The Journal of Immunology
Upregulated IL-7 Receptor a Expression on Colitogenic
Memory CD4+T Cells May Participate in the Development
and Persistence of Chronic Colitis
Tamako Shinohara,*,1Yasuhiro Nemoto,*,1Takanori Kanai,†Kaori Kameyama,*
Ryuichi Okamoto,* Kiichiro Tsuchiya,* Tetsuya Nakamura,* Teruji Totsuka,*
Koichi Ikuta,‡and Mamoru Watanabe*
We have previously demonstrated that IL-7 is essential for the persistence of colitis as a survival factor of colitogenic IL-7Ra–
expressing memory CD4+T cells. Because IL-7Ra is broadly expressed on various immune cells, it is possible that the persistence
of colitogenic CD4+T cells is affected by other IL-7Ra–expressing non-T cells. To test this hypothesis, we conducted two adoptive
transfer colitis experiments using IL-7Ra2/2CD4+CD252donor cells and IL-7Ra2/23 RAG-22/2recipient mice, respectively.
First, IL-7Ra expression on colitic lamina propria (LP) CD4+T cells was significantly higher than on normal LP CD4+T cells,
whereas expression on other colitic LP immune cells, (e.g., NK cells, macrophages, myeloid dendritic cells) was conversely lower
than that of paired LP cells in normal mice, resulting in predominantly higher expression of IL-7Ra on colitogenic LP CD4+cells,
which allows them to exclusively use IL-7. Furthermore, RAG-22/2mice transferred with IL-7Ra2/2CD4+CD252T cells did not
develop colitis, although LP CD4+T cells from mice transferred with IL-7Ra2/2CD4+CD252T cells were differentiated to CD4+
CD44highCD62L2effector-memory T cells. Finally, IL-7Ra2/23 RAG-22/2mice transferred with CD4+CD252T cells developed
colitis similar to RAG-22/2mice transferred with CD4+CD25–T cells. These results suggest that IL-7Ra expression on colitogenic
CD4+T cells, but not on other cells, is essential for the development of chronic colitis. Therefore, therapeutic approaches targeting
the IL-7/IL-7R signaling pathway in colitogenic CD4+T cells may be feasible for the treatment of inflammatory bowel dis-
eases. The Journal of Immunology, 2011, 186: 2623–2632.
to current understanding, IBD is caused by inappropriate re-
sponses of the activated immune system to intestinal commensal
bacteria in patients with a genetically susceptible background.
Above all, effector CD4+T cells including Th1, Th2, and Th17
are highlighted in the pathogenesis of IBD, because some groups
have reported the association between genes involved in the
Th17/IL-23 pathway and IBD (5, 6). Alternatively, we have
nflammatory bowel disease (IBD) is characterized by idio-
a persistent course with lifelong recurrence (1–4). According
investigated the possibility that long-lived memory CD4+T cells
are the main cause of the persistence of IBD and have proved
the importance of IL-7 for the maintenance system of memory
CD4+T cells in chronic colitis (7).
IL-7 is a stromal cell-derived cytokine that is secreted by fetal
liver cells, stromal cells in the bone marrow, and the thymus and
other epithelial cells, including intestinal goblet cells (8, 9). Re-
cently, IL-7 has emerged as a critical key cytokine involved in
controlling the survival of peripheral resting CD4+T cells, in-
cluding naive and memory cells, but not effector cells, and their
homeostatic turnover proliferation (8–15). The effect of IL-7 on
CD4+T cells is controlled by the expression of the specific
receptors for IL-7, the state of differentiation of the T cells, the
available concentration of IL-7, and whether there is concomitant
TCR signaling (16, 17).
In contrast to the role of IL-7 in naiveand memory CD4+T cells
in the resting state, the pathologic role of IL-7 in chronic immune-
mediated diseases, such as autoimmune diseases and IBD, re-
mains largely unclear. We have previously demonstrated that 1)
IL-7 is constitutively produced by intestinal epithelial cells, espe-
cially by goblet cells (18); 2) IL-7 transgenic mice developed chron-
ic colitis that mimicked histopathologic characteristics of human
IBD (19); 3) colonic lamina propria (LP) CD4+IL-7RahighT cells
in RAG-22/2mice in which colitis was induced by adoptive
transfer of CD4+CD45RBhighT cells have characteristics of col-
itogenic memory T cells (20); 4) the selective elimination of
CD4+IL-7RahighT cells by administrating toxin-conjugated anti–
IL-7Ra mAb completely ameliorated ongoing colitis in TCR-a–
deficient mice (21); and 5) IL-7 is essential for the persistence of
colitis by showing that IL-72/23 RAG-12/2mice transferred
with colitogenic LP CD4+T cells did not develop colitis (22).
*Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical
and Dental University, Tokyo 113-8519, Japan;†Division of Gastroenterology and
Hepatology, Department of Internal Medicine, Keio University School of Medicine,
Tokyo 160-8852, Japan; and‡Laboratory of Biological Protection, Department of
Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-
1T.S. and Y.N. contributed equally to this work.
