Targeted therapy to the IL-2R using diphtheria toxin and caspase-3 fusion proteins modulates Treg and ameliorates inflammatory colitis.

Abstract

Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD4(+)CD25(+)Foxp3(+) T cells in mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25(+) T cells in vitro, with lower toxicity of IL2-cas to Foxp3(+) T cells. These data infer that targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25(+) Treg. The IL2-cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.

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Targeted therapy to the IL-2R using diphtheria
toxin and caspase-3 fusion proteins modulates Treg
and ameliorates inflammatory colitis
Shai Yarkoni1, Yuval Sagiv1, Ayelet Kaminitz2, Daniel L. Farkas3
and Nadir Askenasy2,3
1GASR Biotechnology Ltd., Kfar-Saba, Israel
2Frankel Laboratory for Experimental Bone Marrow Transplantation, Schneider Children’s
Medical Center of Israel, Petach Tikva, Israel
3Minimally Invasive Surgical Technologies Institute and Department of Surgery, Cedars-Sinai
Medical Center, Los Angeles, CA, USA
Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients
display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a
therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a
diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran
sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate)
toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented
colon shortening. While both chimeric proteins eradicated CD41CD251Foxp31T cells in
mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally
protective and increased fractional expression of Foxp3. Similar effects of the fusion
proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas
increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD251
T cells in vitro, with lower toxicity of IL2-cas to Foxp31T cells. These data infer that
targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models
of inflammatory colitis, despite depletion of CD251Treg. The IL2-cas fusion protein is
particularly relevant to inflammatory bowel disease, as direct internalization of toxic
moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.
Key words: Apoptosis.Colitogenic T cells.IL-2 fusion proteins.
Inflammatory bowel disease.Treg
Introduction
Development of specific immune therapies to inflammatory
bowel disease (IBD) is difficult within a complex pathophysio-
logic process [1–3]. One of the immunological abnormalities in
human forms of IBD, Crohn’s disease (CD), and ulcerative colitis
(UC) is altered sensitivity of pathogenic lymphocytes to apoptosis
[4–6]. T cells located in the lamina propria of CD patients often
present markers of memory T cells [4] and display relative
resistance to multiple pathways of apoptosis [7–9]. These cells
cycle faster [10] and are sensitive to caspase-8 activation [11],
but possess intrinsic defects in control of the apoptotic cascade
[5, 11, 12]. Resistance of enteric T cells to apoptosis has been
attributed to elevated levels of FLIP (caspase-8 inhibitor) in UC
patients [11] and disturbed ratio between pro- and anti-apoptotic
factors in CD patients [8].
Under this scenario it would be useful to design therapeutic
strategies that induce apoptosis in reactive (colitogenic) T cells.
Correspondence: Dr. Shai Yarkoni
e-mail: shai@GASR-bio.com
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
DOI 10.1002/eji.200839190Eur. J. Immunol. 2009. 39: 2850–2864 Shai Yarkoni et al.
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IL-2 is an attractive therapeutic target, considering that IL-2
withdrawal and CD2/CD28 stimulation induce apoptosis in
normal enteric T cells [7]. Neutralization of IL-2 is particularly
relevant toIBD considering
active CD lesions contain increased levels of mRNA encoding IL-2
and its cognate receptor [13], and these cells proliferate
robustly in response to this cytokine [8]. Furthermore, sporadic
symptomatic exacerbations have been observed in IBD patients
after administration of IL-2 [14]. Attempts to treat IBD using
neutralizinganti-IL-2monoclonal
modest responses [15, 16], possibly because of the relative
resistance of lymphocytes from CD patients to apoptosis
upon IL-2 withdrawal [8]. Targeting this cytokine may not
be an optimal approach, as IL-2 has multiple and pivotal func-
tions in immune homeostasis [17], and monoclonal antibodies
against IL-2 prolong its biological activity and stimulate CD81
T cells [18]. As in other inflammatory conditions, IL-2 plays an
important role in propagation of the immune reaction against
enteric mucosa [7, 8, 13] and also in maintenance of the activity
of Treg [19, 20].
Anotheroptionis to
the IL-2 receptor (IL-2R), which might provide more specificity to
pathogenic T cells. We chose two fusion proteins composed of
IL-2 conjugated to pro-apoptotic molecules. Uptake of the
fusion proteins is mediated by internalization of the IL-2R-ligand
complex [21]. The first fusion protein is an IL-2 conjugate of
diphtheria toxin (IL2-DT,Ontak),
synthesis, leading to cell death [22]. This agent was found
to be effective in amelioration of autoimmune disorders
including diabetes,rheumatoid
through depletion of the effector T cells [23–25], and was
shown to be particularly toxic to colitogenic cells in vitro [26].
The second fusion protein is an IL-2 conjugate of caspase-3
(IL2-cas), which was recently shown to provide protection
in ananimalmodel of experimental
chimericmolecule lowered
decreased IL-1b and TNF-a production, and decreased the Bcl-2/
Bax ratio, which correlated with amelioration of the course of
disease.
Immunomodulation by selective elimination of IL-2R1cells
has two possible outcomes. On the one hand, expression of high
levels of the IL-2Ra chain (CD25) is one of the phenotypic
characteristics of subsets of Treg [28], and IL-2 signaling is
essential to attain effective suppressive function of these cells
[17, 19, 20]. Selective depletion of CD25highTreg might intensify
the inflammatory reaction, leading to eruption of aggressive
disease. Evidence from various experimental models points to the
importance of CD41CD251Foxp31Treg in prevention of IBD
[29]. First, mice deficient in IL-2 and the a (CD25) or b (CD122)
chains of the IL-2R display a variety of autoimmune disorders,
including gastritis and colitis, as features of their generalized
lymphoproliferative state [30–33]. Second, eruption of IBD is a
feature of the immune deregulation caused by IL-2 neutralization
in adult mice [34], implying that the underlying cause of colitis in
IL-2 deficiency is dysfunction of Treg [35]. Third, adoptive
that mucosalT cellsfrom
antibodies haveshown
targetthe cellsexpressing
which inhibits protein
arthritis,and encephalitis
colitis [27].This
myeloperoxidaseactivity,
transfer of CD251Treg abrogates the onset of inflammation in a
variety of autoimmune disorders, including gastritis and colitis
[36–41], resolve already established inflammatory colitis [42],
and prevent the occurrence of such reactions induced by
thymectomy in the neonate [43]. On the other hand, upregula-
tion of CD25 is also a characteristic of T-cell activation [17]. As
IBD is caused by accumulation of pathogenic cells that are
relatively resistant to apoptosis [8], potent killing activity
against cytotoxic T cells might ameliorate the course of the
disease [27].
