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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2963-2970
Role of CD28 in Acute Graft-Versus-Host Disease
By
Xue-Zhong Yu,
Paul J. Martin, and
Claudio Anasetti
From the Division of Clinical Research, Fred Hutchinson Cancer
Research Center, Seattle, WA; and the Department of Medicine, Division
On Oncology, University of Washington, Seattle, WA.
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ABSTRACT |
Because CD28-mediated T-cell costimulation has a pivotal role in the
initiation and maintenance of T-cell responses, we tested the
hypothesis that CD28 is critical for the development of
graft-versus-host disease (GVHD). We compared the in vivo effects of
CD28 / T cells transplanted from B6 donor with the
CD28 gene deleted by homologous recombination with those of
CD28+/+ T cells transplanted from wild-type C57BL/6
(B6) donor. Fifty million CD28/ or
CD28+/+ splenocytes from B6 mice were transplanted into
unirradiated (B6 × DBA/2)F1 (BDF1) recipients. Unlike
CD28+/+, CD28/ T cells from
B6 mice had lower levels of proliferation and interleukin-2 production,
had a limited ability to generate cytotoxic T lymphocytes against the
recipient, and did not induce immune deficiency, despite survival in
the recipient for at least 28 days. The ability to prevent rejection
was reduced by the absence of CD28, because as many as 1.0 × 107 CD28/ CD8+ cells were
needed to prevent rejection of major histocompatibility complex (MHC)
class-I incompatible marrow in sublethally irradiated (550 cGy) bm1
recipients, whereas 8.0 × 105 CD28+/+
CD8+ T cells were sufficient to produce a similar effect,
indicating that CD28 on donor CD8+ cells helps to
eliminate host immunity. Two million CD4+
CD28/ or CD28+/+ T cells were
transplanted into sublethally irradiated (750 cGy), MHC class-II
incompatible (B6 × bm12)F1 recipients. With CD28/
cells, 44% of the recipients died at a median of 20 days compared with
94% at a median of 15 days with CD28+/+ cells
(P < .001). Two million CD8+
CD28/ or CD28+/+ T cells were
transplanted into sublethally irradiated (750 cGy), MHC class-I
incompatible (B6 × bm1) F1 recipients. With CD28/
cells, 25% of the recipients died at a median of 41 days compared with
100% at a median of 15 days with CD28+/+ cells
(P < .001). (B6 × bm12)F1 and (B6 × bm1)F1 mice surviving after transplantation of CD28/ cells recovered
thymocytes, T cells, and B cells in numbers and function comparable
with that of irradiation-control F1 mice. We conclude that CD28
contributes to the pathogenesis and the severity of GVHD. Our results
suggest that the severity of GVHD could be decreased by the
administration of agents that block CD28 function in T lymphocytes.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ACUTE GRAFT-VERSUS-host disease (GVHD)
continues to be the major complication of allogenic bone marrow
transplantation, producing immune deficiency, infection, organ damage,
and death. GVHD is initiated by mature donor T cells that recognize
minor or major histocompatibility antigens of the
recipient.1 Efficient T-cell activation requires antigen
recognition and costimulation.2,3 Antigen recognition is
mediated by the interaction between T-cell receptor (TCR) and antigen
peptides presented by major histocompatiblity complex (MHC) molecules
on antigen-presenting cells. The best characterized costimulatory
system involves the CD28 molecule on T cells and its ligands, B7-1 and
B7-2 on antigen-presenting cells. CTLA-4, a structural homologue of
CD28, shares the ability to bind B7 molecules.4,5 The
B7/CD28 interaction delivers a positive signal, whereas the B7/CTLA-4
interaction delivers a negative signal for T-cell
activation.6-8 Blocking CD28 costimulation can inhibit
T-cell responses in a variety of in vitro and in vivo systems.9-14 CTLA-4Ig or anti-B7 antibodies, which bind to
B7 and block the interaction of B7 with CD28 and CTLA-4, have been shown to ameliorate GVHD in a variety of murine models, suggesting that
CD28 activation is involved in the pathogenesis of
GVHD.15-19
To avoid the limitation that treatment with CTLA-4Ig or anti-B7
antibodies might not completely block the interaction of B7 with CD28,
or might sustain GVHD by blocking the interaction of B7 with CTLA-4, we
and others have used T cells from donors with a deletion of the CD28
gene produced by homologous recombination.20 Recently,
Speiser et al21 reported that splenocytes from
CD28/ donors were comparable with those from
CD28+/+ donors in their capacity to cause acute lethal GVHD
in MHC-mismatched recipients, suggesting that CD28 costimulation is not
necessary for GVHD lethality. Thus, the role of CD28 in GVHD remains
controversial. We have compared the effects of
CD28/ and CD28+/+ T cells from
C57BL/6 (B6) donors in unirradiated H2-incompatible (B6 × DBA2)F1
(BDF1) recipients and in irradiated H2 class-I-incompatible (B6 × bm1)F1 and H2 class-II-incompatible (B6 × bm12)F1 recipients. We found that CD28/ donor
T cells had markedly reduced ability to cause GVHD.