Received for publication January 11, 2010. Accepted for publication December 14,
This work was supported in part by grants-in-aid for Scientific Research, Scientific
Research on Priority Areas, Exploratory Research and Creative Scientific Research
from the Japanese Ministry of Education, Culture, Sports, Science and Technology;
the Japanese Ministry of Health, Labor and Welfare; the Japan Medical Association;
the Foundation for Advancement of International Science; the Keio Medical Science
Foundation; and the Research Fund of Mitsukoshi Health and Welfare Foundation.
Address correspondence to Dr. Mamoru Watanabe, Department of Gastroenterology
and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,
Tokyo 113-8519, Japan. E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this article: IBD, inflammatory bowel disease; LP, lamina
propria; MFI, mean fluorescence intensity; SP, spleen; TEM, effector-memory T; Treg,
regulatory T cell; TSLP, thymic stromal lymphopoietin; WT, wild type.
We hypothesize that the dysregulated IL-7/IL-7Ra pathway is
critically involved in the pathogenesis of animal models of chronic
colitis and human IBD, although IL-7 seems to be strictly regu-
lated at a constant level as a homeostatic cytokine to maintain the
number of CD4+memory T cells in the body.
IL-7R consists of the a-chain (CD127) and the cytokine re-
ceptor g-chain (IL-2Rg; CD132), which is shared by the common
g-chain family cytokines (IL-2, IL-4, IL-9, IL-15, and IL-21) (14,
15). Because IL-7Ra is broadly expressed on CD4+T and NK
cells, macrophages, dendritic cells, fibroblasts, and epithelial cells
(14, 15), the persistence of colitogenic memory CD4+T cells may
be affected by those cells in the form of “IL-7 competition”. To
assess this possibility, we attempted to clarify the link between
the expression of IL-7Ra on various cells in the whole body in nor-
mal and colitic conditions and the pathogenesis of chronic colitis.
In this study, we prove that IL-7Ra expression on CD4+T cells,
but not on other cells (NK cells, granulocytes, macrophages, and
dendritic cells), is essential for the development of colitis by use
of an adoptive transfer colitis model using IL-7Ra2/2donor cells
and IL-7Ra2/23 RAG-22/2recipient mice.
Materials and Methods
Female C57BL/6 mice were purchased from Japan CLEA (Tokyo, Japan).
C57BL/6-background RAG-22/2mice were obtained from Taconic Farms
(Hudson, NY). C57BL/6-background IL-7Ra2/2mice have been de-
scribed previously (23). IL-7Ra2/2mice were intercrossed with RAG-22/2
mice to generate IL-7Ra2/23 RAG-22/2mice in the Animal Care Fa-
cility of Tokyo Medical and Dental University. Mice were maintained
under specific pathogen-free conditions in the Animal Care Facility of
Tokyo Medical and Dental University. Female donors and recipients were
used at 6-12 wk of age. All experiments were approved by the regional
animal study committees and were performed according to institutional
guidelines and home office regulations.
Purification of T cell subsets
CD4+T cells were isolated from spleen cells of IL-7Ra2/2or C57BL/6 mice
using the anti-CD4 (L3T4)-MACS system (Miltenyi Biotec, Auburn, CA)
then labeled with PE-conjugated anti-mouse CD4 (RM4-5; BD Pharmingen,
San Diego, CA) and FITC-conjugated anti-CD25 (7D4; BD Pharmingen).
Subpopulations of CD4+cells were generated by two-color sorting on
a FACSAria (Becton Dickinson). All populations were .97.0% pure on
reanalysis. To isolate LP CD4+T cells, the entire colon was opened longitu-
dinally, washed with PBS, and cut into small pieces. The dissected mucosa
was incubated with Ca2+, Mg2+-free HBSS containing 1 mM DTT (Sigma-
to Ficoll-Hypaque density gradient centrifugation (40%/75%). Enriched LP
CD4+T cells were obtained by positive selection using anti-CD4 (L3T4)
MACS magnetic beads. The resultant cells when analyzed by FACSCalibur
contained .95% CD4+cells.
In vivo experimental design
The role of IL-7Ra in the development and persistence of murine chronic
colitis was investigated through a series of in vivo experiments.
Experiment 1. To assess the necessity of IL-7Ra on donor CD4+cells in the
development of colitis, we performed cell transfer experiments using wild
type (WT) and IL-7Ra2/2mice as donors. RAG-22/2mice were injected
i.p. with 3 3 105splenic CD4+CD252T cells obtained from normal 8-wk-
old WTand IL-7Ra2/2mice. As a negative control, RAG-22/2mice were
transferred with CD4+CD252T cells (3 3 105) and CD4+CD25+regula-
tory T cells (Tregs; 1 3 105).