In this study we monitored the impact of IL-2 targeted therapy
on the immune profile of mice in models of experimental
colitis. We found an overall protective effect of the IL-2 fusion
proteins in prevention of colitis and amelioration of established
disease induced by dextran sodium sulfate (DSS). However
administration of these fusion proteins resulted in remarkable
differences in the immunological status of healthy and sick mice,
with IL2-DT inducing pronounced lymphopenia. In variance, the
protective effect achieved with IL2-cas was accompanied by
increased Foxp3 expression without reducing the cellularity of
lymphoid organs.
Results
Effect of IL-2-targeted therapy on acute toxic colitis
Two models of toxic colitis were experimented in BALB/c mice
(Fig. 1A): a single intracolonic dose of 5mg trinitrobenzene
sulfonic acid (TNBS) and 5% DSS in drinking water for 7 days.
Administration of 5mg TNBS resulted in death of the untreated
mice (n56) [44], which was prevented in 75% of the mice
(n58) by daily injections of 50ng/g IL2-DT and 90ng/g IL2-cas
(Fig. 1B). Administration of DSS in drinking water resulted in
transient morbidity without mortality in a control group injected
with 0.2mL PBS on a daily basis (n510). IL-2-targeted therapy
was administered at doses of 10 and 50ng/g IL2-DT, and 90
and 450ng/g IL2-cas (n56–9). The combined clinical score
ofmice treated withlow
(cumulative doses of 0.63 and 3.15mg/g, respectively) were
significantly lower than those of mice treated with low and high
doses of IL2-DT (cumulative doses of 70 and 350ng/g,
respectively) (Fig. 1C). The protective effect of IL2-cas over
acute DSS-induced colitis corresponded to those recently
reported colon protection at doses of 220–910ng/g [27].
However, the two fusion proteins differentially modulated the
individual parameters of the clinical disease scores. Weight loss
(9.573% in untreated mice) was aggravated by administration
of IL2-DT (p o 0.005) and was ameliorated by IL2-cas (Fig. 1D),
while colon length was preserved by both fusion proteins
administered at the higher doses, but not at the lower doses
(Fig. 1E). Thus, at the therapeutic doses that prevented colon
shortening, the clinical score was dissociated with a remarkably
superior outcome of mice treated with IL2-cas as compared with
IL2-DT.
andhigh dosesofIL2-cas
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Immunological consequences of IL-2-targeted therapy
The primary enteric insult induced by DSS is primarily chemical
(toxic) injury, accompanied by secondary lymphocytic infiltra-
tion. Administration of agents that target cells expressing IL-2R
questioned the effect of the fusion proteins on the immune
system. A striking observation at autopsy was the small size of
spleens from mice treated with IL2-DT (Fig. 1F), confirmed to
treatment
7
DSS
analysis
s
0
8
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12
ease activity index
Dise
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TNBS IL2 cas
% survival%
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012345678
Days
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mm)
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ED
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IL2-cas
p<0.001
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140
splenocytes
% s
p<0.001
GF
0
20
40
Sick
IL2-DT
HD
IL2-cas
LDHD
LD
Control
Figure 1. Disease score in acute colitis with IL-2R-targeted therapy. (A) Mice received one dose of 5mg TNBS into the colon or 5% DSS in drinking
water and were treated with IL2-DT and IL2-cas starting at day 0. Analysis was performed 1 day after discontinuation of DSS and treatment.
(B) Survival of mice after TNBS administration (n56) and treatment with 50ng/g IL2-DT and 450ng/g IL2-cas (n58). (C) Disease activity index
calculated according to the parameters listed in Table 1 in mice treated with low dose (LD) and high dose (HD) of IL2-DT (70 and 350ng/g,
respectively) and IL2-cas (0.63 and 3.15mg/g, respectively). Data show mean7SD. (D) Percent changes in body weight summarized for the
experimental groups (n510). (E) Colons were harvested at the experimental end point and length was measured from the rectum to the ileo-coecal
valve. Colon length is presented for each individual mouse and error bars represent SD for the groups. (F) Significant differences in spleen size
were observed in untreated mice (sick) and treatment with IL2-DT or IL2-cas concurrent with DSS administration. (G) Percent changes in total
spleen cellularity associated with DSS-colitis (sick) and IL-2-targeted therapy as compared with healthy BALB/c controls (n59–10). Data show
mean7SD.
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result from 25–60% (dose-dependent) reduction in spleen
cellularity (p o 0.001 versus sick mice, Fig. 1G). Notably, a sub-
therapeutic dose of IL2-DT that did not protect the colon also
reduced spleen cellularity (p o 0.001 versus untreated mice). The
responses of splenocytes to proliferative stimuli were assessed
1 day after discontinuation of IL-2-targeted therapy (day 8) using
the CFSE dilution analysis (n54). Treatment with IL2-cas was
associated with increased responses to a mitogenic stimulus
provided by concanavalin A (p o 0.001 versus sick mice), whereas
treatment with IL2-DT did not significantly alter the proliferative
responses of residual splenocytes as compared with sick mice.
However, both IL-2 conjugates increased the responses to a low
dose of 20 units IL-2 in culture (p o 0.001 versus sick mice). Used
as a general parameter of immune responsiveness, the competent
proliferative responses suggest that the mice were not function-
ally immunosuppressed by both IL-2 fusion proteins.
