytometry and
analyzed by Flowjo software, as detailed in the legend to Figure 1.
Five thousand gated P1CTL cells were analyzed. The increased
proliferation of CD28+/ T cells over that of the
CD28/ T cells was reproduced in 3 independent
experiments.
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In the spleens, CD28+/ T cells proliferated substantially
faster than the CD28/ T cells. The difference was more
striking in mice bearing J558-Neo tumors than in those that bore
J558-B7 tumors. Similar results were observed in the draining lymph
nodes (data not shown). Thus, CD28 can enhance the clonal expansion of
P1CTL in vivo. It is worth noting that in this experiment, the division
of CD28+/ T cells was not accelerated in the J558-B7
tumor-bearing mice. However, in other experiments, B7-1 on tumor cells
enhanced T-cell division (data not shown). This was perhaps influenced
by the number of tumor cells in the spleen given that considerable
variations in the number were observed among individual
mice.46 J558 tumor cells induce direct priming and
cross-priming in this model.46 Direct priming by tumor
cells requires the expression of B7 on the tumor cells. However,
cross-priming is mediated by B7-expressing host APCs and may not
necessarily be enhanced by B7 expression on the tumor cells. It is
therefore not surprising to observe variations with regard to the
effect of tumor-expressed B7 on the rate of T-cell division in vivo.
To address the role of B7-CTLA4 interactions in T-cell clonal
expansion, we injected anti-B7-1 and anti-B7-2 mAbs into the J558-Neo
tumor-bearing mice on days 0, 1, and 2 of adoptive transfer of
CFSE-labeled T cells. On day 3, T-cell division was analyzed by flow
cytometry. Interestingly, the division of CD28/ T cells
was neither enhanced nor reduced by an effective dose of anti-B7-1 and
anti-B7-2 mAbs (Figure 5A-B). The mAbs
blocked B7-1 and B7-2 as they substantially reduced the proliferation of WT T cells (Figure 5C). The difference between the anti-B7 and
control immunoglobulin groups (Figure 5) was not as dramatic as that
between the CD28+/ and CD28/ groups
(Figure 4). It is possible that the blockade by anti-B7 is incomplete
in vivo. Nevertheless, the complete lack of effect of anti-B7 on the
division of CD28/ T cells supports the conclusion that
B7-CTLA4 interaction does not play a significant role in T-cell
division in vivo.
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| Figure 5.
B7-CTLA4 interaction does not contribute to T-cell
proliferation in vivo.
CD28+/+ or CD28/ P1CTL were labeled with
CFSE and injected into mice that had J558-Neo tumors. On days 0, 1, and
2, the tumor-bearing mice were injected with a mixture of either
control rat/hamster immunoglobulin or anti-B7-1 and anti-B7-2 mAbs
intraperitoneally (100 µg/antibody per mouse per injection). On day
3, spleen (A, C) or tumor (B) cells were harvested and analyzed. (A)
Effect of anti-B7 mAb on the division of CD28/ P1CTL
accumulated in the spleen. (B) Effect of anti-B7 mAb on the division of
tumor-infiltrating P1CTL. (C) Anti-B7 mAb blocks the division of
CD28+/+ T cells. Data shown were CFSE intensity of gated
V8+ T cells, as measured by flow cytometry, and were
analyzed by Flowjo software, as detailed in legend to Figure 1. Five
thousand gated P1CTL cells were analyzed. Data shown are representative
of 3 independent experiments.
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Role for CTLA4 in cognate destruction of tumor cells by
CTL
We have recently demonstrated that B7-1 also plays a major role
for the effector function of P1CTL for several lineages of P1A-expressing tumors.47 To analyze the function of CD28
and CTLA4 at the effector phase, we injected J558-B7 and J558-Neo tumor
cells at separate flanks of the same RAG-2/ mice. Some
of the tumor-bearing mice were then adoptively transferred with either
CD28+/ or CD28/ P1CTL (Figure
6A). As shown in Figure 6B, J558-Neo and
J558-B7 grew at comparable rates in RAG-2/ mice that
received no T cells. The comparable growth kinetics of the 2 tumor
cells in RAG-2/ mice have been verified in more than 20 experiments (data not shown). Thus, NK cells by themselves do not
preferentially reject J558-B7 tumors in syngeneic mice. In mice that
received CD28+/ P1CTL, J558-B7 tumors failed to develop,
whereas J558-Neo tumors grew progressively. This is consistent with our
previous report that WT P1CTL preferentially rejects J558-B7 tumors
over J558-Neo tumors.47
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| Figure 6.