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MATERIALS AND METHODS |
Mice.
B6, BDF1, B6.C-H2bm12 (bm12), and
B6.C-H2bm1 (bm1) mice were purchased from the
Jackson Laboratory (Bar Harbor, ME). (B6 × bm12)F1 and (B6 × bm1)F1 mice were bred at Fred Hutchinson Cancer Research Center
(FHCRC, Seattle, WA). Founders for the Ly5-congenic
B6.SJL-Ly5a Ptprca Pep3b
(B6-Ly5.1) strain were provided by Dr David Myers (Sloan Kettering Institute, New York, NY). B6 CD28-deficient mice were generated through
homologous recombination in embryonic stem cells by Shahinian et
al.20 CD28-deficient mice were bred onto a B6 background for five generations. Homozygous CD28/ mice
were kindly donated to us by Dr Craig Thompson (University of Chicago, Chicago, IL). All the mice used in this report were housed
in microisolator cages.
T-cell purification.
For purification of CD4+ or CD8+ T cells by
positive selection, a magnetic cell separation system was used
according to the manufacturer's instructions (Miltenyi Biotech Inc,
Auburn, CA). Lymph node cells were incubated with biotin-conjugated
anti-CD4 monoclonal antibodies (MoAb) (hybridoma GK 1.5; ATCC,
Rockville, MD) or anti-CD8 MoAb (hybridoma 2.45; ATCC) for 15 minutes
at 4°C. After two washes, cells were incubated with
streptavidin-MicroBeads for 15 minutes at 4°C. After washing, the
cell suspension was passed through a VS+ separation column
placed in the magnetic field. The VS+ column was washed two
to three times with 0.5% bovine serum albumin (BSA)/phosphate-buffered
saline (PBS) and then removed from the magnetic field. Retained cells
were flushed from the VS+ column with 0.5% BSA/PBS. The
isolated cells were passed over a second VS+ column to
increase the purity of enriched cells. After separation, cells were
stained with phycoerythrin-conjugated streptavidin and analyzed by flow
cytometry. The purity of CD4+ or CD8+ cells
ranged from 94% to 99%.
Transplantation.
Fifty million spleen cells were resuspended in PBS and injected via the
tail vein into 8- to 11-week-old unirradiated BDF1 recipients. (B6 × bm12)F1 or (B6 × bm1)F1 recipients mice were exposed to
700 cGy of irradiation (60Co source) at 20 cGy per minute.
Two million CD4+ or CD8+ cells from
CD28/ or CD28+/+ B6 donors were
suspended in PBS and injected via the tail vein into 8- to 9-week-old
irradiated (B6 x bm12)F1 or (B6 x bm1)F1 recipients within 24 hours
after irradiation. Mice were monitored twice weekly for weight loss and
manifestations of GVHD. For experiments in which engraftment was an
endpoint, marrow was depleted of T cells by complement-mediated lysis
with antibodies specific for Thy-1, CD4, and CD8 as described
previously.22 Five million T-cell-depleted marrow cells
from B6-Ly5.1 donors were injected into bm1 recipients exposed to 550 cGy of irradiation with no T cells added to the graft or with graded
numbers of CD8+ cells from CD28/
or CD28+/+ donors added to the graft.