Analysis of the fractional distribution of T cells in the spleens
and mesenteric LN revealed a modest fractional increase in CD81
T cells in spleens of mice treated with IL2-DT (Fig. 2A). However,
normalization of cell fractions against absolute spleen cellularity
showed significant depletion of CD81T cells at the therapeutic
dose of IL2-DT (p o 0.001, Fig. 2B). In variance, both IL-2 chimeric
proteins decreased the fraction of CD41T cells in mesenteric LN
(Fig. 2C), which was translated to a marked decrease in CD41
T-cell subset (p o 0.001) in the spleens of mice treated with IL2-DT
(Fig. 2D). These data indicate: (i) Induction of toxic colitis with
DSS does not induce significant changes in T-cell subsets, except a
trend of decreased CD41T-cell fraction in the mesenteric LN;
(ii) IL2-DT causes severe lymphodepletion even at sub-therapeutic
doses and (iii) variations in fractional and absolute distribution of
CD41and CD81T cells do not correlate with severity of the
disease. These data raised the question of whether the fusion
proteins targeted preferentially T cells with suppressive activity.
Impact of IL-2-targeted therapy on Treg
The high affinity a chain of the IL-2R (CD25) and the
transcription factor Foxp3 are considered to be specific markers
of naturally occurring and adaptive CD41Treg [17, 28, 45, 46].
Targeted IL-2 therapy might selectively deplete and/or disrupt
the function of this suppressive subset, an undesired event in the
context of autoimmune colitis. Therefore, we analyzed the impact
of the fusion proteins on cells expressing CD25 and Foxp3 at the
experimental end point, 1 day after discontinuation of therapy
(Fig. 3A). The fraction of CD41CD251T cells was decreased in
the mesenteric LN of sick animals, and was virtually eradicated by
administration of both IL-2 fusion proteins (p o 0.001, Fig. 3B),
without affecting this cell subset in the spleen. However, the
absolute numbers of splenic CD251T cells were markedly
decreased (p o 0.001) in mice treated with IL2-DT but not with
IL2-cas (Fig. 3C). To determine the function of residual splenic
CD41CD251T cells after administration of IL-2 fusion proteins,
these cells were immunomagnetically isolated and evaluated in
an MLR suppression assay. Proliferation of CD41CD25?T cells
was determined from CFSE dilution with and without addition at
50
50
p<0.001
30
40
Healthy
Sick
IL2-DT HD
IL2-cas HD
p<0.05
A
60
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100
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D8+
% CD
20
40
n splenic
Changes in
te numbers
CD8+ absolut
0
Spleen Lymph Nodes
0
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IL2-DT
HD
IL2-cas
LD HD
LD
Control
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IL2-cas HD
p<0.05
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p<0.001
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100
0
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Spleen Lymph Nodes
% CD4+
%
0
20
40
es in splenic
Change
solute numbers
CD4+ abs
IL2 DT IL2-DT
HD
IL2 casIL2-cas
LDHD
LD
Control Sick
Figure 2. The effect of IL-2R-targeted therapy on T-cell subsets. Fractional distribution of CD81(A) and CD41(C) T cells in the spleens and
mesenteric LN of healthy controls, after DSS administration (sick), and high doses (HD) of IL2-DT and IL2-cas (n56–9). Changes in the absolute
numbers of CD81(B) and CD41(D) T cells in the spleens of mice treated as detailed above and with low doses (LD) and high doses (HD) of IL2-DT
and IL2-cas, normalized against spleen cellularity in naı ¨ve BALB/c mice (n56). Data show mean7SD. p-Values were calculated using t-test.
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a 1:1 ratio of residual CD41CD251T cells from mice treated with
the IL-2 fusion proteins (n54). The proliferation index of CD41
CD25?T cells was reduced from 2.770.4 to 1.270.3 (p o 0.005)
and 1.770.3 (p o 0.01) by addition of CD41CD251T cells from
mice treated with IL2-DT and IL2-cas, respectively. Thus, residual
splenic CD41CD251T cells retained suppressive activity in vitro
after IL-2-targeted therapy.
The chimeric proteins variably affected Foxp3 expression
within the CD41T-cell subset (Fig. 3D). While IL2-DT decreased
the fraction of CD41Foxp31cells in the spleen (p o 0.05) and
particularly in the LN (p o 0.001), IL2-cas depleted these cells only
in the LN (p o 0.001). These variations translated into marked
reduction in absolute numbers of CD41Foxp31T cells in mice
treated with IL2-DT (p o 0.001), as opposed to an increase in their
A
140
p<0.001
C
Healthy
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IL2-DT HD
IL2-cas HD
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p<0.001
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% CD25+ / CD4+
p<0.001
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s
CD25+ absolute numbers
C
Changes in splenic
IL2-DT
HD
IL2-cas
LDHD
LD
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p<0.001
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E
0
Spleen Lymph Nodes
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D
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enic FoxP3+
Changes in sple
umbers
absolute nu
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HD
IL2-cas
LDHD
LD
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/ CD4+
% FoxP3+/
p<0.05
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0
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25+FoxP3+ / CD4+
% CD2
0
20
in absolute numbers
Changes
enic CD25+FoxP3+
of sple
IL2-DT
HD
IL2-cas
LDHD
LD
Control Sick
Figure 3. Effect of IL-2R-targeted therapy on cells expressing CD25 and Foxp3. (A) CD25 and Foxp3 in spleens of healthy and mice administered
DSS: healthy controls, untreated mice (sick) and mice treated with high dose IL2-DT and IL2-cas (n56). Percent of CD25 (B), Foxp3 (D), and CD25/
Foxp3 (F) in CD41T cells in the spleen and mesenteric LN of naı ¨ve, sick, and treated mice (each experimental group includes six to nine mice).
Fractional changes were normalized against the total numbers of CD41T cells in the spleens to determine changes in absolute numbers of cells
expressing CD25 (C), Foxp3 (E), and CD25/Foxp3 (G) (each experimental group includes six mice). Data show mean7SD.
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numbers
Accordingly, the subset of CD41CD251Foxp31T cells was
diminished by both agents in the LN (p o 0.005), but was increased
after IL2-cas treatment in the spleen (p o 0.005, Fig. 3F). These
differential effects were even more accentuated in analysis of the
absolute numbers of CD41CD251Foxp31T cells, which were
elevated after IL2-cas therapy (p o 0.001, Fig. 3G). Notably, the
fractions and absolute numbers of CD41CD251Foxp31T cells
were reduced by induction of colitis as compared with naı ¨ve
BALB/c controls (Figs. 3F–G). While these data suggested that
colon shortening is not directly related to the fraction of CD41
CD251Foxp31T cells, IL2-cas not only preserved but also
increased the fraction of CD41CD251Foxp31T cells in the
spleen.