B7-dependent rejection of J558 tumor: role of CD28 and CTLA4.
(A) Diagram of experimental design. J558-B7 and J558 tumor cells were
injected at separate flanks of the RAG-2/ BALB/c mice.
These mice then either received no T cells or they received
CD28+/ or CD28/ T cells on the day of
tumor injection. The tumor incidence (top panels) and growth kinetics
were monitored. (B) Tumor rejection by 5 × 106 of
CD28+/ or CD28/ P1CTL. Groups that
received no T cells or CD28+/ T cells had 3 mice per
group; the group that received CD28/ T cells consisted
of 5 mice. The sizes of J558-Neo and J558-B7 tumors in mice that
received CD28/ P1CTL were significantly different
between day 12 and day 22 (2-sided P value between .05 and
.0001 by Student t test).
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In mice that received 5 × 106 CD28/ T
cells, we observed a reduction of tumor incidence and growth kinetics,
though B7+ tumors still developed in 60% of the mice
(Figure 6B). Thus, the expression of B7-1 on the tumor is a significant
growth disadvantage. To substantiate the function of B7-CTLA4
interaction, we increased the number of CD28/ T cells
3-fold, to 15 × 106/mouse. As shown in Figure
7, an increase in the amounts of
CD28/ P1CTL leads to a complete rejection of
B7+ tumor cells, whereas growth of the B7
tumor cells is only slightly affected. Selective elimination of
B7+ tumors by the CD28/ T cells indicates
that B7-CTLA4 interaction promotes tumor rejection.
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| Figure 7.
Tumor rejection by 15 × 106 of
CD28/ P1CTL.
The group that received no T cells consisted of 4 mice, whereas the
group that received CD28/ T cells consisted
of 5 mice. Differences in the sizes of J558-Neo and J558-B7 tumors were
statistically significant from day 17 on (Welch t test,
2-sided P value between .05 and .03).
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The failure to reject the J558-Neo tumors can be attributed to a
lack of CTL maturation or to a requirement for B7-1 at the effector
phase. To bypass the requirement for B7-1 at the inductive phase, we
mixed the J558-B7 and J558-Neo cells before injection into
RAG-2-deficient mice, which then received purified
CD28/ P1CTL intravenously. In RAG-2-deficient mice
that received no T cells, all J558-Neo tumor cells were
B7 and all J558-B7 tumor cells were B7+
(Figure 8A), as expected. In mice that
received a mixture of J558-Neo and J558-B7, both types of cells were
presented at an approximate 1:1 ratio when T cells were not present
(Figure 8Bi-ii, for 2 examples). In mice that received either
CD28+/ or CD28/ P1CTL, the overwhelming
majority of the surviving tumor cells were devoid of B7 expression
(Figure 8Biii-iv and 8Bv-vi, respectively; 2 cases presented for each
group). The failure to eliminate B7 tumor cells that
colocalized with the B7+ cells demonstrates a critical role
for B7 in the cognate destruction of tumor cells by CTL. Because the T
cells were devoid of CD28, B7 must have interacted with CTLA4 to
promote the cognate destruction of tumor cells. The increased efficacy
of CD28+ T cells could have resulted from a role for CD28
in effector function or from an increased number of T cells in the
tumors (data not shown).
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| Figure 8.
Selective elimination of B7+ tumor cells by P1CTL from a
mixture of B7+ and B7 tumor cells: flow
cytometric analysis of the composition of B7+ and
B7 tumors in the presence or absence of P1CTL.
Single viable cell suspensions were prepared from freshly isolated
tumors and were stained with anti-B7-1 mAb hybridoma supernatants
(bold lines) or medium as control (thin lines). (A) J558-B7 or J558
tumor cells in separate mice. (B) J558-Neo and J558-B7 cells were mixed
before injection. Tumors in panels i-ii were from mice that received no
T cells; those in panels iii-iv received 5 × 106 of
CD28+/ T cells, and those in panels v-vi received
5 × 106 of CD28/ T cells.
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Mutant B7-1 that selectively binds CTLA4 promotes tumor rejection
in vivo
Given the potential contribution of CD28 in T-cell
development,48,49 the signaling machinery of T cells that
develop in the absence of CD28 can be different from that of wild-type
T cells. Because wild-type B7-1 binds to CD28 and CTLA4, a mutant B7-1
that binds to CTLA4 alone would help to bypass this difficulty. We
recently produced a mutant of murine B7-1 that has a substitution of W
to A at position 88 in the IgV domain. In semiquantitative assays, we
showed that this mutant binds CTLA4, but not to CD28.33,34 To quantitatively determine its binding to CD28 versus CTLA4, we tagged
the wild-type and mutant B7-1 with green fluorescence protein (GFP) and
compared their binding to the 2 receptors. As shown in Figure
9A, based on the density of GFP, the
levels of WT and mutant B7 cells were extremely heterogeneous, with
almost 1000-fold variations in their expression levels. This gave us an
opportunity to examine their receptor bindings over a large dose range.