Flow cytometry.
Spleen cell suspensions from BDF1 recipients were stained with
FITC-conjugated anti-CD4, anti-CD8, or anti-B220 MoAbs (Pharmingen, San
Diego, CA) and biotin-conjugated anti-H2Dd (hybridoma
34-5-8S; ATCC), followed by phycoerythrin-conjugated streptavidin
(Southern Biotechnology, Birmingham, AL). Two-color flow cytometric
analysis was performed on a FACScan using LYSIS II software (Becton
Dickinson, San Jose, CA). Recipient-derived cells were distinguished
from donor cells by H2Dd expression. For analysis of
engraftment by flow cytometry, peripheral-blood lymphocytes (PBL) were
stained with FITC-conjugated anti-CD3 MoAb and biotinylated anti-Ly5.1
MoAb followed by phycoerythrin-conjugated streptavidin as described
previously.22
Detection of the neomycin-resistance gene by polymerase chain
reaction (PCR).
Engraftment of CD28/ T cells was documented
by PCR for the neomycin-resistance gene (neo), which disrupts
the CD28 gene.20 DNA was extracted from spleen cells by
using the IsoQuick nucleic acid extraction kit (ORCA Research Inc.,
Bothell, WA) according to the manufacturer's instruction. An aliquot
of 0.5 µg of DNA was used for PCR amplification. The following
primers were used for PCR: (A) Neo, 5'-CAAGATGGATTGCACGCAGG, and
3'-CCCGCTCAGAAGAACTCGTCAACT CGCCC; (B)  -actin,
5'-TGACGGGGTCACCCACACTGTGCCCATCTA, and
3'-CTCTTCGACACGATGCAGCGGGACCTGAAG. Amplification was performed through
35 cycles of denaturation at 94°C for 40 seconds, annealing at
60°C for 40 seconds, and extension at 72°C for 60 seconds on
GeneAmp PCR system 9600 (Perkin Elmer, Foster City, CA). PCR products
were electrophoresed in 2% agarose gels and visualized with ethidium
bromide. The sensitivity of the detection by PCR is 1 in 10,000 cells.
Cell culture and proliferation.
Single-cell suspensions from spleens of BDF1 mice were depleted of red
cells by hypotonic lysis. Splenocytes were resuspended at a
concentration of 1.0 × 106 cells/mL and cultured in
200-µL aliquots in 96-well plates in complete RPMI-1640 medium
containing 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 15 mmol/L
HEPES, 1 mmol/L sodium pyruvate, 5 × 105 mol/L 2-ME,
100 U/mL penicillin and 100 µg/mL streptomycin. Spontaneous ex vivo
proliferation was assessed by adding 1 µCi/well [3H]TdR
for 4 hours at the end of the culture. Concanavalin A
(ConA)-activated T-cell proliferation or
lipopolysaccharide (LPS)-activated B-cell proliferation
were measured by 8-hour incorporation of [3H]TdR at the
end the culture. DNA was harvested onto glass-fiber filters and
quantitated in a liquid scintillation counter (Topcount, Meridian, CT).
Interleukin (IL)-2 release.
Spleen cells were resuspended at a concentration of 5 × 106/mL and cultured in 2 mL aliquots in 24-well plates in
complete RPMI-1640 medium alone or in the presence of 5 µg/mL ConA
(Sigma, St Louis, MO). Culture supernatants were harvested after 24 hours and frozen at  20°C until assay. Spontaneous IL-2
production was assayed as the ability of supernatants to stimulate
proliferation of the IL-2-dependent cell line CTLL-2, as measured by
[3H]TdR incorporation. Ten-fold dilutions of supernatants
were cultured with CTLL-2 cells (5000/well) for 48-hours in 96-well
plates, with of 1 µCi of [3H]TdR during the last 18 hours. To block the response of CTLL-2 cells to IL-4, 5 µg/mL
anti-IL-4 MoAb (hybridoma 11B11, ATCC) was added to the culture. IL-2
production after ConA stimulation was quantified by sandwich ELISA
using purified rat antimouse IL-2 MoAb JES6-1A12 for capture, and
biotinylated rat antimouse IL-2 MoAb JES6-5H4 (Pharmingen) for
detection. IL-2 concentration was calculated with reference to standard
curves constructed using recombinant murine IL-2 (Genzyme, Cambridge,
MA).
