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Department of Immunology, National Children’s Medical Research Center, Tokyo, Japan;
Department of Oral and Maxillo-facial Surgery, Tokyo Medical and Dental University, Tokyo, Japan;
Division of Pathobiology,
Division of Haematology, Department of Internal Medicine, and
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Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression
by CD4+ and CD8+ T cells and anti-host
cytolytic activity. Thus, blockade of CTLA-4 greatly enhanced
CD8+ T cell expansion, and this may result in the
regulation of consequent Th2-mediated humoral immune responses. These
findings suggest a new approach for regulating IgE-mediated allergic
immune responses by blockade of CTLA-4 during a critical period of Ag
sensitization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A graft-vs-host disease (GVHD) can be caused in inbred F1 mice by the injection of T cells of parental origin. The injection of C57BL/6 (B6) splenocytes into F1 mice (referred as B6 GVH) results in an "immunosuppresive" acute GVHD characterized by an anti-host cell-mediated Th1 cytokine-driven disease (20, 21, 22). By contrast, the injection of splenocytes from the BALB/c or DBA/2 parent into F1 mice (referred as BALB/c GVH or DBA GVH) results in an "immunostimulatory" chronic GVHD characterized by an autoantibody-mediated Th2 cytokine-driven disease (23, 24, 25). Blockade of both CD28 and CTLA-4 signals by a combination of anti-CD80/CD86 mAbs or CTLA-4Ig fusion protein efficiently inhibited both types of GVHD (26, 27, 28, 29). We recently demonstrated the regulatory function of the CTLA-4-CD80/86 pathway in the development of acute GVHD (30). Treatment with anti-CD80/86 mAbs in the CD28-independent B6 GVH model induced by donor T cells lacking CD28 expression exacerbated the clinical manifestations of acute GVHD and increased T cell responses against host alloantigen. However, the function of CTLA-4 in the development of chronic GVHD induced by Th2-dominant immune responses has not been investigated. In this study, we have examined the effects of initial blockade of CTLA-4 signals after donor T cell transfer in a parent into F1 BALB/c GVH model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c (H-2d) donor mice and (BALB/c x C57BL/6)F1 (CBF1, H-2d/b) recipient mice were purchased from SLC (Hamamatsu, Shizuoka, Japan). Donors were 6–8 wk old and recipients were 8–10 wk old at the time of transfer.
Monoclonal Abs
A hybridoma-producing anti-mouse CTLA-4 mAb (UC10.4F10.11,
hamster IgG) was generously provided by Dr. Jeffrey A. Bluestone
(University of Chicago, Chicago, IL). This mAb was purified from
ascites as described (31). Fab of anti-CTLA-4 mAb were
prepared as described (7), the purity was confirmed by
SDS-PAGE analysis, and the similar enhancing activities between intact
mAb and Fab were confirmed in allogeneic MLR responses in vitro as
described (7). The pyrogen level was <0.01 ng/µg
protein as determined by a Limulus amebocyte lysate assay.
Hybridomas producing mAbs against I-Ab,d,q
(M5/114, rat IgG2b), CD24 (HSA, J11d, rat IgM), and CD8 (3.155, rat
IgM) were obtained from American Type Culture Collection (Manassas, VA)
and were used as culture supernatants. Monoclonal Abs against the
following Ags were used for immunofluorescence analysis: CD3 (145-2C11,
hamster IgG), CD4 (RM4-5, rat IgG2a). CD8
(53-6.7, rat IgG2a),
CD45R/B220 (RA3-6B2, rat IgG2a), CTLA-4 (UC10-4F10, hamster IgG),
H-2Kb (AF6-88.5, mouse IgG2a), IFN- (XMG1.2,
rat IgG1), and IL-4 (BVD4-1D11, rat IgG2b). All FITC-, PE-, and
peridinin chlorophyll-a protein (PerCP)-conjugated mAbs were obtained
from PharMingen (San Diego, CA). Immunofluorescence, flow cytometry,
and data analysis were performed using FACSort and CellQuest software
(Becton Dickinson Immunocytometry Systems, San Jose, CA), as described
previously (32).
