Histone deacetylase inhibitor suberoylanilide
hydroxamic acid reduces acute graft-versus-host
disease and preserves graft-versus-leukemia effect
Pavan Reddy*†, Yoshinobu Maeda*, Kevin Hotary*, Chen Liu‡, Leonid L. Reznikov§, Charles A. Dinarello§,
and James L. M. Ferrara*
*Departments of Internal Medicine and Pediatrics, University of Michigan Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0942;
‡Department of Pathology, University of Florida College of Medicine, P.O. Box 100275, Gainesville, FL 32610-0275; and§Department of Medicine,
University of Colorado Health Sciences Center, B168, 4200 East Ninth Avenue, Denver, CO 80262
Contributed by Charles A. Dinarello, January 23, 2004
Acute graft-versus-host disease (GVHD) and leukemic relapse are the
two major obstacles to successful outcomes after allogeneic bone
marrow transplantation (BMT), an effective therapy for hematolog-
ical malignancies. Several studies have demonstrated that the dys-
regulation of proinflammatory cytokines and the loss of gastrointes-
tinal tract integrity contribute to GVHD, whereas the donor cytotoxic
responses are critical for graft-versus-leukemia (GVL) preservation.
at low doses exhibits antiinflammatory effects by reducing the
production of proinflammatory cytokines. Using two well character-
ized mouse models of BMT, we have studied the effects of SAHA on
to day ?7 after BMT reduced serum levels of the proinflammatory
cytokines and decreased intestinal histopathology, clinical severity,
and mortality from acute GVHD compared with vehicle-treated ani-
mals. However, SAHA had no effect on donor T cell proliferative and
cytotoxic responses to host antigens in vivo or in vitro. When mice
of SAHA did not impair GVL activity and resulted in significantly
improved leukemia-free survival by using two different tumor and
donor?recipient combinations. These findings reveal a critical role for
histone deacetylase inhibition in the proinflammatory events con-
may provide a strategy to reduce GVHD while preserving cytotoxic
T cell responses to host antigens and maintaining beneficial GVL
malignant diseases. The therapeutic potential of allogeneic BMT
relies on the graft-versus-leukemia (GVL) effect, which eradi-
cates residual malignant cells by immunologic mechanisms (1).
Unfortunately, GVL effects are closely associated with graft-
versus-host disease (GVHD), the major complication of alloge-
neic BMT (1). The pathophysiology of GVHD is known to
involve donor T cell interactions with host antigen presenting
(cytokine storm) and cellular effectors that cause target organ
damage (2). Several convergent lines of evidence now implicate
a network of proinflammatory cytokines such as IFN-?, tumor
necrosis factor ? (TNF-?), and IL-1 as critical mediators of both
experimental and clinical GVHD (3, 4). Clinical and experi-
mental observations have demonstrated the importance of do-
nor lymphocytes for a GVL response (1, 5). Attempts to reduce
GVHD by T cell depletion have led to significant relapse of
malignancy (because of the loss of GVL), failure of engraftment,
and a higher rate of infections (1, 6).
Chromatin remodeling by acetylation?deacetylation of his-
tones plays an essential role in the regulation of gene expression
(7, 8). Two classes of enzyme affect the acetylation of histones:
histone acetyltransferases and histone deacetylases (HDACs)
llogeneic bone marrow transplantation (BMT) is an effec-
tive therapeutic option with a curative potential for many
and open the compacted chromatin, allowing the binding of
transcription factors and promoting transcription. By contrast,
HDACs decrease the acetylation of histone lysine tails and
thereby condense chromatin structure and repress gene tran-
scription (7, 8). HDAC inhibitors result in hyperacetylation and
modify gene expression either positively or negatively, depend-
ing on the context in a cell-specific manner (8).
Suberoylanilide hydroxamic acid (SAHA) contains the hy-
droxamic acid moiety that binds to the zinc-containing pocket in
the catalytic site of HDAC enzymes and thus causes their
reversible inhibition (9). In general, SAHA increases the ex-
pression of several genes thought to be constitutively suppressed
in tumor cells. Micromolar concentrations of SAHA are re-
quired for antitumor effects, whereas at nanomolar concentra-
tions SAHA reduces the secretion of inflammatory cytokines,
such as TNF-?, IFN-?, IL-1?, and IL-12 (10, 11). Because low
concentrations of SAHA exhibit significant antiinflammatory
properties, and given the central role of proinflammatory cyto-
kines in the pathogenesis of acute GVHD, we investigated the
effects of SAHA administration in well characterized murine
models of allogeneic BMT. We hypothesized that administration
of SAHA during the amplification of the proinflammatory
cascade early in the time course of allogeneic BMT would
suppress cytokine production and reduce systemic GVHD with-
out eliminating donor T cell responses to host alloantigens that
could be important for the beneficial GVL effect.