Antihost cytotoxicity.
Antihost cytotoxicity in BDF1 chimeras was measured directly, without
in vitro restimulation, by testing spleen cells from the chimeras as
effectors against 51Cr-labeled P815 (H2d) or
EL-4 (H2b) targets. Spleen cells were added to U-bottom
96-well plates to achieve E:T ratios of 6, 12, 25, 50, and 100:1 with
2.0 × 103 targets/well. The plates were centrifuged
at 1000 rpm for 2 to 3 minutes and then incubated at 37°C for 4 to
5 hours. Chromium released into the supernatant was measured by
Topcount (Packade Inc, Meriden, CT). The percent cytotoxicity was
calculated as (experimental release spontaneous
release)/(maximal release spontaneous release) × 100%.
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RESULTS |
CD28 contributes to alloreactive T-cell expansion and IL-2 production
in unirradiated hosts.
Parental spleen cells (5.0 × 107) from B6 donors were
injected into unirradiated BDF1 recipients as described
previously17,23 to compare the ability of
CD28/ and CD28+/+ T cells to
mount a graft-versus-host (GVH) reaction. Two days after transplant,
H2d-negative donor CD4 T cells, CD8 T cells, and B cells
were detected in the spleen of recipients transplanted with either
CD28+/+ or CD28/ cells
(Fig 1). At the same time, spontaneous ex
vivo proliferation of splenocytes was higher in recipients of
CD28+/+ cells than in recipients of
CD28/ cells (Fig
2A). Also, much more IL-2 was produced spontaneously by splenocytes
obtained from recipients transplanted with CD28+/+ cells as
compared with CD28/ cells (Fig 2B). Fifteen
days after transplantation, the degree of donor chimerism was markedly
different in the two groups (Fig 1). The numbers of donor
CD4+ T cells, CD8+ T cells, and B cells were
significantly higher in recipients of CD28+/+ cells than in
recipients of CD28/ cells
(Table 1). Because it was difficult to
detect donor cells in recipients of CD28/
cells by flow cytometry analysis beyond 15 days after transplant, we
tested for the presence of the neo gene by PCR and showed that CD28/ donor cells persisted in the recipients
for at least 28 days (Fig 3A). These
results indicated a much lower degree of donor T-cell proliferation and
effector function in response to alloantigen in the absence of CD28,
although CD28/ cells did engraft and persist
after transplantation.
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| Fig 1.
CD28 expression by donor T cells affects the degree of
donor chimerism after transplantation of B6 splenocytes into
unirradiated BDF1 mice. Two and 15 days after injection of donor cells,
splenocytes from normal BDF1 controls and from BDF1 recipients of
CD28+/+ or CD28/ donor cells were
stained for recipient-specific (H2Dd) class-I antigen and
for CD4, CD8, or B220 and analyzed by two-color flow cytometry. Results
were similar with three mice in each group.
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| Fig 2.
Spontaneous ex vivo proliferation and IL-2 production of
donor T cells was reduced in the absence of CD28. Splenocytes were
obtained from control BDF1 mice without transplant and from BDF1
recipients of CD28+/+ or CD28/ cells
2 days after transplantation. Three mice per group were analyzed
separately, and the data represent the mean +/ 1 SD of three
individual mice. (A) Splenocytes were cultured at 2.0 × 105/well for 4 hours in medium, and triplicate cultures
were pulsed with [3H]TdR at the beginning of the culture.