Induction of chronic GVHD and in vivo treatment with anti-CTLA-4 mAb
Single-cell suspensions of splenocytes from BALB/c mice were used as the source of GVHD-causing T cells. Unirradiated CBF1 recipients received 1 x 108 splenocytes diluted in 0.5 ml PBS by i.v. injection. Recipients receiving splenocytes were randomly divided into two groups of 6–12 mice and treated with either control hamster Ig (PharMingen) or anti-CTLA-4 (4F10) mAb. One hundred micrograms of mAb or control Ig per mouse were injected i.p. on day 0, 1, and 2 and then every other day until day 12 posttransfer. In certain experiments, BALB/c splenocytes were treated with anti-I-A, anti-CD24, and anti-CD8 mAbs and rabbit complement to deplete APC and CD8+ T cells. As assessed by flow cytometry, this procedure resulted in <3% contaminating CD8+ T cells and >90% purity of CD4+ T cells. CBF1 recipients received 3 x 107 CD8-depleted T cells and were treated with mAb as described above.
Measurement of serum IgE
Mice were bled from the retro-orbital plexus under ether anesthesia every 3 wk, and serum samples were individually aliquoted and stored at -80°C. The quantitation of total IgE and IgE allotypes was determined by ELISA as described previously (33, 34, 35, 36).
Determination of IgG1 and IgG2a Ab against host alloantigen
EL4 T lymphoma cells (H-2b, 1 x
106 cells/100 µl) were incubated with the
serially diluted (x40, x100, x200, x1000) serum samples (100 µl
each) from either normal or recipient CBF1 mice
for 45 min at 20°C, washed once, and then incubated with either
FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology
Associates, Birmingham, AL) or biotinylated-rabbit anti-mouse IgG2a
Ab (Zymed, San Francisco, CA) for 30 min at 20°C in the dark. For
detection of biotinylated Ab, fluorescence was visualized by
FITC-streptavidin (Dako-Japan, Kyoto, Japan). Samples were analyzed by
flow cytometry. Net mean fluorescence intensity (
MFI) was calculated
by subtracting the MFI of cells stained with the same dilution of sera
from normal CBF1 and the second Ab or
FITC-streptavidin from the MFI of cells stained with recipient
sera.
Multicolor staining for intracellular cytokines and cell-surface Ags
Single-cell suspensions of splenocytes from recipient mice were stimulated with PMA (50 ng/ml; Sigma, St. Louis, MO) and ionomycin (250 ng/ml; Sigma) in the presence of brefeldin A (5 µg/ml) for 4 h. After washing with staining buffer (PBS with 1% FCS and 0.1% sodium azide), cells were incubated with FITC-, PE-, and/or PerCP-conjugated mAb against cell-surface Ags as usual. Cells were fixed with 100 µl of fixation buffer (4% paraformaldehyde in PBS) at 4°C for 20 min and then washed twice with staining buffer. The fixed cells were resuspended in 50 µl of permeabilization buffer (PBS with 1% FCS, 0.1% sodium azide, and 0.1% saponin) with appropriate fluorochrome-conjugated anti-cytokine mAb at 4°C for 30 min. Stained cells were washed twice with permeabilization buffer, resuspended in 200 µl of staining buffer, and then analyzed by flow cytometry.
Measurement of anti-host cytotoxicity
Single-cell suspensions of splenocytes from each group of recipient F1 mice at 2 and 3 wk after transfer were used as effector cells in cytotoxic assays. Cytotoxicity against EL-4 (H-2b) cells was measured by a standard 6-h 51Cr-release assay.