Materials and Methods
Mice and BMT. Female C57BL?6 (B6Ly5.1, H-2b, CD45.2?),
B6D2F1 (H-2bxd, CD45.2?), and BALB?c (H2d) mice were
purchased from The Jackson Laboratory, and B6Ly-5.2 (H-2b,
CD45.1?) mice were purchased from the National Cancer
Institute (Frederick, MD). Mice between the ages of 12 and 20
weeks were used for BMT and in vitro experiments. Mice were
given transplants according to a standard protocol as described
(12). In brief, cell mixtures of 5 ? 106bone marrow cells
supplemented with 2 ? 106splenic T cells from either syngeneic
or allogeneic donors were resuspended in Leibovitz’s L-15
medium (Life Technologies, Grand Island, NY) and trans-
planted into recipients by tail-vein infusion (0.25 ml) on day 0.
The purity of T cells (CD3?) was consistently ?80% after
separation with microbeads by Automacs (Miltenyi Biotec,
Bergisch Gladbach, Germany). Before transplant, host mice
received 13 Gy of total body irradiation (137Cs source) delivered
in two fractions or an 11-Gy single dose. Mice were subsequently
host disease; GVL, graft-versus-leukemia; SAHA, suberoylanilide hydroxamic acid; TNF-?,
tumor necrosis factor ?; HDAC, histone deacetylase; RPA, ribonuclease protection assay.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2004 by The National Academy of Sciences of the USA
March 16, 2004 ?
vol. 101 ?
no. 11 ?
housed in sterilized microisolator cages and received normal
chow and autoclaved hyperchlorinated water for the first 3 weeks
after BMT and filtered water thereafter.
were used as allogeneic BMT donors and 2,000 P815 tumor cells
(H-2dCD45.2, a mastocytoma cell line that is uniformly lethal to
syngeneic animals) were added to the bone marrow (BM)
inoculum on day 0 (13). Death from P815 tumor was defined by
enlargement of the liver and spleen with macroscopic tumor
nodules on postmortem examination or hindlimb paralysis.
GVHD death was defined as the absence of tumor and the
presence of GVHD as determined by the clinical scoring system
described below. Minimal residual tumor was determined in
surviving animals without gross evidence of tumor by using
fluorescence-activated cell sorter analysis of the spleen; sensi-
tivity of tumor detection was 0.2% (13). EL4 leukemia was also
used for the GVL experiments as described (14, 15). EL4 is a B6
MHC Class II?/? T cell leukemia?lymphoma EL4 (H2b) and is
thus syngeneic (H2b) to B6 hosts and allogeneic to BALB?c
(H2d) donors. On day 0, 2,000 EL4 cells were injected into each
recipient along with syngeneic (B6) or allogeneic (BALB?c) BM
and spleen T cells (14). Death caused by EL4 was defined by
enlargement of the liver and spleen, whereas death caused by
GVHD was defined as the absence of tumor and the presence of
Administration of SAHA. SAHA was obtained from Italfarmaco
(Cinisello Balsamo, Italy) as a dry lyophilized powder and was
stored at ?80°C. Vials of SAHA were first dissolved in 50 ?l of
DMSO, diluted in sterile H2O, and heated to boiling for com-
plete dissolution before injection. Recipients of allogeneic BMT
were given 35 mg?kg SAHA i.p. (0.2 ml) once daily from day ?3
to day ?7 (five injections), whereas all the controls received
Systemic and Histopathologic Analysis of GVHD. The degree of
systemic GVHD was assessed by a standard scoring system as
described (12). A clinical index was generated weekly by sum-
mation of five criteria scores: percentage of weight change,
posture, activity, fur texture, and skin integrity (12). Acute
GVHD was also assessed by histopathologic analysis of the ileum
and the ascending colon. Specimens were harvested from ani-
mals on day ?7, processed, stained with hematoxylin and eosin,
and coded for histologic examination. Slides were examined
systematically by a single pathologist (C.L.) in a blinded manner
by using a semiquantitative scoring system as described (12).