Experiment 2. To assess the necessity of IL-7Ra expression on cells of re-
(3 3 105) obtained from WT mice into RAG-22/2mice and IL-7Ra2/23
RAG-22/2mice as recipients. The recipient mice were weighed immediately
after transfer and then three times per week. They were also observed for
transfer for experiment 2 and assessed for a clinical score (24) that is the sum
offour parametersasfollows:hunchingandwasting,0or 1;colonthickening,
0–3 (0, no colon thickening; 1, mild thickening; 2, moderate thickening; 3,
extensive thickening); and stool consistency, 0–3 (0, normal beaded stool; 1,
soft stool; 2, diarrhea; 3, bloody stool) (24). To monitor the clinical signs
during the observation period, the disease activity index is defined as the sum
(0–5 points) of the parameters other than colon thickening.
Tissue samples were fixed in PBS containing 10% neutral-buffered for-
malin. Paraffin-embedded sections (5 mm) were stained with H&E. Two
tissue samples from the proximal and distal parts of the colon were pre-
pared. The sections were analyzed without prior knowledge of the type of
T cell reconstitution or treatment. The area most affected was graded by
the number and severity of lesions. The mean degree of inflammation in
the colon was calculated using a modification of a previously described
scoring system (25) as the sum of three parameters: crypt elongation, 0–3;
mononuclear cell infiltration, 0–3; and frequency of crypt abscesses.
To measure cytokine production, 1 3 105LP CD4+T cells were cultured in
200 ml of culture medium at 37˚C in a humidified atmosphere containing
5% CO2in 96-well plates (Costar, Cambridge, MA) precoated with 5 mg/
ml hamster anti-mouse CD3ε mAb (145-2C11; BD Pharmingen) and 2 mg/
ml hamster anti-mouse CD28 mAb (37.51; BD Pharmingen) in PBS
overnight at 4˚C (24). Culture supernatants were removed after 48 h and
assayed for cytokine production. Cytokine concentrations were determined
by specific ELISA per the manufacturer’s recommendation (R&D Sys-
tems, Minneapolis, MN).
To detect the surface expression of various molecules, isolated splenocytes or
32; 2.4G2; BD Pharmingen) for 20 min followed by incubation with specific
FITC-, PE-, PECy5-, or biotin-labeled Abs for 30 min on ice. The following
mAbs, other than biotin-conjugated anti-mouse IL-7Ra (A7R34; Immuno-
Biological Laboratories (Takasaki Japan), were obtained from BD Pharmin-
gen: anti-CD4 mAb (RM4-5), anti-CD25 mAb (7D4), anti-CD45RB mAb
(16A), anti-CD62L (MEL-14), anti-CD44 mAb (IM7), anti-CD69 mAb
(H1.2F3), and anti–Bcl-2 mAb (3F11). Biotinylated Abs were detected with
PE-streptavidin. Standard two- or three-color flow cytometric analyses were
obtained using the FACSCalibur with CellQuest software. Background fluo-
rescence was assessed by staining with control-irrelevant isotype-matched
mAbs. To analyze the TCRVb family repertoire, splenic cells were double-
stainedwith PE-conjugatedanti-CD4 mAb (RM4-5) and the following FITC-
conjugated mAbs: Vb2; KJ25, Vb3; KT4, Vb4; MR9-4, Vb5; RR4-7, Vb6;
RR3-15, Vb11; MR11-1, Vb12; IN12.3, Vb13; 14.2, Vb14; and KJ23,
Vb17. All Abs were purchased from BD Pharmingen.
For intracellular staining of cytokines, CD4+T cells were cultured for 12
h with ionomycin (500 ng/ml), PMA (50 ng/ml), and BD GolgiPlug (1 ml/
ml BD Pharmingen). After the stimulation, cells were collected and their
surface molecules were stained. Cells were fixed using Cytofix/Cytoperm
Kit (BD Pharmingen) and then stained with PE-conjugated anti–IL-17A
mAb (TC11-18H10; BD Pharmingen) or FITC-conjugated anti–IFN-g
mAb (XMG1.2; BD Pharmingen) for 20 min (26).
We examined the normality of each group. If either group was not normally
distributed, we assessed the difference between two groupsusing the Mann-
Whitney U test. If both groups were normally distributed, we assessed the
variance of population within each group using F test. With homosce-
dasticity of both populations, we assessed the difference between two
groups using the Student t test. Without homoscedasticity, we assessed the
difference using Welch’s t test. We used the program Statcell for all sta-
tistical analysis Differences were considered to be statistically significant
when p , 0.05.
