Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation

Abstract

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.

Full text

Spontaneous Colitis Occurrence in Transgenic Mice with
Altered B7-Mediated Costimulation
1
Gisen Kim,* Olga Turovskaya,* Matthew Levin,* Fergus R. Byrne,
†
John S. Whoriskey,
†
James G. McCabe,
†
and Mitchell Kronenberg
2
*
The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but
also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy
has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7
interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric
protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4
ⴙ
T cells. We show that the
chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell
activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7
double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was
more severe in this context, which was associated with the decreased number of Foxp3
ⴙ
regulatory T cells in transgenic B7 double
knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate
T cell function and the development of inflammatory diseases. The Journal of Immunology, 2008, 181: 5278 –5288.
I
t is well known that T cells play a pivotal role in the patho-
genesis of autoimmune diseases, including inflammatory
bowel disease (IBD)
3
(1). Because optimal T cell activation
requires CD28-mediated costimulatory signals (2– 4), in addition
to the primary signal through the TCR, the CD28 ligands B7-1
(CD80) and B7-2 (CD86) have been reported to be a critical factor
for the induction of T cell mediated autoimmunity (5–7).
Although a complete lack of B7 costimulation abolishes T cell-
mediated colitis (8, 9), it has not been fully elucidated how varying
the level of B7 costimulation affects the pathogenesis of colitis.
Transfer of mouse CD4
⫹
CD45RB
high
T cells to immune deficient
RAG
⫺/⫺
recipients provides a useful model for the T cell-mediated
initiation of IBD (1, 10). We recently reported that a partial re-
duction in costimulation in RAG
⫺/⫺
recipients that lacked either
B7-1 or B7-2 caused accelerated colitis through the enhanced ac-
tivation and differentiation of effector T cells (8). Although the
transferred T cells proliferated comparably to the wild type when
costimulated by a single B7 molecule, either B7-1 or B7-2, the
wild type amount of IL-2 secretion, and subsequent up-regulation
of CTLA-4 expression by the activated T cells, required both B7
molecules. Like CD28, CTLA-4 binds to B7-1 and B7-2, but it
mediates inhibitory signals, as demonstrated by the lethal inflam-
matory disease that occurs in CTLA-4-deficient mice (11–13). Fur-
thermore, in the recent clinical trials for patients with metastatic
melanoma, CTLA-4 blockade caused grade III/IV severe autoim-
munity in several sites, including colitis (14), which strongly sug-
gests a critical role of CTLA-4 in maintaining peripheral tolerance
and in preventing IBD. We therefore concluded that the absence of
a single B7 molecule leads to accelerated colitis induction in part
because of decreased CTLA-4 induction by T cells.
In addition to the regulation that is mediated by the induction of
CTLA-4 expression, B7 molecules further contribute to immune
regulation by participating in the homeostatic maintenance of reg-
ulatory T cells (Treg) that express CD25 and the transcription fac-
tor Foxp3. CD4
⫹
Foxp3
⫹
Treg, which arise both in the thymus (15,
16) and in the periphery dependent upon TGF-
␤
(17), are one of
the most important elements for preventing autoimmunity (18, 19).
The dependence of Treg upon B7-CD28 costimulation is demon-
strated by their reduced number in mice lacking both B7 molecules,
as well as by a report of exacerbated autoimmune disease in CD28-
deficient mice (20). Despite the opposing functions of CTLA-4 and
CD28 interacting with the same B7 molecules, optimal CTLA-4 ex-
pression and the maintenance of Treg both depend on CD28 costimu-
lation and subsequent IL-2 production, suggesting the balance be-
tween CD28-mediated activation and CTLA-4-mediated suppression
is critical for normal immune function.
In this study, we describe a new mouse model of spontaneous
colitis in transgenic mice that express a chimeric, soluble B7-2 Ig
Fc (B7-2 Fc) transgene (Tg). Constitutive expression of the chi-
meric protein in B7-2 Fc transgenic (B7-2 Fc Tg) mice induced
extensive colonic inflammation as well as systemic lymphad-
eonopathy, indicating the critical role of normal B7 interactions in
maintaining immune homeostasis in the mucosae and systemically.
Our experimental results provide insights into the mechanism of T
cell-mediated intestinal inflammation in these mice, and they in-
dicate that the B7-2 Fc Tg mice are a useful model both for study-
ing the roles of the B7 ligand-dependent functions of CTLA-4 and
CD28, as well as for understanding the mechanisms for the initi-
ation and exacerbation of colitis.
*Division of Developmental Immunology, La Jolla Institute for Allergy and Immu-
nology, La Jolla, CA 92037; and
†
Amgen, Thousand Oaks, CA 91320
Received for publication February 6, 2008. Accepted for publication August 13, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant PO1 DK46763 (to
M.K.) and a Career Development Award from the Crohn’s and Colitis Foundation of
America (to G.K.).
2
Address correspondence and reprint requests to Dr. Mitchell Kronenberg, La Jolla
Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037.
E-mail address: mitch@liai.org
3
Abbreviations used in this paper: IBD, inflammatory bowel disease; Treg, regulatory
T cell; Tg, transgene; MLN, mesenteric lymph node; PLN, peripheral lymph node.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
www.jimmunol.org

Materials and Methods
Mice
To generate B7-2 Fc Tg mice, the chimeric sequence was subcloned into
the liver specific apolipoprotein E expression vector. Microinjection, em-
bryo manipulation, and transgene screening were conducted as previously
described (21–23). C57BL/6, B7-1
⫺/⫺
B7-2
⫺/⫺
, CD28
⫺/⫺
, Igh-6
⫺/⫺
,
TCR
␤
⫺/⫺
, IFN-
␥
⫺/⫺
, and RAG1
⫺/⫺
mice on the C57BL/6 background
were purchased from The Jackson Laboratory and bred to the B7-2 Fc Tg
mice at the La Jolla Institute for Allergy and Immunology. Mice used in
these studies were backcrossed 3– 6 times to the C57BL/6 background, and
all Tg mice used were littermates hemizygous for the transgene. OT-II
TCR transgenic mice (24) were a gift of Dr. Stephen P Schoenberger, La
Jolla Institute for Allergy and Immunology (La Jolla, CA).
Abs and reagents
mAbs for CD4 (RM4-5), CD8
␣
(53-6.7), CD11b (M1/70), CD19 (1D3),
CD25 (PC61), CD40 (3/23), CD43 (1B11), MHC class II A
b
(AF6-120.1),
B7-1 (16-10A1), B7-2 (GL1), and CTLA-4 (UC10-4F10-11) were pur-
chased from BD Biosciences. Anti-F4/80 (BM8), CD90.2 (Thy-1 allele
specific, 53-2.1) and TCR
␤
(H57-597) were purchased from eBiosciences.