Mixed Lymphocyte Cultures. All culture media reagents were
purchased from GIBCO?BRL. For analysis of proliferative
response in mixed lymphocyte cultures, splenocytes from indi-
FCS?DMEM supplemented with 50 units?ml penicillin, 50
?g?ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate,
0.1 mM nonessential amino acid, 0.02 mM ?-mercaptoethanol,
and 10 mM Hepes (pH 7.75). These cells were normalized for
2 ? 105donor T cells (CD45.1?CD3?) and cultured in flat-
bottomed 96-well Falcon plates (Becton Dickinson) in the
presence of irradiated splenocytes obtained from naive B6D2F1
(host) animals. Proliferative response to host antigen was mea-
sured by a 1205 Betaplate reader (Wallac Oy, Turku, Finland)
after 72 h by incorporation of [3H]thymidine (1 ?Ci; NEN Life
Sciences Products) for the last 24 h of incubation.
51Cr-Release Assays. Tumor targets, 2 ? 106P815 (H-2d) or EL4
(H-2b), were labeled with 100 ?Ci of51Cr sodium salt (NEN Life
Sciences Products) for 2 h. After washing labeled targets were
resuspended and the
51Cr-release assays was performed as
described (13). Allogeneic splenocytes were harvested and nor-
malized for donor CD8 (CD45.1?, CD8?) cells on day ?14.
These preparations were added to quadruplicate wells at varying
effector-to-target ratios and incubated for 5 h. Maximal and
(Sigma) or media alone to targets, respectively.51Cr activity in
supernatants taken 5 h later were determined in an autogamma
counter (Packard) as described (13).
splenocytes from naive or transplanted mice were resuspended
in PBS and stained with FITC-conjugated mAbs to CD45.1 and
phycoerythrin-conjugated CD3?(Pharmingen) for flow cyto-
metric analysis. In brief, cells (0.5 ? 106) were incubated for 20
min at 4°C with mAb 2.4G2 to block nonspecific staining by Fc
conjugated mAbs for 30 min at 4°C. They were subsequently
washed twice with PBS?0.2% BSA and fixed in 1% paraformal-
dehyde. Two-color flow cytometric analysis was performed by
using a FACScan (Becton Dickinson Immunocytometry Sys-
tems; ref. 15).
RNA Isolation and Ribonuclease Protection Assay (RPA). RNA from
the splenocytes harvested from the transplanted mice was ob-
tained as described (16). In brief, the harvested cells were
homogenized in TRIzol reagent (GIBCO) and incubated at
room temperature for 5 min. Cells were then lysed, mixed with
0.2 ml of chloroform, and centrifuged. RNA-containing phase
was transferred into a fresh tube, and RNA was precipitated by
incubating with isopropyl alcohol. The RNA pellet was washed,
air-dried, and dissolved in an appropriate volume of RNase-free
water and incubated at 8°C. The RNA quantity was determined
from OD260Spectronic LR45227 (Milton Roy, Ivyland, PA); and
the quality by RNA electrophoresis. The RPA was carried out
with RiboQuant MultiProbe RPA System per the manufactur-
er’s protocol (Pharmingen). Radiolabeled, single-stranded RNA
probes from template mCK-3b were synthesized by using
[32P]UTP, 3,000 Ci?mmol, including murine RNA and yeast
tRNA (2 ?g) as positive and negative controls, respectively.
template sets were used for the T7 RNA polymerase-directed
synthesis of a highly specific32P-labeled antisense riboprobe set.
The labeled riboprobe set was hybridized to the total RNA at
56°C for 12–16 h. After hybridization, free riboprobes and
at 30°C for 45 min. A proteinase K treatment was used to
inactivate the ribonucleases. The remaining protected fragments
were purified and separated on denatured gels of 5% acrylamide
and 8 M urea. The undigested labeled probe sets were used as
markers. The bands were visualized by phosphoimager and the
quantity determined by the signal intensity of the protected
probe fragment bands by using IMAGEQUANT software from
(Molecular Dynamics). Templates for GAPDH and L32 house-
keeping genes allowed the assessment of total RNA levels for
normalizing the samples (16).
Cytokine ELISAs. Concentrations of TNF-?, IL-1?, and IFN-?
were measured in serum and culture supernatants by ELISA
appropriate standards were purchased from Pharmingen
(IFN-?, IL-1?, and TNF-?). Assays were performed according
to the manufacturer’s protocol. Samples were diluted 1:4, and
samples and standards were run in duplicate. ELISA plates were
read at 450 nm by using a microplate reader (Bio-Rad).