IL-7Ra is expressed on various immune cells in WTand colitic
To first assess the role of the IL-7/IL-7R signaling pathway in
the development of chronic colitis, we analyzed the expression of
2624 COLITOGENIC IL-7Ra–EXPRESSING CD4+T CELLS
IL-7Ra on various immune compartments in colonic LP of normal
C57BL/6 mice (normal mice) and colitic C57BL/6-RAG-22/2
mice previously transferred with WT CD4+CD25-T cells (colitic
mice). First, both normal and colitic LP CD3+CD4+T cells highly
expressed IL-7Ra, but the mean fluorescence intensity (MFI) of
IL-7Ra expression on LP CD3+CD4+T cells from colitic micewas
significantly higher than in normal mice (Fig. 1A, 1B). Conversely,
the MFIs of IL-7Ra expression on colitic LP CD32NK1.1+NK
cells, CD11b+Grhighgranulocytes, CD11b+Grlow/2macrophages,
and CD11b+CD11c+myeloid dendritic cells were significantly
downregulated compared with those from normal mice (Fig. 1A,
1B). In addition, there were no differences in the expression of
IL-7Ra on CD32NKp46+NK22-like cells (27–29) and CD11b2
CD11c+lymphoid dendritic cells (Fig. 1A, 1B). These changes of
expression of IL-7Ra on CD3+CD4+T cells as compared with that
on other compartments (Fig. 1B), suggesting preferential use of
IL-7 by CD3+CD4+T cells in colitic conditions.
Naive CD4+T cells are retained in substantial numbers in
spleens of IL-7Ra2/2mice
Given the evidence that various immune compartments constitu-
tively express IL-7Ra, we next attempted to assess the role of IL-
7Ra expression in the development of chronic colitis induced
by adoptive transfer of CD4+CD252T cells obtained from age-
matched WT or IL-7Ra2/2mice into RAG-22/2mice. It was
particularly interesting that the expression level of IL-7Ra on
colitic LP CD3+CD4+T cells was significantly higher than that of
other compartments in colitic conditions (Fig. 1). Because it is
also known that IL-7/IL-7R signaling is critically involved in
T cell development in thymus and the periphery (9, 10), we first
assessed phenotypic characteristics of splenic CD4+T cells in age-
matched WT and IL-7Ra2/2mice before starting a series of
adoptive transfer experiments. Consistent with previous reports
(23, 30), the absolute cell number of CD3+CD4+T cells recovered
from spleen (SP) of IL-7Ra2/2mice was significantly lower than
that of WT mice (data not shown). Although the ratio of naive
(CD44low/2CD62L+) versus memory (CD44highCD62L2) T cells in
SP of IL-7Ra2/2mice was markedly decreased compared with that
of WT mice, a substantial number of naive CD4+T cells were
retained in SP of IL-7Ra2/2mice (Fig. 2A). In addition, we con-
firmed that SP CD4+T cells of IL-7Ra2/2mice did not express
IL-7Ra, and no differences in the expression of CD69, Foxp3, and
CD25 were found between two groups (Fig. 2A). Of note, Bcl-2
expression in SP CD4+T cells of IL-7Ra2/2mice was significant-
ly lower than that of WT mice (p , 0.05; Fig. 2A), which seemed to
be consistent with previous reports that IL-7 is essential for sur-
vival of CD4+T cells (24). It was also possible that CD4+CD252
donor T cells in SPs of IL-7Ra2/2mice retain restricted clonality
of CD4+T cells because of the dysregulated differentiation of CD4+
T cells in the thymus as compared with that in WT mice. To test this
possibility, we compared TCR Vb repertoires of SP CD4+CD252
T cells from age-matched IL-7Ra2/2and WT mice. Flow cyto-
metric analysis of these SP CD4+cells using a panel of 15 anti-Vb
mAbs showed that the major Vb population was Vb8.1/8.2 in both
groups, and the only significant difference in Vb repertoires be-
tween the groups was Vb8.3 (Fig. 2B).
RAG-22/2mice transferred with IL-7Ra2/2CD4+CD252
T cells did not develop mild colitis
To then assess the role of the IL-7R signaling pathway in the
development of chronic colitis, we used a chronic colitis model
induced by adoptive transfer of SP CD4+CD252T cells from IL-
7Ra2/2or control WT mice into RAG-22/2recipients (Fig. 3A).
obtained from colonic LP of normal and colitic mice. A, Dot plot analysis
shows the IL-7a expression on each fraction of immune cells from colonic
LP of normal and colitic mice. Numerical values on the dot plots and
histograms express the mean percentage of each fraction. B, The bar
graphs show the MFI of IL-7Ra on each immune compartment obtain-
ed from colonic LP of normal and colitic mice. The graph data are the
mean 6 SEM. *p , 0.05. N.S., not significant.
IL-7Ra expression on various immune compartments
from age-matched WT and IL-7Ra2/2mice. A, FACS analysis shows the
expression of CD44/CD62L, IL-7Ra, Foxp3, and Bcl-2 on/in splenic CD4+
T cells. The dotted line in the Bcl-2 histogram shows the baseline of isotype
control. B, Flow cytometric analysis of Vb families on the surface of the
were double-stained with PE-conjugated anti-CD4 mAb (RM4-5) and
a panel of 15 FITC-conjugated Vb mAbs. The percentagevalue of each Vb
is the frequency pooled from three independent experiments (n = 6). The
data are the mean 6 SEM. *p , 0.05. N.S., not significant.