In view of effective targeting of CD251T cells by both fusion
proteins and virtual eradication of CD41CD251Foxp31in the
mesenteric LN, we focused on CD41Foxp31T cells. The profile of
all experimental groups showed an increase in Foxp3 expression in
20% of splenic and 30% of mesenteric CD41T cells in mice treated
with a therapeutic dose of IL2-cas (Fig. 4A). Analysis of fractional
Foxp3 expression in CD41CD251T cells in the spleens of sick mice
suggested dissociated regulation of the transcription factor and
CD25 (p o 0.01, Fig. 4B). Furthermore, while fractional Foxp3
expression in CD41CD251T cells was relatively steady in the
spleens of all mice that were administered DSS, significant changes
were observed in the mesenteric LN. In IL2-cas-treated mice,
70–85% of the CD251T cells expressed Foxp3 (p o 0.01 versus 62%
in healthy mice), suggesting that CD25 expression was indeed
driven by Foxp3. It is possible that CD41CD251Foxp31T cells
were also generated in mice treated with IL2-DT (as seen at a
lower dose), but high-dose therapy with this fusion protein
efficiently removed cells expressing CD25. Notably, Foxp3 is an
essential transcription factor for IL-2R (CD25) expression in the
ontogeny of naturally occurring and adaptive Treg [45, 46].
(p o 0.005) followingIL2-castherapy (Fig. 3E).
Effects of IL-2-targeted therapy in healthy mice
In view of the dynamic changes in CD25 and Foxp3 expression in
mice treated with the two fusion proteins, we questioned whether
the observations were related to the disease or the differential
effects of the fusion proteins. Therefore, the IL-2 conjugates were
administered to healthy BALB/c mice at two consecutive daily
doses of 50ng/g IL2-DT and 450ng/g IL2-cas, and the spleens
and mesenteric LN were examined after 3 days. As seen in mice
with colitis (Fig. 1G), IL2-DT caused a marked reduction in
spleen cellularity (p o 0.001 versus healthy controls), which was
not observed with IL2-cas (Fig. 5A). Similarly, there were no
significant differences in fractions of CD81and CD41T cells in
the experimental groups (data not shown). Administration of
IL2-DT caused a marked reduction in cells expressing CD25
(Fig. 5B), Foxp3 (Fig. 5C), and both markers (Fig. 5D), both in
the spleen and the mesenteric LN. In variance, IL2-cas depleted
CD41CD251T cells (Fig. 5B) whereas it caused a marked
increase in CD41Foxp31T cells in all lymphoid organs (Fig. 5C).
Further analysis showed expression of Foxp3 in 34–40% of CD41
CD251T cells (p o 0.05 versus healthy mice) and expression of
CD25 in only 12–18% of the CD41Foxp31T cells (p o 0.01 versus
healthy and IL2-DT-treated mice). These data show organ-
specific dissociation between expression of CD25 and Foxp3
after administration of IL2-cas and demonstrate that elevated
Foxp3 expression is related to IL2-cas rather than toxic colitis.
Effect of IL2-caspase on lymphocytes in vitro
Both conjugates of IL-2 target cells expressing IL-2R and induce
apoptosis upon internalization of the death moiety. To determine
the impact of the fusion proteins in vitro, an enriched lymphocyte
population containing ?60% CD41T cells and ?30% CD81
T cells was exposed to the two agents for 24h. Cell death and
FoxP3+
FoxP3-
A
80
90
100
60
70
50
istribution
ractional di
F
xP3
of Fo
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HD
IL2-cas
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IL2-DT
HD
IL2-cas
LD
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HD
LD
SpleenLymph nodes
80
100
spleen
lymph nodes
B
40
60
p<0.01
0
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Si k
LDLD
IL2-DT
5+
oxP3+/CD2
% Fo
HD HD
LDLD
IL2-cas
HD
HD
p<0.001
p<0.001
C Control l Sick
Figure 4. Analysis of Foxp3 expression. (A) Relative changes in Foxp3
expression in CD41T cells in the spleens and mesenteric LN of healthy
controls, sick mice, and treatment with low (LD) and high (HD) doses of
IL2-DT and IL2-cas (each experimental group includes six mice). Gray
bars represent percent FoxP31cells in the different experimental
groups. (B) Percent expression of Foxp3 in CD41CD251T cells in the
spleens and mesenteric LN (each experimental group includes six
mice). Data show mean7SD.
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apoptosis were measured by 7-aminoactinomycin-D (7-AAD) and
annexin-V incorporation, respectively. Low concentrations of the
chimeric proteins induced apoptosis in ?20% of the lympho-
cytes, slightly higher than spontaneous death (Fig. 6A). As
expected, both IL-2 conjugates induced apoptosis preferentially
in CD41CD251T cells (p o 0.05, Fig. 6B), with preferential
targeting of cells expressing high levels of IL-2R (p o 0.01,
Fig. 6C). These data documented the apoptotic effect of low
concentrations of the fusion proteins and specificity to cells
expressing the IL-2R. In view of the variations in cells expressing
Foxp3 in mice treated with IL2-cas in vivo, apoptosis was
determined in the CD25low(30% Foxp31) and CD25highsubset
(75% Foxp31). While CD25highFoxp31were more sensitive to
apoptosis under control incubation conditions, IL2-cas induced
apoptosis preferentially in Foxp3-cells within both the CD41
CD25lowand the CD41CD25highsubsets (Fig. 6D). The reduced
sensitivity to apoptosis of CD41CD25highFoxp31T cells may
explain the higher levels of these cells observed in the lymphoid
organs of healthy and sick mice treated with IL2-cas.
Impact of IL2-caspase on chronic colitis
We reasoned that if high fractional expression of Foxp3 is
involved in resolution of toxic colitis, the protective effects would
be more accentuated in a chronic model of established colitis.
Because the colon recovers spontaneously after discontinuation
of DSS, we evaluated several models of chronic colitis other than
the one recently reported to benefit IL2-cas therapy [27]. Mice
were administered three 5-day cycles of DSS interrupted by 3-day
periods of recovery, and IL2-cas was administered during the
second and third cycles of DSS at a dose of 450ng/g every second
day (Fig. 7A). In this model of established colitis, IL2-cas
improved the clinical score (p o 0.01, Fig. 7B), enhanced weight
gain throughout the experimental period of 4wk (Fig. 7C), and
reduced colon shortening (p o 0.005, Fig. 7D).