Results show the proliferative capacity of splenocytes early in GVHD.
(B) Splenocytes were cultured at 5.0 × 106/mL for 24 hours in medium alone, and supernates were assayed for IL-2 by the
proliferation of CTLL-2 cells in the presence of anti-IL-4 MoAb.
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| Fig 3.
CD28/ donor T cells engraft in
both unirradiated and irradiated hosts. (A) DNA was extracted
from splenocytes of normal BDF1 or BDF1 recipients of
CD28/ spleen cell grafts 28 days after
transplant. (B) DNA was extracted from splenocytes of irradiated
(B6 × bm12)F1 mice or (B6 × bm12)F1 recipients of
CD28/ CD4+ cells 103 days after
transplant. (C) DNA was extracted from splenocytes of irradiated
(B6 × bm1)F1 mice or (B6 × bm1)F1 recipients of
CD28/ CD8+ cells 101 days after
transplant. DNA was amplified by PCR as described in Materials and
Methods. Ethidium bromide-stained agarose gels show the DNA bands for
-actin or neo in each sample.
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Unirradiated recipients transplanted with
CD28/ cells do not develop immune deficiency
associated with GVHD.
Profound immune deficiency appears rapidly after induction of the GVH
reaction.24,25 To assess the effect of CD28 on T-cell function, IL-2 production was measured in the culture supernatants of
splenocytes obtained 2, 15, and 45 days after transplantation and
stimulated for 24 hours with ConA. Splenocytes from recipients transplanted with CD28+/+ showed a reduction in IL-2
production by day 2, lack of IL-2 production by day 15, and could not
be tested on day 45 because the recipients were dead from GVHD. In
contrast, splenocytes from recipients transplanted with
CD28/ cells produced IL-2 levels comparable
with those of normal BDF1 splenocytes at all three time points
(Fig 4). We conclude that CD28/ donor T cells are unable to cause GVHD
in this strain combination.
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| Fig 4.
Immune deficiency does not develop in BDF1 recipients
transplanted with CD28/ donor cells. Splenocytes from
CD28+/+ wild type, CD28/ deficient,
normal BDF1 mice, and BDF1 recipients of CD28+/+ or
CD28/ cells 2, 15, and 45 days after transplantation
were cultured for 24 hours with ConA; and IL-2 in the supernatants was
measured by using a sandwich enzyme-linked immunosorbent assay (ELISA)
technique. IL-2 concentrations were calculated with reference to
standard curves. Data represent the average +/ 1 SD of three mice
per group analyzed separately for BDF1 recipients of
CD28+/+ or CD28/ cells. Data on
CD28+/+, CD28/ and normal BDF1 mice
represent the study of one mouse at each time point. *Recipients of
CD28+/+ donor cells died before day 45.
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CD28 contributes to development of antihost cytotoxic effector cells
in unirradiated recipients.
B cells of recipient origin were severely depleted after
transplantation of CD28+/+ cells but remained at normal
levels after transplantation of CD28/ cells
(Table 1). The loss of B cells in recipients of CD28+/+
cells suggested the presence of antihost cytotoxic effectors. Spleen
cells from recipients transplanted with CD28+/+ or
CD28/ cells were tested on day 15 after
transplantation for antihost (H2d) activity in a 4-hour
cytotoxicity assay against 51Cr-labeled targets without
prior restimulation in vitro (Fig 5). Splenocytes from recipients transplanted with CD28+/+ cells
showed cytotoxicity against recipient-type (H2d) targets
but not against donor-type (H2b) targets (Fig 5). In
contrast, splenocytes from normal BDF1 controls and from recipients
transplanted with CD28/ cells showed no
activity against recipient-(H2d) or donor-(H2b)
type targets. These results showed that CD28/
donor T cells had a limited ability to generate cytotoxic effectors against recipient alloantigens, or the cytotoxic effectors could not be
maintained.
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| Fig 5.
