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Prepublished online as a Blood First Edition Paper on January 9, 2003; DOI 10.1182/blood-2002-10-3048.
TRANSPLANTATION
From the University of Minnesota Cancer Center and
Department of Pediatrics, Division of Bone Marrow Transplantation,
Minneapolis, MN; Department of Pathology, Brigham and Women's
Hospital, Boston, MA; the Department of Immunology, Juntendo University
School of Medicine, Tokyo, Japan; and the Department of Microbiology
and Immunology, University of California, San Francisco, CA.
OX40 (CD134) is expressed on activated T cells; its ligand, OX40
ligand (OX40L) is expressed on dendritic cells, B cells, and activated
endothelial cells. To determine how OX40-OX40L interaction affects
graft-versus-host disease (GVHD), we used antagonistic anti-OX40L monoclonal antibody (mAb) or OX40 T cells receiving signals via the antigen-specific
T-cell receptor (TCR) require a second, costimulatory signal to
stabilize cytokine mRNA and induce antiapoptotic
proteins.1,2 Members of the immunoglobulin supergene and
tumor necrosis factor (TNF)/TNF receptor families can function as
costimulatory molecules. The latter include CD40 ligand (CD40L)
(CD154); 4-1BB receptor (CD137); CD30; and OX40 (CD134).3
OX40 is expressed on activated CD4+ and CD8+ T
cells in mice and humans. OX40L (CD134L) is a type II membrane protein
expressed on antigen-presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages, that have been activated by known inductive stimuli such as CD40 or proinflammatory mediators (eg, lipopolysaccharide [LPS]).4-7 In
addition, OX40L expression has been reported on endothelial cells,
microglial cells, and T cells.8-11 Signaling of OX40
receptor on antigen-specific CD4+ T cells results in
production of T-helper type 1 (Th1) and Th2 cytokines,
up-regulation of antiapoptotic proteins,12-16 clonal expansion, and development into memory cells.11,14,17-22
Because OX40 is up-regulated following T-cell activation, studies were
performed to assess the role of OX40-OX40L interaction on
graft-versus-host disease (GVHD), which is mediated by
alloantigen-activated donor T cells. Tittle et
al23 observed that increased numbers of
alloreactive CD4+ T cells that coexpressed OX40 were
present in the peripheral blood, lymphohematopoietic organs, and liver
of nonirradiated F1 rat recipients of parental donor grafts
that were experiencing a GVHD reaction. Chen et al13
showed comparable allogeneic mixed leukocyte reaction
(MLR) responses using OX40L While the rodent studies described above provide evidence that
OX40/OX40L blockade can reduce acute GVHD responses, the mechanisms responsible have not been fully elucidated.The present studies were
undertaken to investigate the potency and mechanisms by which OX40-OX40L interactions regulate GVHD. Additionally, the potential role
of this pathway on the engraftment of pan-T-cell-depleted (TCD)
allogeneic bone marrow (BM) was examined. To strengthen our
conclusions, we used complementary approaches designed to target the
OX40/OX40L pathway: antagonistic and agonistic mAbs, OX40
receptor-deficient (OX40 Mice
mAb preparation
GVHD generation Different GVHD systems were used to analyze the effects of the OX40/OX40L pathway on alloresponses in vivo. In the first type, recipients were heavily irradiated to simulate human transplantation conditions. B10.BR recipients were lethally irradiated with 8.0 Gy total body irradiation (TBI) by x-ray (39 cGy/min) on day 1 followed
on day 0 by the intravenous infusion of pan-TCD BM (0.8 to
2 × 107), accomplished by treatment with anti-Thy1.2
(clone 30-H-12) plus rabbit complement.27 Donor BM cells
were supplemented with splenocytes, purified lymph node (LN) T cells,
or LN T-cell subpopulations from B6 wild-type, B6
OX40 /, 129/Sv OX40L/, B6
CD28/, BALB/c Stat-4/, or BALB/c
Stat-6/ donors, as indicated. To measure
CD4+ T-cell GVHD responses, bm12 recipients were lethally
irradiated (8.0 Gy TBI) and then infused with TCD BM and splenocytes
obtained from B6 donors.27 To determine the effects of
OX40-OX40L interactions on GVHD and graft-versus-leukemia (GVL) induced
by delayed lymphocyte infusion (DLI), B6 recipients were lethally
irradiated (8.0 Gy TBI), reconstituted with TCD BM, and then given
donor B10.BR splenocytes on day 21 after BM transplantation
(BMT)28 along with either irrelevant or anti-OX40 mAb
infusions as described. Some cohorts of mice were challenged
with acute myeloid leukemia cells (C1498 derived from B6 mice) as
previously described.27 To determine the effects
of host OX40L expression on GVHD lethality, BALB/c OX40L/ mice or littermate controls were lethally
irradiated (6.0 Gy TBI) and reconstituted with B6 TCD BM along with
purified LN T cells (0 or 1 × 106) obtained from B6 or
B6 CD28/ donors.