Statistical analyses
Significant differences between experimental groups were analyzed using the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the effect of anti-CTLA-4 mAb treatment, GVHD
was induced by injecting parent BALB/c spleen cells into
CBF1 mice. Recipient mice were injected i.p. with
100 µg of anti-CTLA-4 (4F10) mAb eight times until day 12. The
elevation of serum IgE, which is a characteristic feature for chronic
GVHD, was examined every 3 wk after splenocyte transfer. In the control
Ig-treated mice, the levels of total serum IgE started to increase at 3
wk posttransfer, reached a maximum at 9 wk, and remained high until 15
wk (Fig. 1
). In contrast, the group of
mice treated with anti-CTLA-4 mAb showed an elevation of total IgE
at 6 wk, but an additional enhancement was not observed at later time
points. The serum IgE was decreased to half of that in the control mice
from 9 to 15 wk. Both IgEa and
IgEb allotypes were detected and responded in a
similar manner. IgEa was produced from both donor
BALB/c and host CBF1 cells, while
IgEb was produced by host
CBF1 cells alone. These results suggest that both
donor and host B cells were activated. Autoantibodies against dsDNA in
both groups of mice were increased in parallel with serum IgE (data not
shown). To verify the antagonistic activity of anti-CTLA-4 mAb, Fab
of anti-CTLA-4 mAb were also injected. The use of Fab also
inhibited total serum IgE, although the inhibitory effect was not as
effective as the intact anti-CTLA-4 mAb (Table I). This may come from less binding
abilities or a rapid clearance of Fab.
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). In contrast, the
anti-host IgG2a titer was not elevated at 3 wk, and this was not
affected by anti-CTLA-4 treatment. The IgG2a titer at 12 wk
was slightly enhanced, and the treatment with anti-CTLA-4
mAb minimally reduced this response. These results suggest that Th2
cytokine-dependent IgE and IgG1 production during the late phase of
GVHD was inhibited by the anti-CTLA-4 treatment.
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in splenic CD4+ or
CD8+ T cells at 2 and 8 wk posttransfer was
analyzed by flow cytometry. Although IL-4 was not detected in
CD4+ T cells from normal
CBF1 mice, a clear induction of IL-4 production
was observed in CD4+ T cells in the GVHD mice at
8 wk, but not at 2 wk (Fig. 3). IL-4 was
preferentially observed in donor cells. Treatment with anti-CTLA-4
mAb significantly reduced the percentage of IL-4-producing
CD4+ T cells. IFN- expression in
CD4+ T cells from mice with GVHD was slightly
enhanced at 2 and 8 wk compared with control CBF1
mice. However, the anti-CTLA-4 treatment did not significantly
affect IFN- production at either early or late time points. In
contrast with CD4+ T cells, IFN- expression in
CD8+ T cells was clearly augmented at 2 wk
posttransfer in both control and anti-CTLA-4-treated mice.
Interestingly, a clear reduction of IFN--producing
CD8+ T cells was observed in control mice, but
not in the anti-CTLA-4-treated mice at 8 wk. Our results
demonstrated that the anti-CTLA-4 treatment suppressed the
generation of IL-4-producing Th2 cells but increased IFN- expression
in CD8+ T cells during the late phase of
disease.
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To investigate how Th2-mediated immune responses are inhibited by
anti-CTLA-4 treatment, we examined the early events during or just
after mAb treatment. The number of total splenocytes in control GVHD
mice was increased 3-fold with a peak at 3 wk, whereas splenocyte
expansion was less in the anti-CTLA-4-treated mice (Fig. 4A). Anti-CTLA-4 treatment
seems to eliminate host B cells (Fig. 4B). The percentage of
donor cells (H-2b-) rapidly increased with the
peak at 2 wk and was maintained at 12–15% of total splenocytes (Fig. 4C). The anti-CTLA-4 mAb treatment clearly enhanced
donor cell expansion to 
2-fold at 2–3 wk. The donor cells consist
of >90% CD3+ T cells in the both
anti-CTLA-4-treated and control GVHD mice. An increase in the
absolute number of donor T cells was observed during the first 2 wk
(Fig. 4D), and this was preferentially observed in the CD8
subset (Fig. 4, E and F). Furthermore, the
anti-CTLA-4 treatment strikingly augmented
CD8+ T cell expansion and persistently maintained
an elevated number of CD8+ donor T cells
throughout the experimental period (Fig. 4F). This was
consistent with the maintained IFN- expression at 8 wk (Fig. 3
). We
next investigated whether the donor CD8+ T cells
possessed cytolytic activity against host cells. We measured
cytotoxicity of whole splenocytes against EL-4
(H-2b) target cells at 2 and 3 wk posttransfer.