Donor T cells lacking CD28 cannot generate cytotoxic
effectors against the recipient. Fifteen days after transplantation,
splenocytes from normal BDF1 mice and from BDF1 recipients of
CD28+/+ or CD28/ cells were assayed
directly without in vitro restimulation. Three mice per group were
analyzed separately, and the data represent the mean +/ 1 SD of
three individual mice. The activity of anti-H2d or
anti-H2b cytolytic effectors was measured in a 4-hour
cytotoxicity assay against specific P815 (H2d) or control
EL-4 (H2b) targets.
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Role of CD28 in prevention of rejection by donor CD8+
cells.
In previous studies, we have shown that cytotoxic activity mediated by
donor CD8+ cells is needed to prevent rejection of MHC
class-I-disparate B6-Ly5.1 marrow in sublethally irradiated (550 cGy)
bm1 recipients.22 In this model, recipients uniformly
reject the marrow when the graft does not contain T cells and as few as
8.0 × 105 CD28+/+ CD8+ cells
are sufficient to prevent rejection in virtually all recipients (Table 2). CD28/
CD8+ cells were impaired in their ability to prevent marrow
graft rejection in this model. Rejection was not prevented when 8.0 × 105 or 3.2 × 106 CD8+
cells from CD28/ donors were added to the
graft (Table 2, Experiment 1), but rejection was prevented when 1.0 × 107 CD8+ cells from
CD28/ donors were added to the graft (Table
2, Experiment 2). These results indicate that
CD28/ CD8+ cells have a limited
ability to eliminate MHC class-I-disparate immune cells in vivo.
Role of CD28 in GVHD mediated by CD4+ T cells in
irradiated recipients.
We tested separately the role of CD28 on GVHD mediated by
CD4+ cells against MHC class-II-incompatible (B6 × bm12)F1 recipients and CD8+ cells against MHC
class-I-incompatible (B6 × bm1)F1 recipients. We selected these
donor and recipient combinations because small numbers of purified
CD4+ or CD8+ T cells from B6 mice respectively
induce lethal GVHD in 100% of irradiated (B6 × bm12)F1 or (B6 × bm1)F1 recipients.26
Irradiated (700 cGy) (B6 × bm12)F1 recipients were injected with
2.0 × 106 CD4+ lymph node T cells from
either CD28+/+ or CD28/ B6
donors. Irradiated (B6 x bm12)F1 controls injected with PBS developed
transient pancytopenia, but all recovered and survived longer than 100 days. Recipients of CD28+/+ cells became acutely ill with
GVHD, characterized by progressive weight loss, ruffled fur, and
hunched back, and 15 of 16 (94%) recipients died at a median of 15 days after transplant. In contrast, recipients of
CD28/ cells had less severe and delayed GVHD,
and 7 of 16 (44%) mice died at a median of 20 days
(Fig 6). Thus, the absence of CD28 on donor
T cells was associated with increased recipient survival (P < .001). On day 15, recipients of CD28+/+ cells had a
hematocrit of 15.7% ± 7.6%, significantly lower than the
hematocrit of 27.8% ± 11.1% in recipients of
CD28/ cells (P < .001). Surviving
recipients of CD28/ cells eventually
recovered from GVHD, and regained body weight to the level of
irradiation controls (Fig 6). They also recovered T- and B-cell numbers
and function comparable with those of irradiation controls (Table 3).
To confirm engraftment of donor cells, we tested for the presence of
neo in recipients of CD28/ cells and
found that donor cells persisted beyond 100 days after transplant in
all recipients tested (Fig 3B).
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| Fig 6.
CD4+ CD28/ donor T cells
have a limited capacity to cause GVHD in irradiated (B6 × bm12)F1
recipients. (B6 × bm12)F1 mice were irradiated (700 cGy) and
transplanted with CD28+/+ CD4+ cells or
CD28/ CD4+ cells from B6 donors.
Irradiated (B6 × bm12)F1 mice were injected with PBS alone as
control. (A) Survival curves; (B) Weight curves show the mean body
weight for each group. Data were pooled from two replicate
experiments.
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Role of CD28 in GVHD mediated by CD8+ T cells in
irradiated hosts.