To more specifically determine the effect of OX40-OX40L interactions on
the GVHD capacity of CD4+ or CD8+ T cells, we
used a system in which purified T-cell subsets are given to major
histocompatibility complex (MHC)-disparate, sublethally irradiated recipients.27 This system permits highly
accurate quantification of the degree of GVHD responses as related to
T-cell dose. MHC class II (bm12)- or class I (bm1)-disparate
recipients were irradiated with 6.0 Gy TBI on day 0 from a
137cesium source at a dose rate of 85 cGy/min. At 4 to 6 hours after TBI, purified LN CD4+ or CD8+ T
cells from B6, B6 OX40 To purify LN cells, single cell suspensions of axillary, mesenteric, and inguinal LN cells were depleted of NK cells and enriched for CD4+ or CD8+ T cells by depletion with anti-CD8 (hybridoma 2.43, rat IgG2b, provided by Dr David Sachs, Charlestown, MA) or anti-CD4 (hybridoma GK1.5, rat IgG2b, provided by Dr Frank Fitch, Chicago IL), respectively. Rat mAb-coated T cells were passaged through a goat antimouse and goat antirat immunoglobulin-coated column (Biotex, Edmonton, AB, Canada). The final composition of T cells in the donor graft was determined by flow cytometry and was always found to be at least 94% T cells of the desired phenotype. Hematocrit values were obtained at periodic intervals as an indicator of the possible bone marrow-destructive effects of infused T cells.27 For all GVHD (and engraftment) systems, mice were monitored daily for survival and clinical appearance and weighed twice weekly. Engraftment studies Bm1 or bm12 mice were irradiated with 4.5 or 5.0 Gy TBI, as indicated, by x-ray on day1 and given B6 CD45.2 TCD BM (0.7 to
1 × 107) cells on day 0.27 Recipients were
given irrelevant anti-OX40 or anti-OX40L mAb (200 µg per dose)
intraperitoneally daily from days 1 to +6, then twice weekly through
day 14. Donor or host chimerism was monitored in peripheral blood at 6 weeks and 3 to 4 months after BMT by means of  CD45.2 (clone 104-2, rat IgG2a) and CD45.1 (clone A20-1.7, rat IgG2a), both provided by
Dr U. Hammerling (New York, NY).27 The T-cell, B-cell, and
granulocyte/macrophage constituency of peripheral blood cells was
measured with the use of mAb directed toward CD4 or CD8, CD19, and
Mac1, respectively. All fluorochrome-labeled mAbs, unless
otherwise indicated, were obtained from PharMingen (San Diego,
CA). Cells were first incubated with 2.4G2 to block Fc receptors, and
then incubated with an optimal concentration of fluorochrome-labeled
mAb for 45 minutes at 4°C. Cells were washed 3 times and resuspended
for analysis by 3-color flow cytometry by means of fluorescein
isothiocyanate-, phycoerythrin-, or biotin (along with SA
[streptavidin]-peridinin chlorophyll A protein
[SA-PerCP])-conjugated mAb purchased from PharMingen or Becton
Dickinson (Mountain View, CA). Irrelevant mAb control values
were subtracted from values obtained with relevant mAbs. All results
were obtained by means of a FACSCalibur (Becton Dickinson). Forward-
and side-scatter settings were gated to exclude red cells and debris,
and 1 × 104 cells were analyzed for each determination.
Statistical analyses Group comparisons of continuous data were made by Student t test. Survival data were analyzed by life-table methods by means of the Mantel-Peto-Cox summary of chi-square.29 Actuarial survival and relapse rates are shown. Probability (P) values of .05 or lower were considered significant.
OX40-OX40L interaction regulates GVHD lethality To determine the role of OX40-OX40L interactions in regulating GVHD responses, initial studies were undertaken to determine the magnitude of effects on OX40 receptor ligation in heavily irradiated B10.BR (H2k) recipients of B6 (H2b) donor BM and supplemental splenocytes (0, 5, or 15 × 106 per recipient). The administration of an agonistic anti-OX40 mAb significantly increased GVHD severity, as evidenced by accelerated mortality, clinical appearance, and body weight loss (Figure 1A and not shown). On the basis of donor splenocyte dose titrations, anti-OX40 mAb infusion accelerated GVHD by at least 5-fold (Figure 1A).