Analysis of the CD41T-cell subset revealed elevated levels of
CD41CD251T cells in the LN of mice treated with IL2-cas
(po0.01, Fig. 7E), decreased levels of CD41Foxp31cells in the
spleens, and elevated levels in the mesenteric LN (p o 0.001,
Fig. 7F). Accordingly, IL2-cas therapy decreased splenic contents
(p o 0.001 versus untreated mice) and increased mesenteric
contents of CD41CD251Foxp31T cells (p o 0.001 versus untreated
mice, Fig. 7G). Taken together these data show that IL2-cas
administration increased Foxp3 expression in sick and healthy
mice, which persisted over a period extending from 3 days in
healthy mice to 4wk in mice with chronic colitis. Comparative
analysis showed a decrease in fractional Foxp3 expression in
splenic and mesenteric CD41CD251T cells of mice with chronic
colitis and wide variations in IL2-cas treated mice: a minor subset
of CD251T cells expressing Foxp3 in the spleen and almost
absolute expression in the mesenteric LN (Fig. 7H). The corre-
lation between the transcription factor and IL-2R in the mesen-
teric LN and increased Foxp3 expression in the acute model and
healthy mice suggest that Foxp3 induced CD25 expression. Thus,
organ-specific modulation of the putative CD41CD251Foxp31
100
120
140
p<0.001
A
15
20
Control
IL2-DT
IL2-cas
p<0.001
p<0.001
B
40
60
80
tes
% splenocyt
5
10
D4+
% CD25+/CD
0
20
PBS IL2-DTIL2-cas
0
Spleen Lymph Nodes
15
20
Control
IL2-DT
IL2-cas
p<0.001
p<0.001
C
6
8
10
Control
IL2-DT
IL2-cas
p<0.001
p<0.001
D
xP3+/CD4+
% CD25+Fox
5
10
+/CD4+
% FoxP3
2
4
0
SpleenLymph Nodes
0
Spleen Lymph Nodes
Figure 5. Influence of IL-2 fusion proteins on healthy mice. (A) Spleen cellularity in control mice infused with PBS and administration of two doses
of 50ng/g IL2-DT or 450ng/g IL2-cas (n56). (B) Percent expression of CD25 in CD41T cells. (C) Percent expression of Foxp3 in CD41T cells.
(D) Percent expression of CD25 and Foxp3 in CD41T cells.
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Treg by IL2-cas included increased levels in the spleens of mice
with acute colitis (Fig. 3F) and in the mesenteric LN of mice with
chronic colitis (Fig. 7F).
Discussion
Three aspects of the data presented in this study are evaluated:
(i) IL-2-targeted therapy improves the outcome of DSS-induced
colitis; (ii) this therapy affects the immune system differentially
in the acute and chronic phases of colitis, and (iii) targeting IL-2R
with two conjugates, diphtheria toxin and caspase-3, has variable
effects on the immune system.
DSS induces toxic injury that disrupts the mucosal barrier and
exposes the enteric wall to luminal flora [47], activates resident
macrophages that secrete pro-inflammatory cytokines, and
attract secondary inflammation [48]. Initial stages of toxic colitis
are largely independent of lymphocyte activity, as demonstrated
by induction of the disease in mice deficient in T cells [49].
However, all models of colitogenic toxins converge to common
pathways that involve lymphocytic responses superposed on
macrophage-initiated inflammation in immunocompetent mice
[1–3, 50]. In general, T-cell responses are either of the Th1 or
Th2 types, with an apparent predominance of Th1 cytokines in
early stages of colon inflammation and a mixed Th1/Th2 pattern
in latent stages of the disease [2]. Withdrawal of the toxin is
followed by gradual recovery of mucosal integrity mediated by
physiological repair mechanisms [50]. The detrimental effect of
lymphocyte-mediated inflammation in acute toxic colitis is
exemplified by the rescue of 75% of the mice from a lethal dose of
TNBS by IL-2-targeted therapy.
Depletion of effector cells appears to be an essential ingre-
dient of therapeutic approaches to IBD, a disorder characterized
by continuous generation of colitogenic T cells [51] that accu-
mulate in the target organ and its draining lymphatics [1–3, 8].
Fusion proteins targeting the IL-2R achieved therapeutic effects
in mice with toxic colitis: both IL-2 fusion proteins prevented the
development of colitis in response to acute injury caused by DSS
and TNBS, and IL2-cas improved the outcome of established
disease (induced by DSS). The protective effect of the IL-2
chimeric proteins is attributed primarily to direct killing of
pathogenic cells, alleviating the lymphocytic component of
inflammation. In contrast, monoclonal antibodies against CD25
that inhibit without killing the target cells (including Treg
subsets) [52] were found inefficient in treatment of UC patients
[15, 16]. In sick mice, the fusion proteins displayed organ-specific
effects. While CD251T cells were evenly affected in spleens and
LN of healthy mice, these cells were predominantly depleted in
40
50
Annexin-V
7-AAD
A
40
50
Control
IL2-DT
IL2-cas IL2-cas
p<0.005
B
20
30
20
30
% apoptosis
p<0.05
0
10
ControlIL2-casIL2-DT
CD4+
Control IL2-DT IL2-cas
CD8+
sis
death/apoptos
%
0
10
CD4+CD25-
CD4+CD25+
40
50
Control
IL2-DT
IL2-cas
p<0.01
C
40
50
FoxP3+
FoxP3-
D
10
20
30
is
% apoptosi
p<0.05
10
20
30
is
% apoptosi
0
CD4+CD25low
CD4+CD25high
0
CD4+CD25low
IL2-cas
CD4+CD25high
IL2-cas Control Control
Figure 6. IL-2 fusion proteins induce apoptosis in vitro. An enriched population containing 60% CD41and 30% CD81of T cells was incubated with
IL2-DT and IL2-cas for 24h in vitro. (A) Cell death and apoptosis as determined by incorporation of 7-AAD and annexin-V, respectively.