Biotinylated Abs against CD8
␣
(53-6.7), CD11b (M1/70), CD11c (N418),
B220 (RA3-6B2), TER-119 (TER-119), NK1.1 (PK136), and CD25 (7D4)
for CD4
⫹
T cell negative selection were purchased from eBiosciences,
except anti-CD25 (BD Biosciences). To purify B7-2 Ig Fc chimeric pro-
tein, conditioned medium from CHO cells expressing the extracellular do-
main of murine B7-2 fused to human IgG1 Fc was concentrated 15-fold
using a Pellicon ultrafiltration device (Millipore) fitted with a 30 kD Mo-
lecular Weight Cut Off screen channel cassette. Filtered concentrate was
bound to protein A-Sepharose at ⬃10 mg/ml resin, then washed with sev-
eral volumes 20 mM HEPES, 100 mM NaCl (pH 7.3) before elution with
neutral elution buffer (Pierce). Eluted fusion protein was dialyzed vs two
40⫻ volumes of column wash buffer 24 h each, then filtered through 0.2
mm. Purified material was tested for endotoxin using Pyrotell LAL vials
(Associates of Cape Cod) and contained 0.2 to 0.5 EU/mg.
Flow cytometric analysis
To isolate cells from the large intestine, tissues from the rectum to the
cecum were opened longitudinally, washed with HBSS (Invitrogen),
minced thoroughly, and then incubated with RPMI-1620 medium contain-
ing 1.5 mg/ml Collagenase type IV (Sigma Aldrich), supplemented with
10% FCS, 2-ME, and penicillin-streptomycin-glutamine (Life Technolo-
gies) for 20 –30 min at 37°C. Digested cells were filtered through a metal
mesh and resuspended with 35% Percoll, followed by centrifugation at
1700 rpm for 20 min. The cell pellets were further run over a 44 –70%
discontinuous Percoll gradient at 2400 rpm for 30 min. Cells recovered
from the interface were used as large intestinal cells for immunostaining.
Foxp3 was detected with the anti-mouse/rat Foxp3 staining kit (FJK-16s)
(eBiosciences), according to the manufacturer’s protocol. For intracellular
cytokine staining, ex vivo isolated or cultured cells were restimulated with
PMA (2 ng/ml) and ionomycin (0.5
␮
g/ml) for 4 h, with Brefeldin A added
for the last 2 h before staining with the BD Cytofix/Cytoperm Fixation/
Permeabilization Solution Kit (BD Biosciences).
Histological scoring
For colitis scoring, three samples of 2–3 mm were obtained from the distal,
middle, and proximal portions of the large intestine, and were processed for
H&E staining. Samples were coded and scored in a blinded fashion. Each
specimen was scored according to a simplified scoring system: 0 ⫽ normal
without any focal inflammation; 1 ⫽ focal infiltration of mononuclear
cells without myeloid cell accumulation; 2 ⫽ focal inflammatory cell
infiltration without affecting villous structure; 3 ⫽ focal accumulation of
inflammatory cells causing epithelial hyperplasia or disturbed villous struc-
ture; 4 ⫽ severe diffuse inflammation with epithelial hyperplasia and crypt
abscesses; and 5 ⫽ apparent defects or ulceration of the epithelial layer in
addition to the findings for score 4.
Ig isotype measurements
Ig isotypes in sera were detected using standard capture ELISA according
to the manufacturer’s protocol (Southern Biotechnology Associates), with
biotinylated anti-Ig Abs and streptavidin-labeled HRP (Jackson Immuno-
Reseach Laboratories) for detection.
CD4
⫹
T cell transfer
For transfer colitis experiments, CD4
⫹
CD45RB
high
cells (99.9% pure, con
-
taining ⬍0.1% CD4
⫹
CD25
⫹
) were sorted on a FACS Vantage SE with the
FACS DiVa option (BD Biosciences) after the preisolation of CD4
⫹
cells
using CD4 (L3T4) Microbeads (Miltenyi Biotec) according to the manu-
facturer’s protocol. Five ⫻ 10
5
sorted cells were resuspended with PBS and
injected i.v. to RAG1
⫺/⫺
or B7-2 Fc Tg
⫹
RAG1
⫺/⫺
recipients. In some
experiments, 100
␮
g B7-2 Ig Fc protein was injected i.p. three times a
week starting just after the transfer of donor cells.
Attenuation of colitis with CD4
⫹
CD25
⫹
Treg
CD4
⫹
cells were negatively selected using a combination of 6 biotinylated
mAbs (anti-CD8
␣
, CD11b, CD11c, NK1.1, B220, TER-119) and anti-
biotin microbeads (Miltenyi Biotec). CD4
⫹
T cells (92–95% purity) were
further separated into CD25
⫹
and CD25
⫺
populations using a biotinylated
anti-CD25 mAb and anti-biotin microbeads (75– 85 and 96 –98% purity,
respectively). One ⫻ 10
6
CD4
⫹
CD25
⫹
or CD4
⫹
CD25
⫺
cells were in
-
jected to randomly selected 4-wk-old B7-1
⫺/⫺
B7-2
⫺/⫺
B7-2 Fc Tg
⫹
mice
once per week for 4 wk, and the mice were analyzed at ages 9 to 10 wk old.
FIGURE 1. Spontaneous colitis in B7-2 Fc Tg mice. A–C, Diffuse in-
flammation of the whole large intestine (A), splenomegaly (B), and en-
larged MLN (arrows in C) in tissues from a B7-2 Fc Tg
⫹
littermate (right)
compared with a B7-2 Fc Tg
⫺
mouse (left). D, Severe mucosal inflamma
-
tion in the large intestine (LI) of the B7-2 Fc Tg
⫹
mouse. E, A character
-
istic enlarged lymphoid aggregate in the large intestine of a B7-2 Fc Tg
⫹
mouse. F, CD19 staining of the organized lymphoid follicles in the large
intestine of the B7-2 Fc Tg
⫹
mouse. G, A mild inflammation in the small
intestine (SI) of the B7-2 Fc Tg
⫹
mouse.