To determine whether the acceleration in GVHD lethality observed with anti-OX40 mAb was dependent upon the use of high-dose lethal irradiation, which induces proinflammatory cytokines, the effect of anti-OX40 mAb was investigated in a setting in DLI.30 DLI in the form of donor splenocyte infusion causes less GVHD mortality than when the same number is infused on the day of BMT.28 B6 recipients were lethally irradiated and reconstituted with B10.BR TCD BM. On day 21 after BMT, some cohorts of mice were given a low dose of donor splenocytes (5 × 106) and irrelevant or anti-OX40 mAb (beginning on day 20 after BMT).27 All mice surviving until day 28 were challenged with AML cells.27 Mice that received no DLI cells all succumbed to AML by day 50. Mice receiving DLI and irrelevant mAb experienced GVL but eventually died of AML. In contrast, recipients given both DLI and anti-OX40 mAb all died of GVHD by day 28, prior to AML cell infusion (Figure 1B). Thus, anti-OX40 mAb given later after BMT substantially increased GVHD mediated by low-dose DLI. While administration of an agonistic anti-OX40 mAb clearly has a potent
effect on GVHD acceleration, engagement of the OX40 receptor by mAb may
overestimate the magnitude of effect that is physiologically conferred
by the binding of OX40 to OX40L during the process of GVHD generation.
Therefore, complementary studies were performed in which donor
splenocytes were obtained from wild-type or OX40 As further evidence that the OX40/OX40L pathway affects GVHD, 2 additional approaches were conducted that targeted the OX40L component
of the pathway. In the first, lethally irradiated B10.BR recipients
were infused with B6 TCD BM, supplemental splenocytes (0 or
25 × 106), and either irrelevant or anti-OX40L mAb
(Figure 2A). Anti-OX40L mAb-treated
recipients had 35% long-term survival, as compared with 0% in the
controls. In the second approach, BALB/c OX40L
OX40-OX40L interactions have a more pronounced effect on CD4+ T-cell- than on CD8+ T-cell-mediated alloresponses in both GVHD and alloengraftment During GVHD induction in lethally irradiated B10.BR recipients of B6 BM and supplemental splenocytes (107), we have observed that the OX40 receptor is up-regulated on both CD4+ T cells (22% positive) and CD8+ T cells (20% positive) isolated from thoracic duct lymphatics on day 7 after BMT (data not shown). To determine whether precluding OX40-OX40L binding would have similar effects on CD4+ versus CD8+ T-cell-mediated GVHD, experiments were performed with the use of highly purified T-cell subsets infused into recipients with an isolated MHC class I or class II only disparity. Because residual host T cells remain in the sublethally irradiated recipients, the infusion of agonistic mAbs could affect GVHD responses by stimulating either donor antihost reactions, resulting in increased GVHD, or host antidonor responses, resulting in less GVHD lethality. To avoid this complication, these studies were performed with OX40/ donor T cells.
To assess CD8+ T cells' responses, sublethally irradiated
bm1 recipients were given B6 CD8+ T cells
(0.3 × 106) obtained from OX40+/+ or
OX40
To ascertain the function of OX40/OX40L in a CD4+
T-cell-mediated GVHD system, OX40+/+ or
OX40 The data presented above clearly indicate that OX40-OX40L interactions
preferentially accelerate GVHD induced by CD4+ donor T
cells that encounter MHC class II antigens that are distributed throughout the host microenvironment. We next sought to determine whether OX40 receptor signaling regulates host CD4+ or
CD8+ T-cell-mediated BM graft rejection, a situation in
which BM cells alone serve as the source of alloantigen. Agonistic
anti-OX40 mAb administration resulted in a marked reduction in
engraftment in sublethally irradiated bm12 recipients of donor B6 TCD
BM at 6 weeks (data not shown) or 4 months (Table 1) after BMT. In contrast, a blocking anti-OX40L mAb
increased alloengraftment at 2 different TBI doses. Consistent with the
lack of pronounced effects of OX40-OX40L interactions on modifying
CD8+ T-cell-mediated GVHD, anti-OX40 mAb administration
had only modest effects in influencing alloengraftment in a
CD8+ T-cell-mediated rejection system (Table 1). Moreover,
anti-OX40L mAb showed no evidence of engraftment promoting
properties in this setting (Table 1). These data demonstrate a more
pronounced role for the OX40/OX40L pathway in regulating
CD4+ as compared with CD8+ T cells
alloresponses in vivo.