The Journal of Immunology 2625
As a negative control, RAG-22/2mice were transferred with
a mixture of SP CD4+CD252T cells and CD4+CD25+Tregs
obtained from WT mice. As depicted in Fig. 3B, RAG-22/2mice
transferred with WT CD4+CD252T cells manifested progressive
weight loss from 4 wk after transfer (Fig. 3B). In contrast, RAG-
22/2mice transferred with IL-7Ra2/2CD4+CD252T cells as
well as RAG-22/2mice transferred with a mixture of CD4+
CD252T cells and CD4+CD25+Tregs appeared healthy and
showed a gradual increase of body weight (Fig. 3B). To check
the possibility that mice transferred with IL-7Ra2/2CD4+CD252
T cells develop colitis with delayed kinetics, we observed all
groups of mice until 11 wk after transfer. Eleven weeks after
transfer, RAG-22/2mice transferred with WT CD4+CD252
T cells, but not those transferred with IL-7Ra2/2CD4+CD252
T cells or WT CD4+CD252T cells and CD4+CD25+Tregs, had
enlarged colons with greatly thickened walls (Fig. 3C). The same
mice also showed the enlargement of SP and mesenteric lymph
nodes (Fig. 3C). The assessment of colitis by clinical scores
showed a clear difference between RAG-22/2mice transferred
with WT CD4+CD252T cells and the other two groups (Fig. 3D).
with glandular elongation and massive infiltration of mononuclear
cells in LP of RAG-22/2mice transferred with WT CD4+CD252
T cells (Fig. 3E, middle panels). In contrast, these inflammatory
changes were mostly abrogated, and only a few mononuclear cells
were observed in the LP of the colon from RAG-22/2mice
transferred with IL-7Ra2/2CD4+CD252T cells (Fig. 3E, left
panels) or with a mixture of SP CD4+CD252T cells and CD4+
CD25+Tregs (Fig. 3E, right panels). This difference was also
confirmed by the histologic scores of multiple colon sections:
5.35 6 0.40 in RAG-22/2mice transferred with WT CD4+CD252
T cells versus 1.65 6 0.57 in RAG-22/2mice transferred with IL-
7Ra2/2CD4+CD252T cells and 2.00 6 0.74 in RAG-22/2mice
transferred with a mixture of SP CD4+CD252T cells and CD4+
CD25+Tregs (p , 0.001; Fig. 3F). Further quantitative evaluation
of CD4+T cell infiltration was made by calculating the absolute
cell number of LP CD3+CD4+T cells recovered from the resected
bowels. Significantly fewer CD4+T cells were recovered from the
colonic tissue of RAG-22/2mice transferred with IL-7Ra2/2CD4+
CD252T cells or a mixture of SP CD4+CD252T cells and CD4+
CD25+Tregs as compared with colitic RAG-22/2mice transferred
with WT CD4+CD252T cells (Fig. 3G). We also examined the
cytokine production by LP CD4+T cells. LP CD4+T cells from
RAG-22/2mice transferred with IL-7Ra2/2CD4+CD252T cells
or a mixture of SP CD4+CD252T cells and CD4+CD25+Tregs
produced significantly lower amounts of IFN-g and TNF-a than did
colitic RAG-22/2mice transferred with WT CD4+CD252T cells
upon in vitro stimulation (Fig. 3H).
did not develop chronic colitis. A, RAG-22/2mice were transferred with
splenic CD4+CD252T cells obtained from age-matched WTor IL-7Ra2/2
mice (3 3 105cells per mouse). As a negative control, RAG-22/2mice
were transferred with splenic WT CD4+CD45RBhighT cells (3 3 105cells
per mouse) and CD4+CD25+Tregs (1 3 105cells per mouse). B, Change
in body weight over time is expressed as a percentage of the original
weight. Data are represented as the mean 6 SEM of nine mice in each
group. *p , 0.05, compared with colitic RAG-22/2mice transferred with
CD4+CD252T cells. C, Gross appearance of the colon, SP, and mesenteric
lymph nodes from RAG-22/2mice transferred with IL-7Ra2/2CD4+
CD252T cells (top), RAG-22/2mice transferred with WT CD4+CD252
T cells (middle), and RAG-12/2transferred with WT CD4+CD252T cells
and CD4+CD25+Tregs (bottom). D, Clinical scores were determined at 8
wk after the transfer as described in Materials and Methods. Data are
indicated as the mean 6 SEM of seven mice in each group. *p , 0.001. E,
RAG-22/2transferred with IL-7Ra2/2CD4+CD252T cells
Histologic examination of the colon from RAG-22/2mice transferred with
IL-7Ra2/2CD4+CD252T cells (left), RAG-22/2mice transferred with
WT CD4+CD252T cells (middle), and RAG-12/2transferred with WT
CD4+CD252T cells and CD4+CD25+Tregs (right) at 11 wk after the
transfer. Original magnification 340 (upper) and 3100 (lower). F, His-
tologic scoring at 11 wk after transfer. Data are indicated as the mean 6
SEM of seven mice in each group. *p , 0.05. G, LP CD3+CD4+T cells
were isolated at 11 wk after transfer, and the number was determined by
flow cytometry. Data are indicated as the mean 6 SEM of seven mice in
each group. *p , 0.05. H, Cytokine production by LP CD4+T cells. LP
CD4+T cells were isolated at 11 wk after transfer and stimulated with anti-
CD3 and anti-CD28 mAbs for 48 h. IFN-g and TNF-a concentrations in
culture supernatants were measured by ELISA. Data are indicated as the
mean 6 SD of seven mice in each group. *p , 0.05.