Histone Isolation and Immunoblotting. Histones were isolated as
described (11). In brief, splenocytes were harvested from the
recipient mice 7 days after BMT and lysed in 1 ml of ice-cold
www.pnas.org?cgi?doi?10.1073?pnas.0400380101 Reddy et al.
buffer (10 mM Tris?HCl, pH 6.5?50 mM sodium bisulfite?1%
Triton X-100?10 mM MgCl2?8.6% sucrose) followed by centri-
fugation. Nuclei were washed, pelleted, and resuspended in
ice-cold water. After acidification with H2SO4, samples were
incubated on ice for 1 h. Proteins were precipitated by overnight
incubation (20°C) with 1 ml of acetone and resuspended in
distilled water. Protein concentrations were determined and
normalized with a Bio-Rad protein assay kit, and equivalent
amounts from each group were loaded. Samples in loading
buffer were boiled and electrophoresed in 15% polyacrylamide
gels and transferred to nitrocellulose membranes and blocked in
5% milk?Tris-buffered saline with Tween (TBST) followed by
overnight incubation in polyclonal rabbit antiacetylated histone
H3 (1:1,000). The blots were incubated for 1 h in goat anti-rabbit
horseradish peroxidase (1:25,000 in block) and washed exten-
sively in Tris-buffered saline?TBST; bands were visualized by
using the enhanced chemiluminescence system according to the
manufacturer’s directions (Pierce) (11).
Statistical Considerations. All values are expressed as the mean ?
SEM. Statistical comparisons between groups were completed
by using the nonparametric, unpaired, Mann–Whitney test,
except for analyzing survival data, when the Wilcoxon rank test
SAHA Inhibits Proiflammatory Cytokines After Allogeneic BMT. We
first tested the HDAC inhibitor, SAHA, in a dose–response
experiment for its effects on the secretion of proinflammatory
cytokines in a B6 (H-2b) 3 B6D2F1 (H-2b/d) mouse model of
acute GVHD. Lethally irradiated B6D2F1 mice received BM
and splenic T cells from allogeneic (B6) or syngeneic (B6D2F1)
donors, as described in Materials and Methods. BMT recipients
were injected i.p. with different doses (10, 20, or 35
mg?kg?1?day?1) of either SAHA or vehicle from day ?3 to day
?7. This schedule was chosen to modulate the inflammatory
cascade of acute GVHD that reaches peak severity by day ?7
after BMT in this model but not interrupting the initial donor T
cell interaction with host antigen presenting cells in the first 72 h
after BMT (12, 17, 18). Administration of 10 or 20 mg?kg had
significantly reduced serum levels of TNF-?, IL-1, and IFN-?
(Fig. 1, P ? 0.03). We also analyzed the effect of SAHA
administration on the cellular production of cytokines by phe-
notyping splenocytes on day ?7 after BMT for intracytoplasmic
IFN-? by fluorescence-activated cell sorter analysis. SAHA
reduced the percent of donor cells secreting IFN-? by ?50%
(18 ? 5.6 vs. 39 ? 4.2, P ? 0.03).
SAHA Increases Histone Acetylation After Allogeneic BMT. The hy-
droxamic acid moiety of SAHA binds to the zinc-containing
pocket of HDAC enzymes, preventing the deacetylation of
histones and resulting in hyperacetylation of histones (8). Anal-
ysis of histone H3 acetylation in splenocytes harvested from the
recipient mice 7 days after BMT showed increased acetylation in
mice treated with SAHA, confirming the inhibition of HDAC
enzymes (Fig. 2a). To determine whether SAHA regulated the
transcription of inflammatory cytokine genes, we next deter-
received transplants of 5 ? 106T cell-depleted BM cells and 2 ? 106T cells from either allogeneic (B6) or syngeneic (B6D2F1) donors as in Materials and Methods.
Each F1recipient of the allogeneic cells were injected i.p. with 35 mg?kg?1?day?1SAHA or the diluent control from day ?3 to day ?7 after transplantation. Sera
from the recipient animals (n ? 3 per group) were obtained by retroorbital venous puncture on day 7 after BMT and analyzed, as described in Materials and
Methods. (a) Serum TNF-? levels are reduced in the recipients treated with SAHA. Shown are syngeneic (?) and allogeneic controls (I) vs. SAHA allogeneic (allo)
vs. SAHA-treated allo recipients (
allo controls (I) vs. SAHA-treated allo recipients (
Injection of SAHA inhibits in vivo proinflammatory cytokine production after BMT. B6D2F1 mice were given 1,300 cGy of total body irradiation and
???);*, P ? 0.05. Results from one of two similar experiments are shown. (b) Serum IL1-? levels are reduced. Shown are syngeneic (?), allo controls (I)
???);**, P ? 0.02. Data are from one of two similar experiments. (c) Serum IFN-? levels are reduced. Shown are syngeneic (?),
???);**, P ? 0.02. Data from one of three similar experiments are shown.