OX40L expression on donor T cells does not have a major effect on GVHD lethality Although OX40L expression has been reported on T cells,11 the function of OX40L on in vivo T-cell alloresponses has not been examined. CD4+ T cells (105) from 129/Sv OX40L/ mice or wild-type
littermate controls were given to sublethally irradiated bm12
recipients (Figure 4A). There was no
significant difference in survival of recipients of
OX40L/ versus wild-type (WT) T cells (56%
versus 38%; P = .24). This was reproduced at a higher
T-cell dose (data not shown). These findings are in sharp contrast to
the 100% long-term survival rates obtained with anti-OX40L mAb in the
B6 bm12 system at 2 different CD4+ T-cell doses,
indicating that the major effect of anti-OX40L mAb is not on blocking
T-cell-expressed OX40L from binding with OX40 receptor-expressing
cells. As might be anticipated from our findings on the relative lack
of importance of the OX40/OX40L pathway on CD8+ T-cell
alloresponses, there were no significant differences in median survival
time in sublethally irradiated bm1 recipients of 129/Sv
OX40L/ versus WT CD8+ T cells (Figure 4B).
Moreover, in nonconditioned, NK cell-depleted BALB/c-SCID recipients,
CD4+ T cells obtained from OX40L/ mice and
WT littermate controls had a comparable efficacy in inducing GVHD
lethality (Figure 4C). Under the conditions of these various GVHD
models, we were unable to uncover a major role for OX40L expression on
donor T cells in regulating in vivo GVHD.
GVHD protection induced by OX40/OX40L blockade is not dependent upon Stat-4 or Stat-6 signaling Some studies have proposed that OX40 receptor ligation preferentially supports Th2 differentiation, while others demonstrate that Th1 responses are also affected by the OX40 pathway.12-16,31,32 In some models, Stat signaling can regulate the propensity of T cells to differentiate into Th1 or Th2 cells. To determine whether the anti-GVHD effects of OX40/OX40L blockade were dependent upon Stat signaling, studies were performed with the use of splenocyte donors deficient in Stat-4 (typically Th1-defective) or Stat-6 (typically Th2-defective) signaling pathways.33 B6 recipients were lethally irradiated and reconstituted with BALB/c TCD BM and supplemental splenocytes (25 × 106) from Stat-4/ or
Stat-6/ mice as indicated (Figure
5). Cohorts of mice were given
irrelevant, anti-OX40, or anti-OX40L mAb. Anti-OX40 mAb accelerated and
anti-OX40L mAb inhibited GVHD lethality in recipients of Stat
4/ splenocytes (Figure 5A). Anti-OX40L mAb also
inhibited GVHD lethality induced by Stat-6/ donor
splenocytes (Figure 5B). Owing to the rapid lethality of Stat-6/ splenocytes (88% lethality by day 7 after
BMT), the effects of anti-OX40 mAb on accelerating GVHD lethality
were not observed (Figure 5B). These data indicate that the inhibitory
effect of OX40/OX40L blockade does not depend upon Stat-4 or Stat-6
signaling.
The effects of regulating OX40-OX40L interactions on GVHD lethality does not require CD28 signaling Data from our group and others have shown that CD28/B7 interactions can modify GVHD responses.27,34,35 To determine if the anti-GVHD effects of OX40/OX40L blockade were redundant with CD28/B7 blockade, donor T cells were obtained from B6 CD28/ mice and infused into lethally irradiated
allogeneic recipients reconstituted with TCD B6 BM. B10.BR recipients
received B6 CD28/ splenocytes at
15 × 106 (Figure 6A) or
25 × 106 (Figure 6B), along with either irrelevant,
anti-OX40, or anti-OX40L mAb. At both splenocyte doses, anti-OX40 mAb
accelerated GVHD lethality while anti-OX40L mAb almost completely
inhibited GVHD lethality. In other studies, the infusion of purified B6
CD28/ T cells (3 × 106) into lethally
irradiated, TCD BM-reconstituted BALB/c OX40L/
recipients resulted in a significant survival advantage as compared with BALB/c OX40L+/+ littermate controls (Figure 6C). Taken
together, these data indicate that the effects of the OX40/OX40L
pathway on GVHD lethality in heavily irradiated recipients do not
require CD28 signaling.