2626 COLITOGENIC IL-7Ra–EXPRESSING CD4+T CELLS
Importantly, further flow cytometric analysis revealed that al-
most all the SP and LP CD3+CD4+T cells isolated from all three
groups of mice at 11 wk after transfer were CD44highCD62L2
CD69+effector-memory T (TEM) cells (Supplemental Fig. 1),
indicating that the transferred CD4+CD252T cells could differ-
entiate to activated TEMcells regardless of the expression of IL-
7Ra or the presence or absence of Tregs. These results suggest
that the lack of IL-7Ra prevented the development of colitis
primarily by inhibiting the expansion or survival of colitogenic
CD4+TEMcells in the colon in accordance with the lower ex-
pression of Bcl-2 (Fig. 2A). We found that SP and LP CD4+
T cells isolated from all groups of mice at 11 wk after transfer did
not express IL-15Rb, which is a critical receptor for IL-15 sig-
naling, and thymic stromal lymphopoietin (TSLP) receptor, which
is critical for TSLP signaling via TSLPR/IL-7Ra complex
receptors (Supplemental Fig. 1), indicating that IL-15 and TSLP
may not be involved in this colitis model.
To further assess whether IL-7Ra2/2CD4+T cells are unable
to produce inflammatory cytokines intrinsically or as the result of
a secondary effect from disorder of cell proliferation or mainte-
nance, we performed the following experiments. First, we ac-
cessed ex vivo cytokine production of IL-7Ra2/2or WT SP
CD4+T cells under Th1 polarizing conditions (Supplemental Fig.
2A). As shown in Supplemental Fig. 2B, IL-7Ra2/2SP CD4+
T cells expressed lower levels of IFN-g than did WT SP CD4+
T cells under the Th1 polarizing ex vivo conditions. This finding
was confirmed by the statistical analysis (Supplemental Fig. 2C).
Next, we examined the ability of the IL-7Ra2/2CD4+T cells to
produce inflammatory cytokines under the same inflammatory
conditions as the WT CD4+T cells. For this purpose, the same
number (3 3 105cells per mouse) of Ly5.2+IL-7Ra2/2SP CD4+
CD252T cells and Ly5.1+WT SP CD4+CD252T cells were
cotransferred to RAG-22/2recipients (Fig. 4A). The percentage of
Ly5.2+-derived IL-7Ra2/2T cells in peripheral blood was grad-
ually decreased after transfer, while that of Ly5.1+-derived WT
T cells in peripheral blood was conversely increased, and the
difference was significant 2 wk after transfer (Fig. 4B). Six weeks
after transfer, all mice developed colitis (data not shown). Al-
though the recovered cell number of Ly5.2+SP or LP CD4+
T cells derived from IL-7Ra2/2donors at 6 wk after transfer was
not survive. A, To discern why IL-7Ra2/2CD4+CD252T cells could not induce colitis, we cotransferred the same number (3 3 105) of Ly5.2+IL-7Ra2/2
CD4+CD252T cells and Ly5.1+WT CD4+CD252T cells to Ly5.2+RAG-22/2mice, and we compared the cell number and ability to produce Th1/Th17
cytokines between transferred IL-7Ra2/2and WT cells. PBMCs were collected 1, 2, 3, and 4 wk after the transfer. All mice were sacrificed and analyzed 6
wk after the transfer. B, Percentage of Ly5.1+or Ly5.2+cells in peripheral blood CD3+CD4+cells at each time point were determined by flow cytometry. C,
Percentage of Ly5.1+or Ly5.2+cells in SP and LP CD3+CD4+cells 6 wk after the transfer. D, IFN-g and IL-17 expression in recovered LP CD4+T cells
from IL-7Ra2/2or WT donor mice. LP CD4+T cells were collected from RAG-22/2recipients 6 wk after the transfer; they were cultured with ionomycin,
PMA, and GolgiPlug for 12 h as mentioned in Materials and Methods. IFN-g and IL-17 expression of them were determined by flow cytometry using
intracellular staining methods. CD3+CD4+Ly5.1+cells were considered as CD4+T cells from WT donor mice, while CD3+CD4+Ly5.12cells were
considered as CD4+T cells from IL-7Ra2/2donor mice. Numerical values on the histograms express the mean percentage of each fraction. E, Percentage
of IFN-g+cells and IL-17+cells in LP CD3+CD4+T cells from IL-7Ra2/2or WT donor mice. Data are indicated as the mean 6 SEM of five mice in each
group. *p , 0.05.