Splenocytes treated with anti-CTLA-4 mAb exhibited higher
cytotoxicity at 2 and 3 wk posttransfer compared with untreated GVHD
mice (Fig. 5). However, the percentages
of donor CD8+ T cells in the
anti-CTLA-4-treated mice were 2- to 3-fold higher in the control
mice. Therefore, the higher cytolytic activity was a consequence of the
presence of more CTL rather than a functional change in
CD8+ T cells at the early phase. This conclusion
was also supported by the results of IFN- production as shown in
Fig. 3. These results indicate that the functional ability of
CD8+ T cells was not affected by the
anti-CTLA-4 treatment at the early events, but the anti-CTLA-4
treatment caused a significant increase in the expansion of
CD8+ T cells in these GVHD mice.
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To examine the involvement of donor CD8+ T
cell expansion in the inhibition of Th2 cell responses, we tested the
effect of CTLA-4 blockade in a chronic GVHD model induced by transfer
of splenocytes depleted of CD8+ T cells. The mice
receiving CD8-depleted T cells showed a elevation of serum IgE at 3 wk
(Fig. 6). As compared with the transfer
of whole splenocytes presenting in Fig. 1, the elevation of IgE was
rapidly induced. Interestingly, the anti-CTLA-4 treatment did not
affect IgE production at 3 wk but seemed to slightly augment IgE
production at late time points. The rapid elevation of IgE may result
from the absence of CD8+ T cells, which regulate
Th2 cell activation. This result indicates that suppression of Th2 cell
activation by anti-CTLA-4 treatment requires donor
CD8+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been reported that blockade of CTLA-4 enhanced Ag-specific Th1-mediated immune responses in several experimental autoimmune disease models, including DO11.10 TCR transgenic mice (10, 16), nonobese diabetic mice (12), and experimental allergic encephalomyelitis (13, 14). Similarly, Th2-mediated immune responses to nematode parasite infection can be augmented by anti-CTLA-4 treatment (19). Furthermore, CTLA-4-deficient mice have a fatal lymphoproliferative disorder (42), and blockade of CTLA-4 efficiently induced antitumor responses (11, 43). Altogether, these results indicate that CTLA-4 signals limit T cell activation and expansion.
How does blockade of CTLA-4 in our GVHD model suppress Th2-mediated
immune responses? We observed a moderate but substantial donor
CD8+ T cell expansion at 2–3 wk. CTLA-4 blockade
strikingly augmented the expansion of donor CD8+
T cells, but the effector function of CD8+ T
cells at a single-cell level was not affected. The importance of
CD8+ T cells in the inhibition of IgE was
directly demonstrated by transfer of splenocytes depleted of
CD8+ T cells. This suggests that donor
CD8+ T cells are essential for the effect of
anti-CTLA-4 mAb. Both acute and chronic GVHD share common
initiating events, the recognition of allogeneic MHC class II molecules
by donor CD4+ T cells, which results in increased
IL-2 production during the first 2 days after parental cell transfer
(44, 45). However, the most striking differences, which
determines the direction toward acute or chronic GVHD, are the
engraftment of donor CD8+ T cells, which
terminates B cell hyperactivity (46). Previously, Via et
al. (47) compared T cell chimerism between DBA GVH and B6
GVH mice at 2 wk after transfer and reported the dominant expansion of
CD4+ donor T cells in DBA GVH mice, but, on the
contrary, CD8+ donor T cells in B6 GVH mice.
Thus, the level of engraftment of donor CD8+ T
cells at an early phase plays an important role for regulation of the
consequent development of chronic GVHD. Prior reports have suggested a
regulatory role for CD8+ T cells in the
development of chronic GVHD (46, 48, 49). The
CD8+ T cell expansion caused a reduction in the
number of host cells, especially B cells at the early phase after
transfer, and a suppression of IL-4 producing Th2 cells at the late
phase after transfer. These results suggest two major roles for donor
CD8+ T cells; the first is their capacity to make
IFN-, which inhibits the differentiation of IL-4-producing Th2
cells, and the second is their cytolytic potential, which eliminates
host cells. A regulatory role of CD8+ T cells has
been demonstrated in other Th2-mediated immune responses, which
resulted in reduced IgE Ab production (50, 51, 52, 53). Depletion
of CD8+ T cells in vivo during a critical period
after immunization produced high and persistent IgE responses and led
to decreased production of IFN-, resulting in a net decrease in the
ratio of IFN- to IL-4 (51, 53). Therefore, manipulation
of CD8+ T cells may be beneficial for regulation
of Th2-mediated allergic immune responses. Expansion of donor
CD8+ T cells and augmentation of IFN-
expression at late time points were not observed in control GVHD mice.