WT B7-1 bound to CD28 immunoglobulin and CTLA4 immunoglobulin, with the
level of B7 expression correlating almost linearly with their binding.
As expected, approximately 10-fold more B7-1 was needed to achieve a
comparable binding to CD28 immunoglobulin. Based on the ratio of CTLA4
binding to GFP signal, WT B7 and B7W bound CTLA4 equally well over a
1000-fold dose variation. These results established that mutant B7W
maintains full binding to CTLA4 while lacking any detectable binding to
CD28 over a large range. As expected, when B7W was expressed in the
J558 cells, it also failed to bind CD28 immunoglobulin, though its
binding to CTLA4 immunoglobulin was unaffected (Figure 9A). To test
whether B7-CTLA4 interaction is sufficient to costimulate tumor
rejection, we adoptively transferred CD28+/ P1CTL into
RAG-2/ mice and challenged them at separate sites with
J558-Neo and J558-B7W. As shown in Figure 9B, P1CTL rejected
J558-B7W, but not J558-Neo. Thus, B7-CTLA4 interaction caused the
rejection of J558 tumors.
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| Figure 9.
Mutant B7-1-transfected tumor cells that bind to CTLA4, but not to
CD28, were selectively eliminated by P1CTL.
(A) Characterization of receptor binding of WT and mutant B7-1 by flow
cytometry. COS cells were transiently transfected with plasmid
expressing GFP (i), GFP-tagged wild-type B7 (B7-GFP) (ii), or mutant
B7(W88 A)(iii) were stained with 100 µg/mL of either CD28
immunoglobulin or CTLA4 immunoglobulin mixed with PE-conjugated
goat-antihuman IgG. Two-color flow cytometry was used to determine B7
expression versus receptor binding. Green indicates binding of CTLA4
immunoglobulin; blue, CD28 immunoglobulin; red, control. (B) Receptor
binding and (C) tumorigenicity of J558-Neo and J558-B7W. (B) Binding to
anti-B7-1 mAb (i), CD28 immunoglobulin (ii), and CTLA4 immunoglobulin
(iii). (Ci, iii) (n = 4): tumor incidence (i) and growth kinetics
(iii) of J558-Neo and J558-B7W tumors in RAG-2/ mice
that received no T cells. (Cii, iv) (n = 5): incidence (ii) and
growth kinetics (iv) in the presence of 15 × 106
CD28+/ P1CTL.
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Discussion |
The function of B7-CTLA4 interaction remains
controversial.23,50 Here we used an adoptive transfer
model to evaluate the function of B7-CTLA4 interaction during an in
vivo antitumor CTL response.
A comparison between CD28+/ and CD28/
P1CTL for their proliferative response to tumor antigens revealed a
potent costimulatory function of CD28 in T-cell proliferation, which is
consistent with previous results from this and other
laboratories.33,45,51 In vitro, CD28+/ T
cells require about 100-fold less antigenic peptide than do CD28/ P1CTL in the proliferative response. This
difference can be eliminated by anti-B7-1 and anti-B7-2 mAbs. In
contrast, anti-B7 mAbs have no effect on the proliferative response of
CD28/ T cells. The results of in vivo analysis
corresponded well to the in vitro observations. In the spleen,
CD28+/ T cells divided substantially faster than did the
CD28/ T cells in tumor-bearing mice. Again,
antibody-blocking studies indicate that B7-CTLA4 interaction does not
contribute to T-cell clonal expansion in vitro and in vivo. Thus,
although B7-1 overexpressed on fibroblasts promotes the proliferation
of CD28/ T cells,33 the amount of B7
expressed on host APCs appears insufficient to induce T-cell division
without the participation of CD28.
Because we did not use CTLA4/ T cells for the current
study, it is unclear whether B7-CD28 interaction provided costimulation without the participation of CTLA4-B7 interaction. However, the profound B7-dependent lymphoproliferative disease in CTLA4-deficient mice52,53 indicated that B7-CD28 interaction is sufficient for T-cell clonal expansion.
The most important conclusion from the current study is that
B7-CTLA4 interaction promotes CTL-mediated tumor destruction in vivo.