(B) Differential induction of apoptosis in reference to CD25 expression in CD41T cells. (C) Analysis of cell death in CD41T cells expressing low and
high levels of CD25 exposed to IL2-DT and IL2-cas. (D) Analysis of cell death in reference to Foxp3 expression in the CD25lowand CD25highsubsets
upon exposure to IL2-cas. Data show mean7SD of three independent incubations.
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the LN of mice with acute colitis, as expected for activated cells
with increased sensitivity to apoptosis.
CD41T cells have dual cytotoxic and regulatory functions in
all models of enteritis [53–55]. Although expression of CD25 is
considered to delineate a subset of naturally occurring Treg that
maintain self-tolerance [20], this is also upregulated in activated
lymphocytes [17]. Therefore, targeting of the IL-2R affects both
effector and suppressor arms of the immune reaction. Various
p<0.01
B
analysis
IL2-cas
A
Therapy onsetpy
SickSick
ty index
Disease activit
IL2-cas IL2 cas
8
DSS
cycles
0
5 12 1519 2226
1234
6
8
10
12
14
C
p<0.005
D
0
2
4
SickIL2-cas
weight gain
% w
20
20
25
PBS
PBS
L2-cas
E
PBS
L2-cas
p<0.001
F
Healthy Sick IL2-cas
length (mm3)
Colon
5
10
15
+
% CD25+/CD4+
p<0.01
+
% FoxP3+/CD4+
p<0.001
0
Spleen Lymph Nodes
Spleen Lymph Nodes
4
4
6
8
10
PBS
L2-cas
p<0.001
p<0.001
G
spleen
lymph nodes
H
0
2
SpleenLymph Nodes
+FoxP3+/CD4+
% CD25+
Sick
Acute
P3+/CD25+
% FoxP
Chronic
Control
IL2-cas Sick
IL2-cas
6
6
8
10
0
2
4
80
90
100
110
20
20
25
50
60
70
5
10
15
0
60
80
100
0
20
40
Figure 7. Disease score and immune consequences of IL-2R-targeted therapy on established enteric disease. (A) Mice received four cycles of 5 days
DSS in drinking water interrupted by 2-day intervals. After the second cycle some mice (n59) received 450ng/g IL2-cas at 2-day intervals. On day
28 the mice were sacrificed for end-point analysis. (B) Disease activity index was determined according to Table 1 after two cycles of DSS (therapy
onset) and the subsequent two cycles with therapy (IL2-cas) and without (sick). (C) Weight gain expressed as percent gain in body weight at the
experimental end-point with therapy (IL2-cas) and without (sick). (D) Colon length at the end of the fourth cycle with therapy (IL2-cas) and without
(sick) as compared with age- and gender-matched healthy control mice. (E–G) Changes in percentage of CD25 (E), Foxp3 (F), and CD25/Foxp3 (G) in
CD41T cells in the spleens and mesenteric LN of untreated (sick) and IL2-cas-treated mice. (H) Percent expression of Foxp3 in CD41CD251T cells
in healthy controls, acute DSS-induced colitis (sick) with high dose IL2-cas therapy and the chronic model of DSS-induced colitis (sick) with IL2-cas
therapy every second day during the third and fourth cycles. Data show mean7SD.
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conjugates of IL-2 display different activities, such as inefficient
treatment of experimental colitis by a Pseudomonas exotoxin-A
chimeric molecule [56]. The two cytotoxic molecules evaluated in
this study had distinct effects on the immune profiles of healthy
mice as well as at therapeutic doses that prevented TNBS-induced
mortality and DSS-induced morbidity. A dose of 8mg IL2-DT over
7 days caused severe lymphopenia (55% reduction in spleen
cellularity), a parameter that has been previously overlooked
when IL2-DT was used for therapeutic purposes in other disease
models [25, 57, 58]. However, it is evident from the present data
that lymphopenia is not absolutely necessary to protect the colon
from inflammation secondary to toxic injury: a sub-therapeutic
dose of IL2-DT depleted lymphocytes without ameliorating the
course of disease, while efficient therapy was attained by
administration of IL2-cas without lymphopenia. Furthermore,
non-selective lymphoablation perturbs immune homeostasis,
with particular detrimental consequences in the setting of auto-
immune disorders. The lymphopenic environment reduces
homeostatic control of autoreactive T cells [59, 60], and the
slower recovery of suppressor cells (as compared with naı ¨ve and
pathogenic T cells) creates a period of imbalance that predisposes
to recurrence of autoimmune reactivity [61].
Both IL-2 fusion proteins virtually eradicated the subset of
CD41CD251Foxp31Treg in the mesenteric LN of sick mice and
consistently apoptosis was predominantly induced in cells
expressing high CD25 levels in vitro. A therapeutic effect medi-
ated by deletion of pathogenic cells despite eradication of
Treg is consistent with the therapeutic efficacy of IL2-DT in a
model of experimental autoimmune encephalitis in rats [25].
Similarly, our data corroborate previous reports of particular
toxicity of IL2-DT to Treg subsets [57, 58], which was applied to
abrogate suppressive T-cell activity for vaccination [58] and
augment antitumor immunity [62]. Although the CD41CD251
Foxp31subset of naturally occurring Treg prevents the devel-
opment of pathogenic cells against self-antigens and blocks the
eruption of the autoimmune disorders [29, 35–43, 63], these cells
might be relatively inefficient in suppression of an ongoing
inflammatory process [64]. In view of the therapeutic efficacy of
the fusion proteins used here, Treg appear to play a secondary
role in resolution of experimental inflammatory colitis when
colitogenic cells are effectively targeted. This interpretation is
evidently limited by our focus on CD251Treg, while significant
regulatory contributions might originate from other subsets of
Treg [2].