5279The Journal of Immunology

In vitro CD4
⫹
T cell culture
To test a costimulatory function of soluble B7-2 Ig Fc, CD4
⫹
CD25
⫺
T
cells were isolated from B7-1
⫺/⫺
B7-2
⫺/⫺
spleens by negative selection
using the combination of biotinylated mAbs mentioned above and anti-
biotin microbeads (Miltenyi Biotec). CD4
⫹
CD25
⫺
cells selected from B7-
1
⫺/⫺
B7-2
⫺/⫺
mice were ⬎97% CD4
⫹
CD45RB
high
, containing ⬍0.05%
CD4
⫹
CD25
⫹
cells (data not shown). Before the culture, 96-well plates
were coated with 1.0
␮
g/ml anti-CD3 mAb diluted in PBS for 3– 4 h at
37°C. One ⫻ 10
5
CFSE-labeled T cells were stimulated in the anti-CD3
coated plate for 4 days in the presence or absence of 60
␮
g/ml soluble B7-2
Ig Fc or 0.5
␮
g/ml anti-CD28 (37.51, BD Biosciences). Proliferation and
IL-2 secretion in the culture supernatants were analyzed by flow cytometry
(CFSE dilution) and ELISA. In some experiments, to stimulate T cells with
immobilized B7-2 Ig Fc, 1.0
␮
g/ml anti-CD3 (145-2C11, BD Biosciences)
and 1.0
␮
g/ml anti-human IgG1 Fc (Jackson ImmunoResearch Laborato-
ries) were coated onto 96-well culture plates. Some wells were further
FIGURE 2. Systemic expansion and activation of CD11b
⫹
cells and CD19
⫹
cells in B7-2 Fc Tg mice. A, A representative surface staining for CD11b
together with CD19 (left panel) and F4/80 (right panel). Numbers show the percentage of the population gated as a square among total cells from each
organ. Cells were isolated from spleen, MLN, PLN, and large intestine of B7-2 Fc Tg
⫹
(left of each panel) and Tg
⫺
(right) littermates. B, Total cell numbers
in spleen (top left), CD11b
⫹
spleen cells (top right), total MLN cells (bottom left), and CD19
⫹
cells in MLN (bottom right). Bars represent the averages
from diseased B7-2 Fc Tg
⫹
(left, n ⫽ 7), disease-free B7-2 Fc Tg
⫹
(middle, n ⫽ 6), and B7-2 Fc Tg
⫺
littermates (right, n ⫽ 10). Statistical analyses were
performed using a t test. (ⴱ, p ⬍ 0.05; ns, not significant) (C) I-A
b
, B7-1, B7-2 and CD40 staining of splenic B cells (CD19
⫹
CD11b
⫺
) from diseased B7-2 Fc
Tg
⫹
(solid line) and disease-free B7-2 Fc Tg
⫺
(gray filled) mice. Stainings with isotype control for diseased B7-2 Fc Tg
⫹
were shown as broken lines. D, Increased
serum Ig in B7-2 Fc Tg mice (both diseased and disease-free). The concentrations of serum Ig subclasses as well as total Ig are shown. Each triangle and square
represents individual B7-2 Fc Tg
⫹
and Tg
⫺
mouse, respectively. The differences were significant (p ⬍ 0.05) in all the subclasses (shown as ⴱ).
5280 A NOVEL SPONTANEOUS MODEL OF COLITIS BASED ON TRANSGENIC B7

incubated with 10
␮
g/ml B7-2 Ig Fc for 60 min at room temperature before
adding T cells with fresh medium. In coculture, experiments with
CD4
⫹
CD25
⫹
Treg, for the preparation of APCs, T lmphocytes were
depleted from spleen cells using CD90.2 microbeads (Miltenyi Biotec).
Selected CD90.2
⫺
cells were gamma-irradiated before use. One ⫻ 10
5
CFSE-labeled naive T cells, isolated as mentioned above, and 2.5 ⫻ 10
4
FACS-sorted CD45.1
⫹
CD4
⫹
CD25
high
Treg were stimulated with 1.0
␮
g/ml anti-CD3 in the presence of 5 ⫻ 10
5
APC. The proliferation index
of the CD45.1
⫺
CD90.2
⫹
cells was calculated using FlowJo software. For
T cell restimulation experiments, OT-II OVA specific T cells were primed
for 3 days with 1
␮
M OVA peptide ISQAVHAAHAEINEAGR (Abgent)
in the presence of 5 ⫻ 10
5
irradiated CD90.2
⫺
spleen cells as APC. For
restimulation, primed T cells were restimulated with 0.1
␮
M OVA peptide
in the presence of spleen cells from B6 RAG1
⫺/⫺
mice as APC. On day 3
of the restimulation culture, cells were harvested and processed for intra-
cellular cytokine staining as mentioned above.
Statistical tests
An unpaired t test was used for all statistical comparisons.
Results
Soluble B7-2 Fc transgenic mice develop chronic colitis
Our previous work indicated that reduced costimulation could lead to
accelerated colitis induction in the context of the T cell transfer model
of colitis, in which the recipients are immune deficient. To determine
how the modulation of the amount of B7-mediated costimulation
might affect inflammation in the intestine and elsewhere in intact
mice, we generated and analyzed Tg mice that express a soluble B7-2
Ig Fc fusion protein under the control of a liver-specific promoter. The
constitutively produced soluble B7-2 Ig Fc should interact with its
counterreceptors, CD28 and CTLA-4, to modify CD28-mediated co-
stimulation and/or the B7 ligand-dependent regulatory function of
CTLA-4. B7-2 Ig Fc Tg mice matured normally without early lethal-
ity or severe systemic inflammation. Interestingly, however, some
B7-2 Ig Fc Tg mice exhibited symptoms of chronic inflammatory
bowel disease, such as diarrhea and rectal prolapse, starting between
the ages of 5 to 8 wk. Although the severity of the symptoms was
variable, many of the diseased B7-2 Ig Fc Tg mice survived long
term, carrying the symptoms of chronic colitis. Histopathological
analysis demonstrated severe diffuse inflammation throughout the
large intestine including the cecum of sick mice (Fig. 1A), as well as
a marked enlargement of the spleen (Fig. 1B) and the mesenteric
lymph nodes (MLN) (Fig. 1C). Microscopically, the colonic mucosa
was hypertrophic and severely damaged by an extensive infiltrate of
mononuclear cells and myeloid cells (Fig. 1D). In many cases, the
inflammation involved the whole mucosal layer and lamina propria,
which often contained enlarged lymphoid aggregates or follicles (Fig.
1E) that were composed mostly of B lymphocytes, as indicated by
staining for CD19 (Fig. 1F). The inflammation was restricted mostly
to the large intestine, although mild villous swelling was seen at the
terminal part of the ileum (Fig. 1G). Later in the chronic phase in
some mice, the inflammation progressed toward the submucosal
layer, however, no granulomatous changes, a typical finding in human
Crohn’s disease, were observed. Because the inflammation was con-
tinuous, diffuse, and restricted to the mucosa, the lesions bore a closer
resemblance to those typical of ulcerative colitis.
B cell and myeloid expansions in B7-2 Ig Fc Tg mice
Flow cytometric analyses revealed a marked expansion of CD19
⫹
B cells especially in the peripheral lymph nodes (PLN) and MLN
of the diseased B7-2 Fc Tg (average of 58% among CD45
⫹
cells)
FIGURE 3. Accumulation of activated CD4
⫹
T cells in B7-2 Fc Tg mice. A, Ratio of B cells (CD19
⫹
) to T cells (TCR
⫹
in the spleen, MLN, PLN, and
large intestine). Bars represent ratios in the diseased B7-2 Fc Tg
⫹
(filled) and Tg
⫺
(open) littermates (four mice each). ⴱ, Significant (p ⬍ 0.05) differences.
B, Accumulation of CD4
⫹
T cells in the large intestine of the B7-2 Fc Tg mice. CD4 and CD8 staining for gated TCR
␤
⫹
cells from 8- (left) and 14-wk-old
(right) diseased B7-2 Fc Tg
⫹
mice and disease-free Tg
⫺
littermates (left and right of each panel, respectively) (representative data of four mice analyzed
in each group). C, Representative CD43 (1B11) staining for the CD4
⫹
(left) and CD8
⫹
(right) TCR
␤
⫹
cells isolated from a diseased B7-2 Fc Tg
⫹
mouse
(bold lines) and a disease-free B7-2 Fc Tg
⫺
littermate (gray filled). Stainings for the cells isolated from spleen, MLN, PLN, and large intestine are shown.