irradiated and transplanted with cells from either syngeneic (syn) or allo donors as in Fig. 1. Allo recipients were injected with either SAHA or the control diluent for
5 days, from day ?3 to day ?7 after BMT. (a) On day ?7 after BMT the recipient splenocytes were harvested (n ? 3 per group), and histones were isolated by acid
was extracted from the splenocytes harvested and pooled together on day ?7 (n ? 3 per group; ?, syngeneic; I, allo controls;
were reduced in the recipients treated with SAHA. Shown are controls (I) vs. SAHA allo recipients (
SAHA increases the acetylation of histone H3 and down-regulates the transcription of TNF-? and IFN-? after allogeneic (allo) BMT. B6D2F1 animals were
???, SAHA allo recipients) after BMT as
???);*, P ? 0.05. Results are from one of two similar experiments.
Reddy et al.
March 16, 2004 ?
vol. 101 ?
no. 11 ?
mined the mRNA expression of TNF-? and IFN-? after BMT by
RPA on day ?7 after BMT, as described in Materials and
Methods. Consistent with the increase in histone H3 acetylation,
mice treated with SAHA showed a significant reduction in the
transcription of TNF-? and IFN-? by ?50%. Thus, brief admin-
istration of SAHA after allogeneic BMT increased histone
acetylation and reduced the production of proinflammatory
cytokines at systemic, cellular protein, and transcription levels.
Inhibition of Proinflammatory Cytokine Secretion Decreases the Se-
verity of Intestinal and Systemic GVHD After Allogeneic BMT. TNF-?
is a critical inflammatory mediator of gastrointestinal toxicity
after allogeneic BMT (19), therefore we analyzed whether the
reduction in TNF-? levels seen after SAHA administration
affected the pathology of gastrointestinal tract, a major target
organ of acute GVHD. Samples of small and large bowel were
harvested on day ?7 after BMT and evaluated for histopatho-
logic changes, as described in Materials and Methods. Examina-
tion of small intestine from syngeneic BMT recipients showed
almost complete restoration of normal villous architecture (Fig.
3a). By contrast, recipients of allogeneic BMT and the vehicle
exhibited severe villous blunting, crypt destruction, and epithe-
lial attenuation with intense inflammation of the lamina propria,
all characteristics of acute GVHD (Fig. 3b). SAHA administra-
tion significantly reduced the gastrointestinal tract damage when
compared with allogeneic controls (Fig. 3c), although scores
remained higher than the syngeneic recipients (Fig. 3d; P ?
0.02). We next evaluated the effects of SAHA administration on
the clinical severity and mortality of acute GVHD. SAHA
treatment produced a highly significant survival advantage (Fig.
4a; P ? 0.01) and reduced the severity of clinical GVHD scores
(Fig. 4b; P ? 0.05). All surviving mice showed greater complete
donor chimerism by fluorescence-activated cell sorter analysis
(data not shown), ruling out graft failure or mixed chimerism as
a cause for the GVHD protection (20, 21).
To test the concept that HDAC inhibition by SAHA is a
generalizable strategy for GVHD prophylaxis, we administered
SAHA in a second well characterized mouse model of acute
GVHD directed against both MHC and minor histocompatibility
antigens, BALB?c (H-2d) 3 B6 (H-2b) (15). Recipient B6 mice
were conditioned and received transplants of cells from either
syngeneic (B6) or allogeneic (BALB?c) donors, as described in
P ? 0.02) and reduced its clinical severity compared with controls,
(see Fig. 7, which is published as supporting information on the
PNAS web site). Together these data demonstrate that SAHA
reduced but did not completely eliminate acute GVHD in two
different allogeneic BMT models.
SAHA Administration Does Not Impair T Cell Responses to Host
Antigens After BMT. During GVHD, donor T cells respond to host
histocompatibility antigens within 72 h of injection after BMT (3,
?3 to day ?7 was to permit initial donor T cell activation while
preventing the amplification of the systemic inflammation of acute
IFN-?, can enhance the activation and expansion of T cells (4, 19),
it was possible that inhibition of these cytokines by SAHA admin-
istration could reduce donor T cell responses to host antigens. We
the spleens of BMT recipients. In these experiments we used B6
Ly5.2 (CD45.1) mice as donors. The overall splenic cell counts in
the SAHA- and control-treated allogeneic mice were equivalent
(data not shown). Furthermore, as shown in Fig. 5 a and b, SAHA
did not significantly alter the expansion of donor CD45.1?CD3?
lymphocytes either at day ?7 or ?14, the period of maximal donor
T cell expansion in this model. Proliferation of donor T cells to
irradiated host F1stimulators in vitro was also equivalent between
animals treated with SAHA and control on day ?14 (Fig. 5c).
from C57BL?6 donors as in Fig. 1. Allo recipients were injected with either SAHA or the control diluent for 5 days, from day ?3 to day ?7 after BMT. On days
?7 to ?8, small-bowel samples from BMT recipient mice (n ? 4 per group) were obtained and analyzed microscopically, as described in Materials and Methods.