Irradiated (700 cGy) (B6 × bm1)F1 recipients were injected with
2.0 × 106 CD8+ lymph node T cells from
either CD28+/+ or CD28/ B6
donors. Recipients of CD28+/+ cells became acutely ill with
GVHD, characterized by progressive weight loss, ruffled fur, and
hunched back, and all 14 recipients died at a median of 15 days after
transplant. In contrast, recipients of CD28/
cells had a delayed and less-severe GVHD, and only 3 of 12 (25%) recipients died at a median of 41 days (Fig
7). On day 15, recipients of CD28+/+ cells had a hematocrit
of 5.8% ± 2.6% compared with 26.3% + 4.9% in recipients of
CD28/ cells (P < .001). Surviving
recipients transplanted with CD28/ cells
eventually recovered from GVHD and regained body weight to the level of
irradiation controls (Fig 7). These recipients also recovered T and B
cell number and function comparable to those of irradiation controls
(Table 3). To assess engraftment of donor
cells, we tested for the presence of neo in recipients of
CD28/ cells and found that donor cells
persisted for at least 100 days after transplant in all recipients (Fig
3C).
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| Fig 7.
CD8+ CD28/ donor T cells
have a limited capacity to cause GVHD in irradiated (B6 × bm1)F1
recipients. (B6 × bm1)F1 mice were irradiated (700 cGy) and
transplanted with CD28+/+ CD8+ cells or
CD28/ CD8+ cells from B6 donors.
Irradiated (B6 × bm1)F1 mice were injected with PBS alone as control.
(A) Survival curves; (B) Weight curves show the mean body weight for
each group. Data were pooled from two replicate experiments.
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Table 3.
Recipients of CD28/ Donor Cells
Recovered Normal Numbers of Thymocytes and Normal Numbers and
Function of Splenic T and B Cells*
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| |
DISCUSSION |
In this study we have compared the pathogenicity of
CD28/ and CD28+/+ donor T cells
in GVHD induced by parental B6 grafts in unirradiated BDF1 recipients
and in irradiated MHC class-I (bm1) or class-II (bm12) -incompatible
recipients. In the absence of CD28, donor T cells have markedly reduced
proliferative and cytolytic activity against the recipient and induce
less-severe GVHD resulting in lower fatality. These findings indicate
that CD28 plays an important role in the pathogenesis of GVHD but is
not necessary for development of the disease.
One concern in this study was that donor engraftment could be
compromised by the lack of CD28, especially in B6 BDF1
transplants. Hybrid resistance mediated by natural killer (NK) cells is
particularly effective in this strain combination,26 and
there was the possibility that hybrid resistance could not be overcome
in the absence of donor CD28 despite transplantation of a large number
of splenocytes. We have shown, however, that engraftment of
CD28/ donor T cells was evident at 2 and 15 days after transplantation by flow cytometry, and very low levels of
engraftment were detected at 28 days after transplantation by the more
sensitive PCR assay. In the B6(B6 × bm12)F1 and
B6(B6 × bm1)F1 strain combinations, there is no hybrid
resistance27 and graft rejection was not expected. Results
of PCR assays showed that cells from CD28/
donors persisted in the recipients beyond 100 days after
transplantation. We found that CD28/ cells
have limited ability to prevent rejection of T-cell-depleted marrow
cells in sublethally (550 cGy) irradiated bm1 recipients. However,
CD28/ CD8+ cells can overcome
graft resistance by the host, if administered in sufficiently large
numbers. Therefore, we conclude that CD28 on donor CD8+
cells helps to eliminate MHC class-I-incompatible host-immune cells.