Table I. Histological scores and disease penetrance of B7-2 Fc Tg
mice
a
Histological Scores
No Colitis Mild to Moderate Severe
Strain (0–1) (2–3) (4–5)
B7-2 Fc Tg
⫹/⫺
5/13 (39%) 3/13 (23%) 5/13 (39%)
B7-2 Fc Tg
⫹/⫺
TCR
␤
⫺/⫺
10/10 (100%) 0/10 (0%) 0/10 (0%)
B7-2 Fc Tg
⫹/⫺
Igh-6
⫺/⫺
8/12 (67%) 1/12 (8.3%) 3/12 (25%)
B7-2 Fc Tg
⫹/⫺
CD28
⫺/⫺
10/10 (100%) 0/10 (0%) 0/10 (0%)
a
Maximal score from distal, middle, and proximal part of large intestine was used.
5281The Journal of Immunology

(Fig. 2A). In contrast, the percentage of CD11b
⫹
cells was greatly
increased in the spleen. The CD11b
⫹
cells consisted of both F4/
80
⫹
macrophages and F4/80
⫺
and Gr-1
⫹
(data not shown) gran
-
ulocytes (right panel of Fig. 2A). The migration of both macro-
phages and granulocytes also was characteristic of the inflamed
large intestine. The absolute number of cells in spleen and MLN,
was significantly increased in the diseased B7-2 Fc Tg
⫹
mice,
especially for CD11b
⫹
cells. This increase was evident not only
compared with the B7-2 Fc Tg
⫺
littermates, but also to those B7-2
Fc Tg
⫹
mice that did not bear colitis (Fig. 2B), indicating that
FIGURE 4. Increased disease severity in B7
⫺
B7-2 Fc Tg mice. A, Histological scores of colitis in B7
⫹
(left, circles) and B7
⫺
(right, squares) B7-2 Fc
Tg
⫹
mice (filled) compared with Tg
⫺
littermates (open). (ⴱ, p ⬍ 0.05 between B7
⫹
and B7
⫺
B7-2 Fc Tg
⫹
). B, Number of CD11b
⫹
cells in the spleen
(left) and CD19
⫹
cells in the MLN (right) of diseased B7
⫹
B7-2 Fc Tg
⫹
mice (F), B7
⫺
B7-2 Fc Tg
⫹
mice (f) and B7
⫺
B7-2 Fc Tg
⫺
littermates (䡺).
C, Serum IgG1, IgG2a and IgM concentrations from the B7-2 Fc Tg
⫹
(filled) and B7-2 Fc Tg
⫺
(open) littermates that are B7
⫹
(circles) and B7
⫺
(squares).
ⴱ, Significant (p ⬍ 0.05) differences.
FIGURE 5. A reduced percentage of Treg contributes to the severe colitis in B7
⫺
B7-2 Fc Tg mice. A, Representative stainings for CD25
⫹
Foxp3
⫹
Treg
in the spleen (top) and MLN (bottom) from the B7
⫹
(left) and B7
⫺
(right) background B7-2 Fc Tg
⫹
(left in each) and B7-2 Fc Tg
⫺
(right in each) mice.
B, Percentages of Foxp3
⫹
cells averaged from four mice each from the B7
⫹
or B7
⫺
background, diseased B7-2 Fc Tg
⫹
or B7-2 Fc Tg
⫺
mice. ⴱ, Significant
(p ⬍ 0.05) differences. C, In vitro coculture experiment showing the suppression of T cell proliferation by Treg in the presence or absence of the B7-2 Ig
Fc. CFSE dilutions of responder CD4
⫹
CD45RB
high
cells (gated as CD45.1
⫺
) cocultured without Treg (gray area), with Treg (solid line), and with Treg
in the presence of B7-2 Ig Fc (dotted line) are shown. Numbers indicate the proliferation indexes. D, Injection of CD4
⫹
CD25
⫹
cells but not CD4
⫹
CD25
⫺
cells attenuated the severity of colitis in the B7
⫺
B7-2 Fc Tg mice. Averaged histological scores from three parts of the large intestines of B7
⫺
B7-2 Fc
Tg
⫹
mice injected with CD4
⫹
CD25
⫺
(n ⫽ 4) or CD4
⫹
CD25
⫹
(n ⫽ 4) cells. (ⴱ, p ⬍ 0.05).
5282 A NOVEL SPONTANEOUS MODEL OF COLITIS BASED ON TRANSGENIC B7

these cellular increases were secondary to the development of
inflammation. The expanded B cell population exhibited an ac-
tivated phenotype, including higher expression of MHC class II
A
b
molecules, B7-1, B7-2, and CD40, as shown for splenic
CD19
⫹
CD11b
⫺
cells (Fig. 2C). Serum Ig was significantly in
-
creased for all the subclasses analyzed (Fig. 2D). In accordance
with the histology indicating enlarged lymphoid aggregates and
follicles, flow cytometric analyses also demonstrated that B cells
accumulated in the large intestine (Fig. 2A). These data suggested
that modification of T cell-APC interactions by the soluble B7-2
Fc fusion protein altered systemic immunity causing spontaneous
expansions of B and myeloid cells in addition to an organ-based
inflammation in the large intestine.
Altered activation of CD4
⫹
T cells in B7-2 Ig Fc Tg mice
Because the B7-2 Ig Fc should bind to two receptors, CD28 and
CTLA-4, mostly expressed by T cells, we hypothesized that an
excess amount of soluble B7-2 Ig Fc in the transgenic mice would
modulate positive and/or negative costimulatory signals. Alter-
ation of the activation state of T cells likely would then elicit
dysregulated immune responses, including the expansions of B
cells and myeloid cells that we observed. Consistent with this idea,
despite a pronounced expansion of B cells in the lymphoid organs,
T lymphocytes were predominantly accumulated in the large in-
testine of the diseased B7-2 Fc Tg mice, as determined by the ratio
of CD19
⫹
to TCR
␤
⫹
lymphocytes among the gated CD45
⫹
cells
(Fig. 3A). Although the percentage of T cells was decreased in the
lymphoid organs, the absolute T cell number was not significantly
changed when increases in the total cell number were accounted
for (data not shown). The ratio of CD8
⫹
to CD4
⫹
T cells was
increased in the lymphoid organs of younger B7-2 Fc Tg mice, as
shown in representative data from an 8-wk-old diseased Tg mouse
(Fig. 3B, left panel). This increased CD8/CD4 ratio was not evi-
dent, however, when we analyzed older, chronically diseased, Tg
mice (Fig. 3B, right panel). Despite these dynamic changes in LN
T cell populations, increases in CD4
⫹
T cells in the large intestine
were consistently observed in the diseased Tg mice, regardless of
the age or phase of the disease. We speculate that the relative
increase of CD8
⫹
T cells compared with CD4
⫹
T cells in the
lymphoid organs at early stages may have been caused by an ab-
normal activation and enhanced migration of CD4
⫹
T cells to the
large intestine. I n accordance with this idea, CD4
⫹
T cells were
strongly activated in lymphoid organs and the large intestine in
the early phases of the disease, as indicated by staining for the
activation-induced 130kDa glycoform of CD43 (Fig. 3C).
These data suggested a critical role for activated and migrating
CD4
⫹
T cells in the induction of a spontaneous inflammation in
the large intestine.