The effector mechanism is not clearly understood at present. However,
we do not believe CTL-activated NK cells are the direct effector
because P1CTL select for major histocompatibility complex (MHC) class
Ilow tumor cells in vivo.54 This is consistent
with the direct function of that CTL because it would require tumor
expression of MHC class I. In contrast, CTL could be cross-primed by
host APCs and would not require MHC on tumor cells to produce cytokines
for NK activation. Moreover, because NK cells prefer MHC class
Ilow targets, MHC down-regulation would lead to a growth
disadvantage if NK cells were the effectors. The role for CTLA4-B7
interaction is based on results from 2 experimental approaches. The
first approach involved CD28+/ and CD28/
P1CTL. We found that in the same mice challenged with both
B7-1+ and B7-1 tumor cells,
CD28/ transgenic T cells rejected the
B7-1+, but not the B7, tumors. More
strikingly, when the 2 types of tumor cells were injected as a mixture,
CD28/ T cells selectively eliminated B7-1+
tumor cells while leaving B7-1 tumor cells in the same
sites. Because the B7-1 binding to CD28/ P1CTL can be
completely blocked by anti-CTLA4 mAbs, it is likely that the enhanced
effector function of CD28/ is mediated by CTLA4.
Nevertheless, given the suggestion that a yet unidentified B7 receptor
may exist on T cells,44 this approach alone cannot
formally rule out the possibility that other unidentified B7 receptors
can be responsible. In the second approach, tumor cells expressing
mutant B7-1 that bound to CTLA4, but not to CD28, were also
preferentially rejected by P1CTL that expressed CD28 and CTLA4 and
should have developed normally. This approach not only ruled out the
possibility that P1CTLdeveloped in the absence of CD28differ from
those that develop in the presence of CD28 with regard to their CTLA4
function, it also demonstrated that specificity similar to that of
B7-CTLA4 interaction (and unlike that of B7-CD28 interaction) is
responsible for the effector function. Taken together, the data from
these 2 approaches provided a compelling case that B7-CTLA4 interaction
promotes cognate destruction of tumor cells in vivo.
The mechanism by which B7 promotes the effector function of
tumor-specific CTL remains unclear. Based on our finding that B7-CTLA4
interaction promoted the production of IFN- but not cytotoxicity, it
is possible that the function of CTLA4 is mediated through local
production and effector function of IFN-, which has been shown to be
important for T-cell-mediated tumor immunity.55
To dissect the function of B7 receptors in a defined system, we have
chosen to use an adoptive transfer of transgenic T cells specific for a
natural tumor antigen P1A into immune-deficient mice as our basic
model. As such, our model differs from physiological conditions in
which the frequency of T cells are lower and in which immunity is
provided by interactions among different subset of T
cells.56 However, it is important to emphasize that the conclusion that B7-CTLA4 interaction promotes tumor immunity was first
reached in a nontransgenic mouse model.34 The simplicity of the current model helps to define the subset of T cells and the
stage of immune response at which B7-CTLA4 interaction mediates this
important function. Moreover, because the function of CTLA4 uncovered
in the current study involves CD8 T cells in the absence of CD4 T
cells, it is distinct from the hypothetical role of B7-CTLA4 interaction in the induction and function of CD25+CD4 T
cells.57
In summary, our data revealed a novel function of CTLA4 during the
effector phase of CTL responses. The relation between this function in
the effector phase of CD8 T cells and the previously proposed negative
regulation at the inductive phase of CD4 T cells14,15 is
unclear. Understanding the mechanism of CTLA4-enhanced CTL effector
function may lead to novel approaches to reinvigorate the effector
function of the high number of tumor-specific CTL found in cancer
patients58,59 for optimal antitumor effector function.
| |
Acknowledgments |
We thank Jing Wen for purification of the fusion protein and
Jennifer Kiel for editorial assistance.
| |
Footnotes |
Submitted March 20, 2001; accepted December 3, 2001.
Supported by National Cancer Institute grants CA69091, CA58033, and CA82355.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Yang Liu and Pan Zheng, Department of
Pathology and Comprehensive Cancer Center, Ohio State University
Medical Center, 129 Hamilton Hall, 1645 Neil Ave, Columbus, OH 43210;
e-mail: liu-3{at}medctr.osu.edu and zheng-1{at}medctr.osu.edu.
| |
References |
1.
Linsley PS, Brady W, Grosmaire L, Aruffo A, Damle NK, Ledbetter JA.
Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation.
J Exp Med.
1991;173:721-730[Abstract/Free Full Text] |
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Abstract
Introduction