The distinct effects of diphtheria toxin and caspase-3 on
immune homeostasis in healthy and sick mice point to inherent
differences in their mode of action, resulting in marked variations
in the immune profiles. The most prominent difference was the
preserved fraction of CD25?Foxp31T cells after administration
of IL2-cas, as opposed to depletion of this subset by IL2-DT. These
data are not unprecedented, as high levels of Foxp3 expression
have been generally associated with beneficial effects of immu-
nomodulatory agents in autoimmune disorders, such as IL-2/IL-2
antibody complexes, anti-thymocyte globulin, and complete
Freund’s adjuvant in NOD mice [65–67]. Furthermore, a relative
proportional increase in Foxp3, as a marker associated with the
suppressor activity of Treg, has been previously demonstrated in
studies that targeted IL-2R using anti-CD25 antibodies [68, 69]. It
is evident from the present data that IL2-cas removed most
CD251T cells in initial stages of therapy, followed by a relative
increase in fractional expression of Foxp3 most remarkable
in the spleens of mice with acute colitis and in the mesenteric LN
of mice with chronic colitis. Detailed analysis of the relative
patterns of CD25 and Foxp3 expression in the aftermath of
targeted depletion of CD251T cells is difficult because this
receptor is variably downregulated in naturally occurring
and adaptive Treg without affecting their suppressive capacity
[70, 71]. These dynamic changes indicate that expression of
putative Treg markers is independently regulated under various
conditions, while suppressive function is maintained despite loss
of CD25.
The exact mechanism underlying the high levels of CD41
Foxp31T cells in mice treated with IL2-cas is unknown, though
several possibilities can be envisioned. The first explanation is the
relative resistance of Foxp31cells to apoptosis, which is not
surprising in view of the variable sensitivities of Treg to apoptosis
under different inflammatory environments [72]. Accordingly,
CD251Foxp31T cells were less sensitive to apoptosis induced by
IL2-cas than CD251Foxp3?
cells in culture, and elevated
fractions of CD41Foxp31T cells were observed as early as
3 days after administration of IL2-cas to healthy mice. Consid-
ering that only 50–60% of Foxp31cells co-express CD25,
concomitant depletion of CD251
expansion of Foxp31T cells is expected to selectively enrich the
CD25?Foxp31subset in lymphoid organs. It is tempting to
speculate that internalization of IL2-cas augments expansion
of some cells, as both IL-2 and caspase-3 activation are physio-
logical pathways involved in stimulation of lymphocyte prolif-
eration [73–75]. Another possible mechanism is increased
expression of Foxp3 in CD41T cells, following attenuation of the
immune balance by IL2-cas. In addition to induction of Foxp31
Treg from naı ¨ve CD25?T cells achieved in vitro by CD3/CD28
stimulation in the presence of TGF-b [71, 76], the development of
adaptive Treg within inflamed tissues and the adjacent LN is a
process of physiological significance [17, 77]. Antigen-specific
adaptive Treg arise within the inflammatory cytokine environ-
ment at the site of inflammation [78], where Foxp3 expression is
triggered by injury signals such as nitric oxide [79] and TNF-a
[80]. In addition, other Treg types develop in remote lymphoid
organs and acquire antigen specificity after migration to the
enteric wall [81, 82]. Whether one or several of these mechan-
isms are responsible for the increased Foxp3 expression, these
cells have been generally attributed a beneficial effect in auto-
immune disorders.
Targeting cells expressing IL-2R by fusion proteins offers
several advantages of particular relevance to the treatment of
IBD, despite depletion of Treg. Although it is difficult to extra-
polate animal models to human IBD, there are some similarities
[2, 35, 38] including the aberrant sensitivity to apoptosis in
models of toxic colitis and pathogenic T cells in CD patients [9].
T cells and homeostatic
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The increased levels of IL-2 in enteric mucosa of IBD patients [13]
imply that interruption of the IL-2 signaling pathway is of
potential therapeutic benefit. In this context, targeting of the
IL-2R is superior to IL-2 neutralization because colitogenic cells
are relatively insensitive to apoptosis caused by withdrawal of
this cytokine [8]. The use of fusion proteins for elimination of
cells positive for IL-2R have the distinct advantage of direct
intracellular insertion of toxic moieties that overrides the relative
resistance to apoptosis characteristic of enteric lymphocytes of
IBD patients [4–11, 83–86]. Furthermore, the use of activated
caspase-3 as a toxic moiety is a particular advantage in the
context of IBD, which suggests that this fusion protein may be of
therapeutic value. First, internalization of a downstream caspase
bypasses the reduced susceptibility of colitogenic T cells to
apoptosis mediated by death receptors (Fas) and perforin/gran-
zyme [83], resistance that is partially mediated by expression of
high levels of decoy receptors [84]. Second, IL2-cas sensitizes
pathogenic cells to apoptosis by reducing the Bcl-2/Bax ratio
[27], which is abnormally high in CD patients and contributes to
the resistance of colitogenic cells to apoptosis [8, 85, 86]. Third,
intracellular insertion of IL2-cas overrides the reduced activity of
caspase-3, which is an intrinsic property of mucosal T cells in CD
patients [5, 86] and counteracts the elevated levels of FLIP in UC
patients [11].
Materials and methods
Animal preparation and disease model
Animals used in this study were BALB/c mice purchased from
Jackson Laboratories (Bar Harbor, ME) and housed in a barrier
facility. The Institutional Animal Care Committee approved all
procedures. TNBS (Sigma, St Louis, MO) was administered into
the colon within 150mL of 50% ethanol. DSS (MP Biomedicals,
Irvine, CA) was administered in drinking water at 5%w/v ad
libitum for 7 days. Daily disease scoring included body weight
and occult blood in the stools, and disease activity index was
calculated according to the classification presented in Table 1.
The colons were excised and measured at the experimental end
points. Histological evaluation was performed in cryosections of
the colon counterstained with H&E and observed with an
Axioplan 2 microscope (C. Zeiss).
Treatments
IL-2 conjugated to truncated diphtheria toxin (denileukin diflitox,
Ontak) was purchased from Seragen (San Diego, CA). IL2-cas is a
fusion protein consisting of full-frame IL-2 conjugated to full-
frame caspase-3 (without the leading procaspase segment). The
segments were fused head to tail into a PET-28 plasmid under
control of the T7 promoter and expressed in E. Coli. Protein
retrieved from the inclusion bodies was unfolded, purified under
denatured conditions, and refolded to a biologically molecule.
The IL-2 conjugates were administered intravenously at daily
intervals for 7 days in 200mL PBS, and the control group was
injected with the same volume of PBS to avoid differences in
hydration between the experimental groups (Figs. 1A and 7A).