Colitis induction in Tg mice requires CD28 costimulation
To confirm the idea that the B7-2 Ig Fc chimera induced a spon-
taneous colitis by altering the activation and regulation of T cells,
we bred the B7-2 Fc Tg mice onto the CD28
⫺/⫺
, TCR
␤
⫺/⫺
or B
cell deficient (Igh-6
⫺/⫺
) genetic backgrounds. We assessed colitis
by histological scoring when the mice were between 8 to 10 wk
old. As shown in Table I, none of the TCR
␤
⫺/⫺
B7-2 Fc Tg mice
exhibited any symptoms of chronic colitis, or any other inflam-
matory disease. Furthermore, the CD28
⫺/⫺
B7-2 Fc Tg mice also
did not develop colitis. These data clearly indicated that sponta-
neous colitis in the B7-2 Fc Tg mice is T cell mediated and re-
quires CD28 costimulation. B cells were not required, however, as
4of12Igh-6
⫺/⫺
B7-2 Fc Tg mice developed colitis, although the
disease penetrance was slightly lower than that of Igh-6 wild type
B7-2 Fc Tg mice. Despite a massive cell infiltration in the large
intestine, the enlarged lymphoid follicles and aggregates that were
seen in the Igh-6 wild type B7-2 Fc Tg mice were not seen in the
inflamed B7-2 Fc Tg mice that lack B lymphocytes (data not
shown).
Lack of endogenous B7 molecules exacerbates B7-2 Ig
Fc-induced colitis
We considered the possibility that additional costimulatory signals
potentially provided by the B7-2 Fc fusion protein might be driv-
ing super normal T cell activation in the Tg mice, leading to colitis.
If this were the case, then disease should be ameliorated when the
B7-2 Fc Tg mice were crossed to mice that lack endogenous ex-
pression of both B7-1 and B7-2 (hereafter B7
⫺
mice). Surpris
-
ingly, however, the B7
⫺
B7-2 Fc Tg mice developed a more severe
colitis, with a higher disease penetrance. As shown in Fig. 4A, all
of the eight B7
⫺
B7-2 Fc Tg mice analyzed developed very severe
colitis, as determined by higher histological scores and an ⬃2-fold
increase in CD11b
⫹
cells in the spleen and CD19
⫹
cells in the
MLN, when compared with diseased, B7 wild type B7-2 Fc Tg
mice (Fig. 4B). These data indicate that excessive CD28-mediated
costimulation is unlikely to be the main factor leading to colitis.
However, because colitis pathogenesis required CD28-mediated
costimulation (Table I), it is apparent that the B7-2 Fc protein must
be capable of providing at least some costimulation in vivo.
The amount of serum Ig exhibited a distinct pattern in the B7
⫺
B7-2 Fc Tg mice. IgM was significantly elevated in the B7
⫺
B7-2
Fc Tg mice (n ⫽ 9), even compared with diseased B7 wild type
B7-2 Fc Tg mice (n ⫽ 13) (average 377.7 compared with 214.9
␮
g/ml, p ⫽ 0.042). Serum IgG1 and IgG2a concentrations were
quite low (27.2 and 28.7
␮
g/ml, respectively) in B7
⫺
B7-2 Fc Tg
mice due to impaired costimulation, although they were still higher
than in healthy B7
⫺
mice lacking the B7-2 transgene (10.9 and 3.9
FIGURE 6. The soluble B7-2 Ig Fc accelerates colitis independently of
Treg. A, CD4
⫹
CD45RB
high
cells were injected to RAG1
⫺/⫺
(circles) or
B7-2 Fc Tg
⫹
RAG1
⫺/⫺
(squares) recipients. In a separate group of four
RAG1
⫺/⫺
recipients, the B7-2 Ig Fc (100
␮
g) was injected three times a
week for 2 wk (E). The average relative body weight of each group is
shown. B, Representative histology (⫻40 magnification) of the large in-
testine 6 days post transfer of CD4
⫹
CD45RB
high
cells into B7-2 Fc Tg
⫺
RAG1
⫺/⫺
(left) and B7-2 Fc Tg
⫹
RAG1
⫺/⫺
(right) recipients. Arrows rep
-
resent infiltration of mononuclear cells into the mucosa.
5283The Journal of Immunology

␮
g/ml, respectively) (Fig. 4C). These data indicate that the in vivo
positive effects of the B7-2 Fc fusion protein on CD28 mediated T
cell costimulation in the absence of endogenous B7 expression
were relatively weak, as they were not sufficient to restore B cell
class switching, although they could trigger T cell mediated colitis.
The B7-2 Ig Fc has a minimal affect on Treg function in vitro
One important role of B7 costimulation is to maintain the periph-
eral Treg population (20). As we expected from the data showing
a minimal effect on B cell class switching by the B7-2 Ig Fc, Treg
were severely reduced in diseased B7
⫺
B7-2 Fc Tg mice in spleen,
MLN and PLN (Fig. 5, A and B).
To determine whether the B7-2 Ig Fc inhibited the function of
Treg, we performed conventional cocultures of naive T lympho-
cytes and Treg. In this experiment, CFSE-labeled, sorted
CD4
⫹
CD45RB
high
cells were cultured with T cell depleted irra
-
diated spleen cells as APC, in the presence or absence of sorted,
CD45 congenic CD4
⫹
CD25
high
cells. The T cells were anti-CD3
stimulated, and the proliferation indexes were calculated from the
CFSE dilution (Fig. 5C). The data indicate that the addition of the
FIGURE 7. Weak agonistic and antagonistic properties of the B7-2 Ig Fc chimera. A and B, Soluble B7-2 Ig Fc. CFSE-labeled naive CD4
⫹
T cells were
stimulated for 4 days with 1.0
␮
g/ml plate-bound anti-CD3 mAb. A, CFSE dilutions in the presence (solid line) or absence (gray-filled) of 60
␮
g/ml soluble
B7-2 Ig Fc (top)or0.5
␮
g/ml soluble anti-CD28 mAb (bottom) are shown. B, IL-2 concentration in the supernatant was measured from cultures in Fig.
7A. C and D, Plate-bound B7-2 Ig Fc. CFSE-labeled naive CD4
⫹
T cells were stimulated with plate-bound anti-CD3 or anti-CD3 with B7-2 Ig Fc for 4
days. C, CFSE dilution in the presence (solid line) or absence (gray-filled) of immobilized B7-2 Ig Fc is shown. These data are representative of two
independent experiments. D, IL-2 concentration in the supernatant from a culture in the presence (⫻) or absence (●) of the immobilized B7-2 Ig Fc. E,
The B7-2 Ig Fc inhibits Ab binding to CTLA-4 but not to CD28. Pre-activated CD4
⫹
T cells (for CTLA-4) or naive CD4
⫹
T cells (for CD28) were
incubated with 100
␮
g/ml B7-2 Ig Fc (solid line) for 10 min before staining with PE-conjugated anti-CTLA-4 (top) or anti-CD28 (bottom) Ab. Cells
preincubated with 100
␮
g/ml unconjugated anti-CTLA-4 (for CTLA-4) and naive CD4
⫹
T cells isolated from CD28 deficient mice were used as negative
controls, respectively (shown as broken lines). CTLA-4 or CD28 expression without pre-incubation is shown by gray-filled area. F, OT-II OVA specific
CD4
⫹
T cells were primed and restimulated in the presence of 0.1
␮
M OVA. B7-2 Ig Fc (left), anti-CTLA-4 (middle), or neither of them (right) was added
in both primary and secondary cultures. Intracellular IFN-
␥
staining after a brief activation with PMA; ionomycin in the presence of Brefeldin A is shown.