The role of CD28 on GVHD was apparent in all three strain combinations
tested, although there were notable differences among them. GVHD after
B6BDF1 transplants appeared to be heavily dependent on CD28
costimulation, because CD28/ donor T cells
had marginal alloresponsiveness, lacked antihost cytotoxicity, and
recipients of CD28/ cells survived with no
manifestations of the disease. On the other hand, GVHD after
B6(B6 × bm12)F1 and B6(B6 × bm1)F1
transplants was less dependent on CD28 costimulation, because not all
(B6 × bm12)F1 or (B6 × bm1)F1 recipients of
CD28/ CD4+ or CD8+
cells survived. Irradiation of the recipients might account for this
difference. (B6 × bm12)F1 and (B6 × bm1)F1 recipients
received sublethal irradiation that caused marrow hypoplasia, and
marrow failure was caused by GVHD. The release of proinflammatory
cytokines, such as TNF- and IL-1 induced by irradiation might also
predispose towards the development of GVHD.28-31
Recently, Speiser et al21 found that
CD28/ and CD28+/+ spleen cells
were equally capable of causing lethal acute GVHD, and concluded that
CD28 costimulation is not necessary for the induction of lethal acute
GVHD. The different results between their study and ours presented here
and the studies of others in which B7 blocking agents prevented
GVHD15-19 may reflect differences in genetic disparity between donor and recipient. Speiser et al21 transplanted
cells from B6 mice into irradiated BALB/c recipients or vice versa
under conditions in which recipients transplanted with wild-type T
cells died early after transplantation with severe GVHD. We, and
others,16,17 transplanted splenocytes from B6 mice into
BDF1 recipients or purified T cells from B6 mice into (B6 × bm1)F1 or (B6 × bm12)F1, under conditions in which the GVHD
induced by wild-type T cells was less severe. Blazar et
al15 reported that CTLA-4Ig protected B10.BR/SgSnJ
recipients from GVHD induced by B6 splenocytes under conditions in
which the GVHD induced by wild-type T cells was less severe than the
GVHD observed by Speiser et al.21 It is possible that
costimulatory requirements of T cells may differ in response to
distinct alloantigens and that high TCR affinity for alloantigen might
overcome the requirements for CD28 costimulation, as shown in other
systems.32-34
A defect in the expansion of CD28/ cells
appears to account for their limited ability to induce
GVHD.35 Our data indicated that neither CD4 nor CD8 donor T
cells were able to expand after transplantation in the absence of CD28
costimulation (Table 1). CD28/ donor T cells
had reduced proliferation and IL-2 production in response to recipient
alloantigens and could not generate cytotoxic effectors against the
host. These results are consistent with the observation by Hakim et
al17 that CTLA-4Ig inhibited the expansion of
CD4+ and CD8+ cells from B6 donors in BDF1
recipients, and the results of Blazar et al,18 who observed
that anti-CD80 plus anti-CD86 MoAbs inhibited the expansion of
CD4+ T cells from B6 donors in irradiated bm12 recipients.
Therefore, the lack of CD28 costimulation appears to result
consistently in a decreased T-cell expansion, IL-2 production, and CTL
generation. Because we used CD28/ T cells
rather than B7 inhibitors that bind to both CD28 and CTLA-4, our data
provide definitive evidence that CD28 contributes to T-cell activation
and expansion after recognition of alloantigen in vivo. Because
engagement of CTLA-4 can deliver a negative signal to T cells and
facilitate peripheral T-cell tolerance,6-8 our findings
provide a rationale to investigate whether alloantigen-specific tolerance can be achieved by selectively blocking CD28 costimulation while still allowing CTLA-4 engagement on donor T cells.
| |
FOOTNOTES |
Submitted March 16, 1998;
accepted June 14, 1998.
Supported by Grants No. CA 18209, AI 40680, AI 33484, and HL 55257 from
the Department of Health and Human Services of the National Institutes
of Health, Bethesda, MD.
Address correspondence to Claudio Anasetti, MD, Fred
Hutchinson Cancer Research Center, 1100 Fairview Ave N, D2-100,
Seattle, WA 98109.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs Craig B. Thompson and Patricia Noel for their generous
gift of the CD28-/- mice; Dr Stanley Riddell for providing PCR primers
specific for neo and -actin; Drs Yoshiki Akatsuka and Ming-Tsen Lin for helpful technical advice; and Ms Alison Sell for
assistance in preparing the manuscript.
| |
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Abstract
Introduction
Abstract