Cell preparation
Spleens and LN were harvested and gently minced on a 40mM
nylon mesh in HBSS (Kibbutz Beit Haemek, Israel) to prepare
single-cell suspensions. The cells were aspirated with an 18G
needle to obtain single-cell suspensions. For flow cytometry the
red cells were removed by ammonium chloride lysis for 4min at
room temperature. The reaction was arrested with excess ice-cold
solution and cells were washed. Isolation of lymphocytes was
performed by centrifugation over 1.5mL Lympholyte-M (Cedar-
lane, Ont., Canada) and T cells were collected after immuno-
magnetic depletion using antibodies against MAC-1, GR-1, and
B220. All antibodies were obtained from hybridoma cell cultures
(ATCC). Antibody-coated cells were washed twice with PBS
containing 2% FCS and were incubated with sheep-anti-rat IgG
conjugated to M-450 magnetic beads at a ratio of four to five
beads per cell (Dynal). Conjugated cells were precipitated by
exposure to a magnetic field. The purity of T-cell elution was
reassessed by flow cytometry using primary labeled monoclonal
antibodies against CD4 and CD8.
CD25?and CD251subsets of CD41T cells are isolated from
the spleens and mesenteric LN using the CD41CD251Regulatory
T-cellisolationkit (Miltenyi
Germany). Lymphocytes were mixed with a cocktail of biotiny-
lated antibodies against CD8, CD11b, CD45R, CD49b, and Ter-
119 and incubated with magnetic beads conjugated to anti-biotin
antibody. Elution through a column under a magnetic field
enriched the unlabeled CD41T cells. CD251was stained with
PE-labeled monoclonal antibodies, mixed with anti-PE magnetic
microbeads, and positively selected by passage through a second
column under a magnetic field. Purity was evaluated using
FITC-labeled monoclonal antibodies.
Biotec, Bergisch-Gladbach,
In vitro apoptosis
A concentration of 2?106cells/mL was prepared in DMEM
supplemented with 2mM L-glutamine, 1mM sodium pyruvate,
13.6mM folic acid, 270mM L-asparagine, 548mM L-arginine HCL,
Table 1. Scoring system for the disease activity index
Score
Weight
loss (%)
Stool
consistency
Occult/gross
rectal bleeding
0
1
2
3
4
o1
1–5
5–10
10–20
420
Normal Negative
Loose stool Hemo-occult positive
Diarrhea Gross bleeding
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10mM HEPES, 50mM 2b-Mercaptoethanol, 100mg/mL strepto-
mycin, 100U/mL penicillin, and 5% heat-inactivated FBS (MLR
medium). All the ingredients were purchased from Beit Haemek
and Sigma. Cells were incubated at 371C in a humidified 5% CO2
atmosphere for 24h, with and without 0.6mg/mL IL2-DT and
5mg/mL IL2-cas. Cell death and apoptosis were determined in
cells incubated with 5mg/mL 7-AAD (Sigma) and Annexin-V
(IQ products, Groningen, The Netherlands).
Proliferation assay
Cells were plated in plastic dishes (5?107) and after 45min the
non-adherent lymphocytes were collected and washed. The
lymphocytes were incubated at room temperature for 7min with
10mM CFSE (Molecular Probes, Carlsbad, CA), after which
labeling was arrested by addition of 50% FCS and washed with
PBS. Stained cells were cultured at 371C in a humidified 5% CO2
atmosphere for 3 days in MLR medium containing 1% heat-
inactivated mouse serum. Cells were stimulated with 5mM
concanavalin A (Sigma) and 20 units IL-2 (Peprotech, London,
UK) and were related to unstimulated cells. All proliferation
assays were performed in triplicate. Suppression of T-cell
proliferation was performed in stimulated mixed cultures.
Isolated CD41CD25?T cells were labeled with CFSE and
stimulated with an equal number of CD3/CD28 beads (Invitro-
gen, Oslo, Norway). Proliferation was assessed after 48h with
and without the addition of CD41CD251T cells at a ratio 1:1.
CFSE dilution was analyzed in flow cytometry by gating on the
live lymphocytes and proliferation was quantified the ModFit
software (Verity Software House, Topsham, ME).
Flow cytometry
Cells were labeled by incubation for 45min at 41C with the
appropriate antibodies conjugated to FITC, PE, allophycocyanin,
and peridinin chlorophyll a-protein (BD Pharmingen, San Diego,
CA): CD4 (clone RM 4–5), CD8 (clone 53–6.7), CD25 (clone
PC61.5). Foxp3 was determined following permeabilization and
intracellular staining with a PE-labeled antibody (Foxp3 staining
buffer set NRRF-30, eBioscience, Santiago, CA). Antibodies were
purchased from BD Pharmingen and eBioscience. Measurements
were performed with a Vantage SE flow cytometer (Becton
Dickinson, Franklin Lakes, NJ). Positive staining was determined
on a log scale, normalized with control cells stained with isotype
control antibodies.
Statistical analysis
Data are presented as means7standard deviations for each
experimental protocol. Results in each experimental group were
evaluated for reproducibility by linear regression of duplicate
measurements. Differences between the experimental protocols
were estimated with a post hoc Scheffe t-test and significance was
considered at p o 0.05.
Acknowledgements: This work was supported by the Frankel
Trust for Experimental Bone Marrow Transplantation and Target-
In Ltd.
Conflict of interest: Shai Yarkoni serves as the CEO of GASR
Biotechnology Ltd. and has significant equity in this company.
Yuval Sagiv is an employee of GASR Biotechnology Ltd.
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Abbreviations: 7-AAD: 7-aminoactinomycin-D ? CD: Crohn’s disease ?
DSS: dextran sodium sulfate ? IBD: inflammatory bowel disease ? IL2-
cas: IL-2caspase-3
?
IL2-DT:
trinitrobenzene sulfonic acid ? UC: ulcerative colitis
IL-2diphtheria toxin
?
TNBS:
Full correspondence: Dr. Shai Yarkoni, Lamed-Hei Street 33, Kfar-Saba
44395, Israel
Fax: 19729-765-3960
e-mail: shai@GASR-bio.com
Additional correspondence: Dr. Nadir Askenasy, Frankel Laboratory for
Experimental Bone Marrow Transplantation, Schneider Children’s
Medical Center of Israel, Petach Tikva, Israel
e-mail: anadir@012.net.il
Received: 19/12/2008
Revised: 19/5/2009
Accepted: 6/7/2009
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
Eur. J. Immunol. 2009. 39: 2850–2864 Shai Yarkoni et al.
2864