IL-7Ra2/2CD4+CD252T cells cotransferred with WT CD4+CD252T cells to RAG-22/2mice could produce IFN-g and IL-17, but could
The Journal of Immunology 2627
significantly lower than that from Ly5.1+WT donors (Fig. 4C),
both WT and IL-7Ra2/2donor-derived CD4+T cells could sim-
ilarly express IFN-g and IL-17 in the colitic condition (Fig. 4D).
These results indicate that IL-7Ra2/2cells in the absence of the
neighboring WT cells cannot produce Th1 or Th17 cytokines as
a result of suppression of colitis through a disorder of proliferation
or maintenance, rather than intrinsically impaired ability.
As shown in Fig. 2A, the ratio of naive T cells in SP of IL-
7Ra2/2mice was significantly lower than that of WT mice.
Therefore, the possibility remains that this different ratio of naive-
memory phenotypes of transferred cells might influence the
strength of colitis. To rule out this possibility, we next performed
another transfer experiment using the same number of naive CD4+
T cells (3 3 105cells per mouse) as donor cells. RAG-22/2mice
were transferred with SP CD3+CD4+CD62L+CD442naive T cells
obtained from age-matched WT or IL-7Ra2/2mice (Fig. 5A). As
a negative control, RAG-22/2mice were transferred with SP WT
naive T cells and CD4+CD25+Tregs (Fig. 5A). As expected,
neither mice transferred with IL-7Ra2/2naive T cells nor mice
transferred with naive T cells and Tregs developed colitis as
assessed by gross appearance of the colon (Fig. 5B), clinical (Fig.
5C) and histologic scorings (Fig. 5D, 5E), and the absolute cell
number of LP CD3+CD4+T cells (Fig. 5F) in sharp contrast to the
diseased mice transferred with WT naive T cells, confirming that
IL-7Ra expression on CD4+T cells is essential for the devel-
opment of colitis, regardless of the different ratio of naive and
memory cells in SP of IL-7Ra2/2mice and WT mice. We further
performed an apoptosis assay using annexin V/PI staining in this
transferred with splenic CD3+CD4+CD62L+CD442T cells obtained from age-matched WT or IL-7Ra2/2mice (3 3 105cells per mouse). As a negative
control, RAG-22/2mice were transferred with splenic WT CD3+CD4+CD62L+CD442T cells (3 3 105cells per mouse) and CD4+CD25+Tregs (1 3 105
cells per mouse). B, Gross appearance of the colon, SP, and mesenteric lymph nodes from RAG-22/2mice transferred with IL-7Ra2/2CD3+CD4+CD62L+
CD442(top), RAG-22/2mice transferred with WT CD3+CD4+CD62L+CD442T cells (middle), and RAG-12/2transferred with WT CD3+CD4+CD62L+
CD442T cells and CD4+CD25+Tregs (bottom). C, Clinical scores were determined at 8 wk after the transfer as described in Materials and Methods. Data
are indicated as the mean 6 SEM of each group. *p , 0.05. D, Histologic examination of the colon from RAG-22/2mice transferred with IL-7Ra2/2
CD3+CD4+CD62L+CD442T cells (left), RAG-22/2mice transferred with WT CD3+CD4+CD62L+CD442T cells (middle), and RAG-12/2transferred
with WT CD3+CD4+CD62L+CD442T cells and CD4+CD25+Tregs (right) at 8 wk after the transfer. Original magnification 340 (upper) and 3100
(lower). E, Histologic scoring at 8 wk after transfer. Data are indicated as the mean 6 SEM of each group. *p , 0.05. F, LP CD3+CD4+T cells were isolated
at 8 wk after transfer, and the number was determined by flow cytometry. Data are indicated as the mean 6 SEM of each group. *p , 0.05. G, The expression
of propidium iodide (PI) and annexin V in SP CD4+T cells from RAG-22/2mice transferred with IL-7Ra2/2CD3+CD4+CD62L+CD442T cells, RAG-22/2
mice transferred with WT CD3+CD4+CD62L+CD442T cells, and RAG-22/2transferred with WT CD3+CD4+CD62L+CD442T cells and CD4+CD25+Tregs
at 8 wk after the transfer. H, The percentage of early apoptotic cells (annexin V+PI2) and late apoptotic cells (annexin V+PI2). I, Intracellular staining of
cytokines (IL-17/IFN-g) in the colonic LP CD4+T cells. Numerical values on the dot plots and histograms express the mean percentage of each fraction.
RAG-22/2transferred with IL-7Ra2/2CD3+CD4+CD62L+CD442T cells did not develop chronic colitis. A, RAG-22/2mice were
2628COLITOGENIC IL-7Ra–EXPRESSING CD4+T CELLS
setting. IL-7Ra2/2SP CD4+T cells underwent apoptosis more
frequently than WT SP CD4+T cells (Fig. 5G, 5H), which sup-
ports the hypothesis that expression of IL-7Ra on CD4+T cells
is important for their survival. Furthermore, the expression of
IL-17 and IFN-g in IL-7Ra2/2LP CD4+T cells was markedly de-
creased compared with that in WT LP CD4+T cells (Fig. 5I).