Data for all experiments in the figure are representative of at least two experiments.
5284 A NOVEL SPONTANEOUS MODEL OF COLITIS BASED ON TRANSGENIC B7

B7-2 Ig Fc did not affect the in vitro suppressive function of Tregs
(proliferation indices with Treg ⫽ 2.48 and 2.41, with or without
the B7-2 Ig Fc, respectively, compared with 3.83 without Treg).
As shown above, the B7
⫺
B7-2 Fc Tg mice had a decreased
percentage of Treg compared with their B7 wild type B7-2 Fc Tg
counterparts, likely due to an amount of B7-mediated costimula-
tion that was not sufficient for Treg homeostasis. To determine
whether a reduction in Treg could be an important mechanism for
the enhanced colitis pathogenesis in B7
⫺
B7-2 Fc Tg mice, we
injected CD4
⫹
CD25
⫹
cells once a week for 4 wk, starting when
the mice were 4 wk old. Repeated injections of CD4
⫹
CD25
⫹
cells
were conducted, because an efficient expansion of Treg was not
expected in the B7
⫺
B7-2 Fc Tg mice. As shown in Fig. 5D,
injections of CD4
⫹
CD25
⫹
cells, but not CD4
⫹
CD25
⫺
cells, at
-
tenuated the disease severity to the levels in the B7 wild type B7-2
Fc Tg mice.
The B7-2 Ig Fc acts on naive CD4
⫹
T cells
To address the question if the B7-2 Ig Fc enhances CD4
⫹
T cell
mediated colitis induction independently of Treg, we tested the
effect of the B7-2 Ig Fc in the CD4
⫹
CD45RB
high
T cell transfer
model of colitis. In this model, transfer of naive, CD4
⫹
CD45RB
high
T cells in the absence of Treg induces chronic colitis
in immune deficient RAG1
⫺/⫺
mice by 4 to 8 wk post transfer.
Interestingly, injection of a soluble B7-2 Ig Fc fusion protein to the
recipients after CD4
⫹
CD45RB
high
T cell transfer dramatically ac
-
celerated colitis induction, assessed by weight loss (Fig. 6A) and
severe diarrhea. These data were confirmed by a similar acceler-
ation of disease induction in RAG1
⫺/⫺
recipients that express the
B7-2 Fc transgene (B7-2 Fc Tg
⫹
). In accordance with the T cell
dependence of spontaneous colitis in B7-2 Fc Tg mice, none of the
B7-2 Fc Tg
⫹
RAG1
⫺/⫺
developed colitis in the absence of donor
CD4
⫹
CD45RB
high
T cells (data not shown). Only 6 days after T
cell transfer, however, there was a massive migration of mononu-
clear and myeloid cells in the B7-2 Fc Tg
⫹
RAG1
⫺/⫺
recipients
(Fig. 6B), while only a few mononuclear cells were observed in the
control B7-2 Fc Tg
⫺
RAG1
⫺/⫺
recipients. These data indicated
that the B7-2 Ig Fc could accelerate colitis mediated by naive
CD4
⫹
T cells in the absence of the other arms of the adaptive
immune system, although these mice showed no signs of disease in
the absence of CD4
⫹
T cells.
CTLA-4 blockade by the B7-2 Ig Fc enhances T cell
differentiation
To further analyze the function of the B7-2 Ig Fc protein in T cell
activation, we stimulated naive CD4
⫹
T cells with plate-bound
anti-CD3 mAb in the presence or absence of the soluble B7-2 Ig
Fc. Unexpectedly, there was no effect of the soluble chimera on T
cell proliferation (Fig. 7A) and IL-2 production (Fig. 7B). A similar
experiment using OT-II OVA specific T cells cultured with OVA
peptide and APC also gave no costimulatory effect by the soluble
B7-2 Ig Fc protein. Furthermore, the soluble B7-2 Ig Fc did not
restore costimulation in the culture with B7
⫺
APC (data not
shown). These data indicate that the weak costimulatory activity of
the B7-2 Ig Fc is not sufficient to activate T cells in vitro during
priming when added in a soluble form. To confirm an interaction
between the B7-2 Ig Fc and CD28 causing costimulation, we im-
mobilized the B7-2 Ig Fc together with anti-CD3 mAb in tissue
culture wells. To have the same density of the anti-CD3 mAb in
the presence or absence of the B7-2 Ig Fc, we coated the plate first
FIGURE 8. IFN-
␥
dependent and independent colitis induction in the B7-2 Fc Tg mice. A, Cells were isolated from spleen, MLN and LI (as indicated)
from 8-wk-old diseased B7-2 Fc Tg
⫹
mice (top) or disease-free B7-2 Fc Tg
⫺
littermates. Intracellular stainings of CD4
⫹
TCR
␤
⫹
cells for IFN-
␥
/IL-17 (left)
and IL-4/IL-10 (right) after ex vivo restimulation are shown. Data are representative from eight diseased B7-2 Fc Tg
⫹
and four B7-2 Fc Tg
⫺
mice. B,
Averaged histological scores from B7
⫹
(top)orB7
⫺
(bottom) background IFN-
␥
⫺/⫺
B7-2 Fc Tg
⫹
mice. C, Representative H&E stainings from a middle
part of large intestine of B7
⫹
(left) and B7
⫺
(right) background IFN-
␥
⫺/⫺
B7-2 Fc Tg
⫹
mice. Scale bars showing 1 mm are placed at the left bottom corner
of each figure.
5285The Journal of Immunology

with anti-CD3 mAb together with an anti-human IgG1 Fc mAb to
capture the fusion protein. Some of the wells were further incu-
bated with the B7-2 Ig Fc before adding T cells. Although it was
not effective when soluble, when immobilized, the B7-2 Ig Fc
clearly enhanced T cell proliferation and IL-2 production (Fig. 7,
C and D).
We hypothesized that CTLA-4 blockade by the B7-2 Ig Fc
could be at least part of the mechanism for dysregulated CD4
⫹
T
cell responses and colitis. Preincubation of activated CD4
⫹
T cells
with a B7-2 Ig Fc completely inhibited surface staining with a
PE-labeled anti-CTLA-4 mAb, although it did not block staining
with an anti-CD28 mAb (Fig. 7E). These data indicated that the
B7-2 Ig Fc could bind to CTLA-4 and compete for the binding site
of this blocking mAb, but do not by themselves demonstrate func-
tional role for this interaction.
It has been reported that CTLA-4 induces anergy and inhibits T
cell differentiation. We therefore conducted a culture experiment
to test whether the soluble B7-2 Ig Fc could facilitate T cell dif-
ferentiation, although it has only limited effect on priming. After
priming with irradiated spleen cells, OT-II T cells were rested and
restimulated with total spleen cells from RAG1
⫺/⫺
mice for 3 days.