Cytokines secreted from Th2 cells may inhibit activation of
CD8+ T cells.
In this BALB/c GVH model, we also observed a mild enhancement of CD4+ donor T cell expansion at 1–2 wk as well as CD8+ donor T cells. However, CTLA-4 blockade seems to preferentially affect CD8+ T cells rather than CD4+ T cells. Why does CTLA-4 blockade preferentially affect CD8+ T cells? Differential expression of CTLA-4 by CD4 vs CD8 T cells is unlikely, because both cell types express similar amounts of CTLA-4 after stimulation (7, 8, 54). In contrast with our result, two recent studies using CTLA-4-deficient mice have suggested that CTLA-4 is less important in regulating CD8+ T cells compared with CD4+ T cells (55, 56). In these experiments, highly antigenic viral Ag or peptide-Ag were used for inducing primary CD8+ T cell responses. Varying the priming conditions, such as peptide concentration, antigenic capacity of Ags and costimulatory signals may affect the dependency of CTLA-4-negative signals in primary responses of CD8+ T cells. In addition, the contribution of initial activation of CD4+ T cells in the subsequent CD8+ T cell activation may be another possible explanation. Blockade of CTLA-4 by in vivo administration of anti-CTLA-4 mAb in the early stages of tumor growth enhanced the capacity to generate antitumor T cell responses in which final effector T cells should be CD8+ T cells (11, 43). In both tumor-transplantation and GVHD models, it is possible that an initial enhancement of CD4+ T cell expansion by prevention of CTLA-4 signals may further augment subsequent CD8+ T cell responses. In agreement with this notion, Chambers et al. (57) suggested the possibility that the initial expansion of CD4+ T cells may determine a skewing the CD4/CD8 T cell ratio based on the results of the phenotype in neonatally CD4- or CD8-depleted CTLA-4-deficient mice. CD8+ T cell depletion did not alter the onset of severity of lymphoproliferative disorder, whereas CD4+ T cell depletion completely prevented. Although further studies will be required, we cannot negate a possibility that the initial expansion of CD4+ T cells by CTLA-4 blockade may be responsible for the sequential CD8+ T cell expansion.
In this study, we demonstrate the immunosuppresive effect of CTLA-4 blockade in a murine chronic GVHD model. CTLA-4 blockade at the initial Ag sensitization enhanced expansion of CD8+ T cells in the early response and resulted in the regulation of the consequent Th2-mediated humoral immune response. Our results imply a new approach for regulating IgE-mediated allergic diseases by blockade of CTLA-4 signals during a critical period of Ag sensitization.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Miyuki Azuma, Department of Immunology, National Children’s Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan. E-mail address:
3 Abbreviation used in this paper: CD80/86, CD80 and CD86; GVHD, graft-vs-host disease; B6, C57BL/6; CBF1, (C57BL/6 x BALB/c)F1; MFI, mean fluorescence intensity; PerCP, peridinin chlorophyll protein.
Received for publication May 12, 1999. Accepted for publication November 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, anti-IFN-, or anti-interleukin-4 monoclonal antibodies in chronic autoimmmune graft-versus-host disease. Clin. Immunol. Immunopathol. 63:66.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or control hamster Ig (•). One
hundred micrograms mAb or control Ig per mouse was injected i.p. on day
0, 1, 2, 4, 6, 8, 10, and 12. Total IgE and IgE allotypes in sera were
determined at every 3 wk after transfer. Each plot represents the
mean ± SD and the data are representative of two similar
experiments. *, Statistically different from control group
(p < 0.05).
) and
anti-CTLA-4-treated (
) recipient mice at 2 and 8 wk after
transfer and normal CBF1 mice (
) was assessed by
immunofluorescent staining of intracellular cytokines by flow
cytometry. Cells were stained with either a combination with
FITC-anti-H-2b, PE-anti-IL-4 and
PerCP-anti-CD4, or FITC-anti-IFN-