(a) Syngeneic BMT recipients demonstrate reestablishment of intestinal architecture with villi of near-normal length and without significant cellular infiltration
show severe intestinal toxicity, including surface erosion, villous blunting, epithelial attenuation, and an intense cellular infiltration in the lamina propria. (c)
(I, n ? 4) or SAHA (
and colon from individual animals in each group.*, P ? 0.05, control (I) vs. the SAHA (
Treatment with SAHA results in decreased intestinal histopathology after allogeneic (allo) BMT. B6D2F1 animals received syngeneic BMT or allo BMT
???, n ? 4) from allo donors and of syngeneic donors (?, n ? 4) are shown. Total GVHD score: mean ? SE of the sum of scores for small bowel
???) allo group.
GVHD mortality and morbidity. B6D2F1 mice were given 1,300 cGy of total
T cells from allo B6 donors as in Materials and Methods. Syngeneic recipients
(I, n ? 5) received transplants similarly with cells from F1donors. Each allo
recipient was injected i.p. with either 35 mg?kg?1?day?1SAHA (F, n ? 10) or
the control vehicle (Œ, n ? 10) for 5 days from day ?3 to day ?7. Transplanted
animals were monitored daily for survival and assessed weekly for clinical
of two similar experiments are shown. (a) Percent survival after BMT. F vs. Œ,
*, P ? 0.002 by Wilcoxon rank test. (b) Animals scored for clinical GVHD, as
described in Materials and Methods. Data are expressed as mean ? SEM. F vs.
Œ,**, P ? 0.05 by Mann–Whitney U test from day 7 to day 35.
www.pnas.org?cgi?doi?10.1073?pnas.0400380101Reddy et al.
Furthermore, T cells demonstrated vigorous lysis of host-type P815
(H-2d) tumor targets with only a small reduction in killing at lower
effector?target ratios on day ?14 (Fig. 5d). However, when the
splenocytes harvested after BMT were stimulated in vitro with
lipopolysaccharide, members of the SAHA-treated group secreted
the proliferative responses of donor T cells to F1stimulators taken
7 days after BMT from SAHA- and control-treated animals were
also equivalent (27,899 ? 5,113 vs. 32,549 ? 3,677 cpm, P ? NS).
These data thus demonstrate that SAHA administration at this
dose and schedule after BMT did not affect donor T cell responses
to host antigens, and its ability to reduce GVHD was primarily due
to its inhibition of inflammatory cytokines.
Inhibition of HDACs by SAHA Preserves GVL Activity After Allogeneic
BMT. The maintenance of donor T cell responses to host antigens
after SAHA treatment suggested that this approach might also
preserve the beneficial GVL activity. To test this hypothesis di-
rectly, we added 2,000 P815 (H-2d, CD45.2?) mastocytoma cells to
the BM inoculum to model residual disease at the time of BMT. As
shown previously, when injected intravenously into B6D2F1 (H-
2bxd) hosts, this dose of P815 is uniformly lethal and behaves like a
leukemia, causing massive infiltration and enlargement of the liver
and spleen and hindlimb paralysis from infiltration of the spinal
cord (13). All syngeneic BMT recipients treated with SAHA or the
control showed evidence of tumor infiltration at necropsy and did
not exhibit prolonged survival, ruling out a significant direct
antitumor effect from this dose and schedule of SAHA (Fig. 6a).
In recipients of allogeneic BMT and 2,000 P815 cells, SAHA
administration resulted in 50% survival, a significant survival
advantage compared with controls (Fig. 6a; P ? 0.03). Necropsy
performed either on the day of death or at the end of the
observation period showed no evidence of tumor in the recipients
of allogeneic BMT regardless of SAHA treatment, confirming the
by using a BALB?c 3 B6 allogeneic BMT model of GVL. We
added 2,000 EL-4 (T cell leukemia?lymphoma) cells (H-2b,
CD45.2?) to the BM inoculum on the day of transplant. EL-4 cells
also cause massive infiltration and enlargement of liver and spleen,
and an injection of as few as 500 cells is uniformly fatal (14).