After a third stimulation with PMA and ionomycin for 4 h, T cells
that were cultured during priming and restimulation either with the
B7-2 Ig Fc or an anti-CTLA-4 blocking Ab were more capable of
producing IFN-
␥
detected by intracellular cytokine staining (Fig.
7F). These data demonstrated that the B7-2 Ig Fc efficiently bound
to CTLA-4 and caused a similar effect on T cell differentiation to
generate IFN-
␥
producing cells as a CTLA-4 blocking Ab.
The role of IFN-
␥
in colitis induction
Effector cytokines produced by differentiated T cells are important
for the induction of T cell mediated inflammatory diseases, includ-
ing colitis. Intracellular staining of the ex vivo restimulated
CD4
⫹
TCR
␤
⫹
cells for IFN-
␥
, IL-4, IL-17, IL-10 (Fig. 8
A), IL-5,
IL-13, TNF-
␣
, and IL-2 (data not shown) revealed that a signifi-
cantly higher percentage of CD4
⫹
TCR
␤
⫹
cells in the spleen,
MLN, and large intestine of the diseased B7-2 Fc Tg were capable
of producing IFN-
␥
, compared with those from the transgenic neg-
ative littermates. Increases in other proinflammatory cytokines
were less consistent, and there was no evidence for increased Th2
cytokine production. In accordance with these data, B7-2 Fc Tg
mice that were crossed onto IFN-
␥
deficient background (IFN-
␥
⫺/⫺
B7-2 Fc Tg) did not develop colitis. None of the 18 IFN-
␥
⫺/⫺
B7-2 Fc Tg exhibited any symptom of colitis, which was
confirmed by histological scores for colonic specimens (Fig. 8B,
top). Interestingly, however, IFN-
␥
⫺/⫺
B7-2 Fc Tg that also are
deficient for B7-1 and B7-2 developed severe colitis, as shown in
Fig. 8C. Interestingly, this colitis exhibited different histological
features represented by a vigorous accumulation of mononuclear
cells in the submucosal area as a prominent feature. These data
indicate that while IFN-
␥
is important for the induction of colonic
inflammation, this requirement is diminished if the host lacks the
elements needed for normal immune regulation, including Tregs.
Therefore endogenous B7 molecules are critical factors for the
maintenance of Treg and the integrity of immune tolerance.
Discussion
In this study, we characterized a new model for mouse colitis that
exhibited some symptoms similar to human IBD. The constitutive
production of a soluble mouse B7-2-human IgG1 Fc fusion protein
by liver cells induced chronic colitis and systemic lymphadenop-
athy. The inflammation observed was a diffuse colitis that involved
the entire large intestine, from the cecum to the rectum, but that
was mostly restricted to the large intestine. Histological analysis
demonstrated a very severe mucosal inflammation with crypt ab-
scesses and enlarged lymphoid follicles and aggregates. Although
these features are often observed in human ulcerative colitis, we
are not proposing that this model provides a completely faithful
representation of human ulcerative colitis.
In the B7-2 Fc Tg mice, there were indications of systemic
immune activation, including an increased number of myeloid
cells in the spleen, a marked expansion and activation of B cells in
the lymphoid organs, and elevated serum Ig. The increased B cell
activation was caused by abnormal T cell activation and differen-
tiation. Although B cell activation and Ig may have contributed to
disease severity, B lymphocytes were dispensable for colitis in-
duction. On the contrary, the absence of colitis in the TCR
␤
⫺/⫺
and CD28
⫺/⫺
B7-2 Fc Tg mice, and the effects of the B7-2 Fc in
the transfer model, clearly indicated that the colitis induction in the
B7-2 Fc Tg mice required CD4 T cell activation in the presence of
B7-CD28 costimulation. We cannot exclude the possibility that
other factors contribute to colitis pathogenesis, including interac-
tion of the Fc portion of the transgene with Fc receptors expressed
innate immune cells. Clearly, however, this cannot by itself cause
disease in the absence of CD4
⫹
T cell activation.
Because B7-2 binds to both CD28 and CTLA-4, which play
opposite but interdependent roles in T cell activation, we examined
three hypotheses to understand the mechanism for dysregulated
CD4
⫹
T cell activation, differentiation and eventually, inflamma
-
tion. The first hypothesis was that a B7-2 Ig Fc might give an
additional and excess costimulatory signal to T cells through
CD28, which might enhance T cell activation and survival. The
strongest argument against this is the finding of increased disease
severity and penetrance in B7
⫺
B7-2 Fc Tg mice. This led us to
consider whether the B7-2 Fc protein had any agonistic activity.
The presence of colitis in the B7
⫺
B7-2 Fc Tg mice, in contrast to
the absence of disease in the CD28
⫺/⫺
B7-2 Fc Tg mice, clearly
demonstrated an agonistic function of the B7-2 Ig Fc in vivo. Our
in vitro experiments also were consistent with an agonistic func-
tion, but only when the B7-2 Ig Fc was immobilized on APC. We
speculate that the fusion protein could associate with APC by a
relatively weak affinity between the human IgG1 Fc and mouse Fc
receptors. However, the costimulatory signal generated by the
B7-2 Ig Fc in vivo in the B7
⫺
B7-2 Fc Tg mice was not sufficient
even for the basal level functions of B7 costimulation, required for
maintaining B cell Ig class switching (25) and a normal population
of Treg (20). In summary, the evidence suggests that the fusion
protein has weak costimulatory activity, but the dissociation be-
tween the total intensity of CD28 costimulation and the disease
severity, comparing B7-2 Fc Tg mice on the B7
⫺
and B7
⫹
back
-
grounds, indicated that an excess of B7-CD28 costimulation was
not the main mechanism for the induction of colitis.
Because both B7-1 and B7-2 bind to CTLA-4 with a much
higher affinity than they do to CD28 (26, 27), it would be reason-
able to propose that the soluble B7-2 Ig Fc has a greater affect on
CTLA-4 function than on CD28 function. The B7-2 Ig Fc bound
to CTLA-4, as demonstrated by the inhibition of CTLA-4 Ab
staining. Therefore it is possible that the B7-2 Ig Fc acted primarily
by attenuating the function of Treg, by blocking constitutively ex-
pressed CTLA-4 (28, 29). There is abundant evidence that
CD4
⫹
Foxp3
⫹
Treg are essential for the maintenance of peripheral
tolerance. The role of CTLA-4 in the function of Treg is contro-
versial. In one study, it was reported that constitutively expressed
CTLA-4 is required for the function of Treg, because injections of
anti-CTLA-4 in the recipients accelerated colitis induction in the
CD4
⫹
CD45RB
high
transfer model, but only in the presence of
Treg (29). However, in a more recent study, Treg isolated from the
5286 A NOVEL SPONTANEOUS MODEL OF COLITIS BASED ON TRANSGENIC B7

CTLA-4 deficient mice successfully prevented colitis in the same
model (30).