The present study provides definitive data indicating that
OX40-OX40L interactions are critical for CD4+ and far less
critical for CD8+ T-cell alloresponses in vivo. The
beneficial effects of blockade of the OX40/OX40L pathway did not
require CD28 signaling. Although some studies have shown that the
OX40/OX40L pathway is a more potent regulator of Th2 than Th1
responses, suggesting that this pathway may be preferentially affected
by Stat-6 versus Stat-4 signaling, our studies using splenocytes
obtained from Stat-4 We have conclusively demonstrated that the OX40/OX40L pathway is an
important regulator of GVHD in a variety of GVHD models with different
pathophysiological mechanisms. These data extend those of Tsukada et
al,25 who used a single donor/recipient strain
combination (C57BL/6 Our studies extend the literature by demonstrating the importance of the OX40/OX40L pathway in regulating GVHD by using 4 distinct and complementary approaches: agonistic and antagonistic mAbs and genetic deletion of either the OX40 receptor or OX40L. Three GVHD systems in which recipients were lethally irradiated and given MHC-disparate donor T cells were used to determine the effects of OX40/OX40L on GVHD induced early in the post-BMT period by both CD4+ and CD8+ T cells. Since the injury induced by heavy irradiation can lessen the requirement for T-cell costimulation early in the post-BMT period, additional studies using a DLI model were performed in which the OX40 receptor was purposefully engaged by agonistic mAb later in the post-BMT period, at a time when proinflammatory cytokine release and irradiation-induced tissue injury have subsided. In each experimental setting, the OX40/OX40L pathway had a major biological effect in regulating GVHD lethality. We show in both GVHD and alloengraftment systems that the
dominant effect of the OX40/OX40L pathway is on alloreactive
CD4+ and not CD8+ T cells. Although the
majority of the literature has focused upon CD4+ T-cell
responses, several studies have shown that this pathway also regulates
CD8+ T-cell responses. Chen et al13 observed
decreased CD4+ and CD8+ T-cell proliferation in
OX40L Our data indicate that the beneficial effects of blocking the
OX40/OX40L pathway does not require CD28 signaling. It has been shown
that these 2 costimulatory pathways can provide nonredundant functions
as assessed in vitro since OX40 receptor expression can be induced on T
cells from CD28 Compelling evidence exists that OX40 signaling can support CD4+ T cells to develop into Th2 cells.31,41,42 However, not all studies have shown the generation of Th2 responses to have an absolute dependence on the OX40/OX40L pathway.11 In our studies, GVHD lethality was lessened by the administration of anti-OX40L mAb to recipients of Th2- as well as Th1-defective donor splenocytes, indicating that the GVHD-preventive effect of anti-OX40L mAb was not strictly due to the preferential induction of Th2 responses, as has been suggested by other GVHD studies.28 Our studies indicate that the regulation of GVHD lethality by the OX40/OX40L pathway does not depend upon either Stat-4 (Th1) or Stat-6 (Th2) signaling. Although OX40L expression has been reported on activated T cells, we
did not observe differences in alloresponses in vitro as measured with
the use of OX40L Although an agonistic anti-OX40 mAb was not well tolerated when given 3 weeks after BMT along with DLI, it is possible that anti-OX40 mAb could be used to induce antitumor effects after BMT when combined with either no DLI or lower DLI cell doses. Such an approach may be of particular benefit in an autologous setting because signaling via OX40 can break peripheral tolerance, which may be therapeutically helpful in patients with residual post-BMT hematological malignancy that may continue to be tolerized by tumor antigens.43 Our studies indicate that the OX40/OX40L pathway has a broad importance in GVHD induction. Several but not all studies in humans have suggested that OX40 up-regulation can precede acute GVHD generation and may be a marker of steroid-resistant acute GVHD or chronic GVHD.44-47 Regardless of whether the up-regulation of OX40 receptor is of prognostic significance, interruption of the OX40/OX40L pathway early in the post-BMT period warrants testing as an approach to prevent GVHD and allogeneic BM graft rejection.
Submitted October 18, 2002; accepted December 23, 2002.
Prepublished online as Blood First Edition Paper, January 9, 2003; DOI 10.1182/blood-2002-10-3048.
Supported in part by NIH grants ROI AI34495, ROI CA72669, ROI HL63452, and R37 HL56067.
B.R.B. and A.H.S. contributed equally to this study.
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: Bruce R. Blazar, University of Minnesota Hospital, Box 109 Mayo Bldg, 420 SE Delaware St, Minneapolis, MN 55455; e-mail: blaza001{at}tc.umn.edu.
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Top
Abstract
Introduction
) or OX40
) donors. Data from 2 replicate experiments with similar results are pooled. In each
instance, targeting of the OX40 receptor had a significant impact
on survival rates.
) or anti-OX40L (
) mice or OX40L+/+
(
) littermate controls, as indicated. Data from 2 replicate
experiments with similar results are pooled. Groupwise comparisons
revealed that P = .24. (B) Sublethally irradiated
bm1 recipients (n = 5 per group) were given highly purified
CD8+ T cells (1 or 3 × 106, as indicated in
parentheses) from either 129/Sv OX40L