Syngeneic BMT recipients again showed 100% mortality with
evidence of tumor infiltrating into liver and spleen regardless of
SAHA treatment (Fig. 6b). By contrast, allogeneic recipients
injected with SAHA showed 50% survival on day ?50 that was
significantly greater than controls (Fig. 6b; P ? 0.01) with no
evidence of tumor in the liver and spleen on necropsy. Thus, the
maintenance of T cell function by HDAC inhibition with SAHA
separate BMT tumor models.
properties at higher concentrations and have recently been shown
to suppress proinflammatory cytokine secretion at much lower
concentrations (10, 11). Our data show that brief inhibition of
HDAC enzymes with SAHA from day ?3 to day ?7 after
allogeneic BMT significantly suppressed serum levels of TNF-?,
IL-1, and IFN-? and thus modulated the cytokine storm and
reduced acute GVHD by clinical and histologic parameters. How-
ever, SAHA treatment did not alter T cell activity to host antigens
either in vivo or in vitro and preserved GVL effects, resulting in
significantly higher leukemia-free survival in two separate models.
The induction of acute GVHD is a direct consequence of donor
T cell responses to host alloantigens and the dysregulation of
inflammatory cytokine cascades (4). Inflammatory cytokines, such
as IL-1 and TNF-?, are now known to play a central role in the
pathogenesis of many transplant-related complications in clinical
initiated by BMT-conditioning regimens allows passage of lipo-
5.1, CD 45.2) or allogeneic (allo) (B6D2F1) recipients injected with SAHA or the vehicle as in Materials and Methods. Splenocytes were harvested from the
recipients on days ?7 (a) and ?14 (b) after BMT (n ? 4 per group) and labeled with anti-CD3 phycoerythrin and anti-CD45.1 FITC. The number of donor T cells
(CD45.1?CD3?) were determined [allo plus control (I) vs. allo plus SAHA (
T cells at both time points. Data from one of three similar experiments are shown. (c) Harvested splenocytes normalized for donor T cells (CD45.1?and CD3?)
were restimulated in quadruplicate with irradiated naive host (syngeneic B6 Ly5.2 and allo F1) splenocytes in mixed lymphocyte reaction cultures for 48 h.
Proliferation was determined by incubation of cells with [3H]thymidine (1 ?Ci per well) for an additional 24 h. T cells from SAHA-treated animals showed
after BMT. Splenocytes harvested from allo animals on day ?14 after BMT were pooled (n ? 3 per group), normalized for donor CD8?cells, and used in a
51Cr-release assay. Cytotoxic T lymphocyte activity against allo targets (P815) in control (I) and SAHA (
syngeneic targets (EL-4) by both groups (Œ and ?) occurred. Data from one of two similar experiments are shown.
Effect of SAHA administration on donor T cell functions after BMT. B6 Ly5.2 (CD45.1?) donor cells were transplanted into irradiated syngeneic (B6 Ly
???), P ? NS]. Recipients of syngeneic BMT (?) demonstrated lower numbers of donor
???) groups was similar, whereas no significant lysis of
free survival. (a) B6D2F1 mice receiving transplants of T cells from syngeneic
vehicle (Œ, n ? 10) as described in Fig. 1. P815 cells (2,000) were added to the
BM inoculum at day 0, as described in Materials and Methods. Animals were
allo plus SAHA (‚) vs. allo plus vehicle (Œ) and P ? 0.02 for allo plus SAHA (‚)
vs. syngeneic plus SAHA (?). Data shown are from one of two similar exper-
TCD BM and 2 ? 106splenic T cells from allo BALB?c donors and were treated
with SAHA (‚, n ? 11) or control (Œ, n ? 10) or from syngeneic B6 donors and
were injected with control (I, n ? 10) or SAHA (?, n ? 11) as in Materials and
day 0.**, P ? 0.05 for ‚ vs. Œ and P ? 0.01 for ‚ vs. ?.
Treatment with SAHA preserves GVL activity and improves leukemia-
Reddy et al.