Regardless of the role of CTLA-4 in Treg function, our
CD4
⫹
CD45RB
high
transfer experiment, either with coinjections of
the B7-2 Ig Fc, or in the B7-2 Fc Tg RAG1
⫺/⫺
recipients, clearly
demonstrated that the B7-2 Ig Fc can enhance colitis induction
even in the absence of Treg. It should be noted that disease de-
veloped in these recipients as early as 1 wk post transfer, well
before we have observed the generation of adaptive Treg, or
Foxp3
⫹
cells, from the naive CD4 T cell population (data not
shown). Furthermore, in the B7-2 Fc Tg B7
⫹
mice, the
CD4
⫹
Foxp3
⫹
Treg number was increased significantly, with an
equivalent level of Foxp3 expression, compared with wild-type
littermates. Because the expression level of Foxp3 is directly cor-
related with the regulatory function of Treg (19), it was expected
that the Treg in the B7-2 Fc Tg B7
⫹
mice would be functionally
normal. In accordance with this, the addition of B7-2 Ig Fc did not
affect the regulatory capacity of Treg in a coculture system. In
summary, these data led us to conclude that Treg are quantitatively
and qualitatively normal in the B7-2 Fc Tg B7
⫹
mice, and there
-
fore that impaired Treg function is not the main cause of colitis
induction.
The last hypothesis we examined was that the B7-2 Ig Fc might
enhance naive T cell differentiation, by affecting the T cell auton-
omous regulatory function of CTLA-4. A number of studies dem-
onstrated that the engagement of CTLA-4 molecule suppresses
IL-2 expression and also restricts T cell clonal expansion by lim-
iting cell cycle progression (13, 31–33), which forms a checkpoint
before T cells undergo differentiation into effector cells. The re-
sults from in vitro experiments demonstrated that the addition of
anti-CTLA-4 blocking mAb, and to a similar extent, the addition
of the B7-2 Ig Fc, significantly enhanced the induction of IFN-
␥
producing cells after secondary culture with RAG1
⫺/⫺
spleen
cells. These data, together with the data indicating that the soluble
B7-2 Ig Fc does not give excess costimulation to T cells, suggested
that the blocking of CLTA-4 by the B7-2 Ig Fc could be a principal
mechanism for colitis induction. In the context of colitis models,
the importance of the cell autonomous regulation of activated T
cells by CTLA-4 has recently been demonstrated in the CD4 T cell
transfer model (8). The importance of CTLA-4 expression in co-
litis prevention is further demonstrated by the findings in cancer
patients that developed intestinal inflammation following treat-
ment with CTLA-4 blocking Abs.
The physical form of the B7-2 ectodomain in solution might be
a key mechanism for explaining the blocking rather than agonistic
function of the fusion protein when interacting with CTLA-4. Both
B-1 and B7-2 are known to exhibit a complex dimer interface that
is critical for a stable ligand-receptor interaction and signaling
(34). However, in solution, the receptor binding domain of human
B7-2 is exclusively monomeric (35), although it still binds tightly
to monomeric and homodimeric CTLA-4. This is in contrast to
B7-1, which is in a rapid equilibrium between monomer and dimer
(36). We speculate that an excess amount of the soluble form of the
B7-2 Ig Fc in the Tg mice, with the B7-2 ectodomain essentially
in monomeric form at the ends of the Ig Fc arms, may inhibit the
binding between dimeric endogenous B7 molecules and CTLA-4,
which would block the inhibitory signal to activated T cells.
Recent studies demonstrated that the ligation of B7 molecules
by CTLA-4 could constitute a reverse signaling pathway by which
B7 molecules signal to APC to induce immune suppression
through the induction of IDO (37). IDO is a tryptophan-degrading
enzyme that could possibly suppresses T cell proliferation by caus-
ing a deprivation of the essential amino acid tryptophan. However,
IDO deficient mice do not exhibit an evident autoimmune pheno-
type, including no evidence for colitis (data not shown). Therefore,
while the reverse signaling mechanism could contribute to patho-
genesis, the impaired T cell autonomous regulation of proliferation
and differentiation is likely to be the major factor responsible for
colitis.
There are several explanations for the surprising finding that the
B7-2 Fc Tg mice deficient for endogenous B7 molecules exhibited
a much more severe disease phenotype. First, although we do not
have evidence indicating that the B7-2 Ig Fc affects the regulatory
function of Treg, the number of Treg is severely reduced compared
with Tg mice on the wild type background, probably because the
homeostasis of Treg is dependent upon the normal level of B7-
CD28 costimulation. Consistent with the importance of reduced
Treg number in the B7
⫺
B7-2 Fc Tg mice, injections of Treg could
diminish disease severity in these mice. Second, the level of
CTLA-4 on cells other than Treg should be lower in the B7
⫺
B7-2
Fc Tg mice, because the maximal level of CTLA-4 expression
requires full B7-CD28 costimulation (8, 38). Therefore, blocking
of the suppressive function of CTLA-4 by the B7-2 Ig Fc should
be more efficient in the B7
⫺
B7-2 Fc Tg mice. Lastly, as men
-
tioned above, ligation of endogenous B7 molecules leading to the
induction of IDO could also be a disease-limiting factor, although
by itself IDO deficiency does not cause colitis.
Although colitis in B7 wild-type B7-2 Fc Tg mice required
IFN-
␥
, disease that developed in the IFN-
␥
⫺/⫺
B7
⫺
B7-2 Fc Tg
mice indicated that the requirement for particular proinflammatory
cytokines critically depends upon the expression of costimulatory
molecules and regulatory elements in the host. In the absence of
IFN-
␥
in the B7
⫺
mice, despite severe inflammation and a mark
-
edly enlarged colon, mucosal mononuclear cell infiltration and hy-
perplasia were decreased. The comparative analysis of inflamma-
tion in the presence or absence of IFN-
␥
should allow us to dissect
the roles of IFN-
␥
in the cascade of events leading to chronic
inflammation in the intestine.
In conclusion, the chronic colitis spontaneously induced the
B7-2 Fc Tg mice provides a new and very useful model for dis-
secting the mechanisms of inflammatory disease induction in the
intestine. Advantages of this model are that disease occurs spon-
taneously in the absence of any genetic deficiency, but also, dis-
ease can be studied in an acute situation following T cell transfer.
In this model, disease penetrance and severity are determined by
the balance between progressive factors, such as proinflammatory
cytokines, and disease limiting factors, such as Treg. In the B7
wild-type B7-2 Fc Tg mice that exhibited an intermediate disease
phenotype and penetrance, endogenous elements controlled dis-
ease manifestation and severity. The lack of a progressive factor,
such as IFN-
␥
, abolished the disease. In contrast, the lack of en-
dogenous B7 molecules exaggerated the disease, perhaps due to a
deficiency in Treg. Although the significance of soluble B7 mol-
ecules in the context of normal physiology has not been deter-
mined, a soluble, truncated form of B7-2, capable of binding to
both CD28 and CTLA-4, can be detected in human serum (39).
Further studies of the amount and variability of these soluble mol-
ecules could help in understanding the pathogenesis of some forms
of IBD.
Disclosures
G. Kim, O. Turovskaya, M. Levin, and M. Kronenberg have no conflicts of
interest, while authors F. Byrne, J. Whoriskey, T. Horen, and J.
McCabe are employees of Amgen, Inc.
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5288 A NOVEL SPONTANEOUS MODEL OF COLITIS BASED ON TRANSGENIC B7