March 16, 2004 ?
vol. 101 ?
no. 11 ?
by mononuclear cells that have been primed by IFN-?. These
proinflammatory cytokines further damage gut mucosa and per-
petuate the translocation of additional lipopolysaccharide into the
systemic circulation, thus establishing a positive feedback loop for
progressive gastrointestinal tract injury and systemic inflammation
matory cytokines, including TNF-? and IFN-?, have been associ-
ated with increased GVHD severity after allogeneic BMT (22),
strategies aimed at inhibiting these cytokines individually have not
yielded encouraging clinical results (22, 23). In the current study,
inhibition of HDAC enzymes with SAHA reduced GVHD, sug-
gesting that perhaps inhibiting several proinflammatory cytokines
simultaneously might be a possible strategy for this intractable
complication of BMT.
our study are consistent with several recent reports of this
activity in other disease models. For example, the combination
of HDAC inhibitors trichostatin and butyrate have been shown
to reduce IFN-? production by T cells (24). Leoni et al. (10) have
demonstrated significant reductions of TNF-?, IL-1?, and IFN-?
in experimental endotoxemia and ConA-induced hepatic injury
models with lower doses of SAHA than usually used as an
antitumor agent. Trichostatin has also been recently shown to
reduce the inflammation and clinical manifestations of systemic
lupus erythematosus in a murine model (11). Another recent
study demonstrated that inhibition of HDAC enzymes reduced
the activation of both CD4 and CD8 T cells and lessened the
severity of autoimmune diabetes (25). However the lack of
significant effect on donor T cell responses to host antigens after
BMT at the dose and schedule used in our experiments shows
that this level of HDAC inhibition was not immunosuppressive
despite its clear antiinflammatory effects. In support of this
differential mechanism of action, consistent low concentrations
of SAHA (100 nM) did not significantly alter T cell proliferation
in a mixed lymphocyte reaction (25,911 ? 2,835 vs. 29,357 ?
3,843 cpm, P ? NS) but reduced TNF-? secretion by naı ¨ve
splenocytes after lipopolysaccharide stimulation. Furthermore,
SAHA has been shown to have no effect on anti-CD3?-induced
T cell activation at nanomolar concentrations in vivo (10). Taken
together with our study, these data would suggest that the extent
of HDAC inhibition may produce qualitatively different effects
in terms of tumor cell proliferation?apoptosis, T cell activation?
are needed to further delineate the potential differences.
Histone acetylation is critical in regulating gene expression for
suppression of proinflammatory cytokines remain to be deter-
mined, the inhibition of cytokines by SAHA temporally correlated
with enhanced acetylation of H3 histones. These parallel findings
suggest that chromatin remodeling may play a role in decreased
cytokine production perhaps by increasing the expression of genes
that down-regulate cytokine production, such as the CIS?SOCS?
SSI family proteins (26). It is also possible that HDAC inhibitors
could increase the production of antiinflammatory cytokines such
as TGF-? (27) or regulate nitric oxide (NO) secretion that could
of other nonhistone proteins might also indirectly contribute to the
determine whether these multiple potential mechanisms of action
contribute to the differential effects observed in cell death, prolif-
eration, and cytokine secretion.
The toxicity of GVHD is difficult to separate from GVL
effects, a well recognized benefit of immunotherapy for malig-
nancies (1). Inflammatory cytokines significantly contribute to
the toxicity of GVHD but have a more limited role in the
eradication of residual leukemia, which is primarily mediated by
donor cytotoxic T lymphocytes and NK cells in experimental
models by using established tumor cell lines (5). The mainte-
nance of such lines for extended periods in vitro may cause
significant shifts in tumor cell biology, and, thus, they may not
accurately reflect all aspects of spontaneously occurring malig-
nancies, but such tumor systems have been useful in elucidating
effector mechanisms of immunotherapy and GVL activity (13,
14, 28). Thus, SAHA administration in two different tumor
models leads to disruption of inflammatory cytokine cascades
while maintaining cytotoxic T lymphocytes and thereby success-
fully separates GVHD and GVL. HDAC inhibitors can reduce
the proliferation of transformed cells in vitro and in vivo (9, 29),
but at the lower doses used in our study, SAHA did not
independently reduce tumor-related mortality in the syngeneic
controls even though it increased H3 acetylation (data not
shown). Experiments are needed to determine whether the
concentrations of SAHA sufficient for antitumor activity will
also suppress immunologically mediated GVL effects.
In summary, our findings demonstrate a role for the HDAC
inhibitor SAHA in reducing the early inflammatory responses
contributing to GVHD while preserving donor T cell responses
and GVL effects. Because SAHA is relatively nontoxic and is
currently being tested in phase I?II clinical trials (30), it may
soon be testable in allogeneic BMT recipients as an antiinflam-
matory adjunct to standard GVHD prophylaxis and treatment.
This work was supported by National Institutes of Health Grants K08
AI052863-01 (to P.R.) and CA 49542 (to J.L.M.F.).
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