IL-7Ra2/23 RAG-22/2mice transferred with CD4+CD252
T cells developed colitis
To further assess the role of IL-7/IL-7R signaling in the devel-
opment of chronic colitis, we next focused on IL-7Ra expres-
sion on non-T cells, such as APCs and NK cells that reside in
RAG-22/2recipients, because it is possible that IL-7 is compet-
itively used by various IL-7Ra–expressing immune compart-
ments, and the competition may affect the development of chron-
ic colitis. To test this hypothesis, WT CD4+CD252T cells were
transferred into RAG-22/2or IL-7Ra2/23 RAG-22/2mice (Fig.
6A). As a negative control, a mixture of WT CD4+CD252T cells
and CD4+CD25+Tregs was transferred into RAG-22/2mice (Fig.
6A). When CD4+CD252T cells were transferred into the control
RAG-22/2mice, the recipients, as expected, rapidly developed
severe wasting disease associated with clinical signs of severe
colitis, in particular, weight loss, persistent diarrhea and occa-
sionally bloody stool and anal prolapses, in sharp contrast to
healthy RAG-22/2mice transferred with a mixture of CD4+
CD252T cells and CD4+CD25+Tregs (Fig. 6B). When CD4+
CD252T cells were transferred into the IL-7Ra2/23 RAG-22/2
mice, the recipients also developed severe wasting chronic colitis
(Fig. 6B). These RAG-22/2and IL-7Ra2/23 RAG-22/2mice
transferred with CD4+CD252T cells, but not RAG-22/2mice
transferred with a mixture of CD4+CD252T cells and CD4+
CD25+Tregs, had enlarged colons with significantly thickened
walls accompanied with enlarged SPs and mesenteric lymph
nodes 8 wk after transfer (Fig. 6C). Consistent with this finding,
clinical scores of RAG-22/2and IL-7Ra2/23 RAG-22/2mice
transferred with CD4+CD252T cells were significantly higher
than those of RAG-22/2mice transferred with a mixture of CD4+
CD252T cells and CD4+CD25+Tregs (Fig. 6D). No significant
difference in clinical scores was found between RAG-22/2and
IL-7Ra2/23 RAG-22/2mice transferred with CD4+CD252
T cells, although the score of IL-7Ra2/23 RAG-22/2mice
tended to be higher than that of RAG-22/2mice transferred with
CD4+CD252T cells (Fig. 6D).
Histologic examination showed that tissue sections from RAG-
22/2and IL-7Ra2/23 RAG-22/2mice transferred with CD4+
CD252T cells were characterized by inflammatory infiltrate,
epithelial hyperplasia, crypt cell damage, and goblet cell de-
pletion, in contrast to RAG-22/2mice transferred with a mixture
of CD4+CD252T cells and CD4+CD25+Tregs, which showed no
features of colitis (Fig. 6E). This difference was also confirmed by
histologic scoring of multiple colon sections (Fig. 6F). Consistent
with the histologic assessment, the numbers of LP CD4+T cells
recovered from RAG-22/2and IL-7Ra2/23 RAG-22/2mice
transferred with CD4+CD252T cells were similar to each other
but significantly higher than that from noncolitic RAG-22/2mice
transferred with a mixture of CD4+CD252T cells and CD4+
CD25+Tregs (Fig. 6G). Cytokine production by LP CD4+T cells
is depicted in Fig. 6H. LP CD4+T cells from RAG-22/2and IL-
7Ra2/23 RAG-22/2mice transferred with CD4+CD252T cells
produced significantly higher levels of IFN-g and TNF-a than did
those from the control mice transferred with a mixture of CD4+
CD252T cells and CD4+CD25+Tregs (Fig. 6H).
Flow cytometric analysis revealed that the LP CD4+T cells
isolated from all groups of mice at 8 wk after transfer were
CD44highCD62L2CD69+TEMcells (Supplemental Fig. 3A), indi-
cating that the transferred CD4+CD252T cells could differenti-
ate to activated TEMcells regardless of the presence or absence of
IL-7Ra on non-T cells in the RAG-22/2recipient mice. In-
tracellular analysis further showed that almost the same fraction
of LP CD4+T cells from both RAG-22/2and IL-7Ra2/23 RAG-
22/2mice transferred with CD4+CD252T cells had differentiated
to IFN-g–producing Th1 or IL-17–producing Th17 (Supplemental
Fig. 3B). In contrast, the expression of IFN-g in LP CD4+T cells
from RAG-22/2mice transferred with a mixture of CD4+CD25+
and CD4+CD252T cells was markedly reduced as compared with
the groups with colitis (Supplemental Fig. 3B).
To further clarify whether the lower number of CD4+CD252
T cells in the transfer experiment makes this difference significant,
RAG-22/2mice and IL-7Ra2/23 RAG-22/2mice were trans-
ferred with 3 3 105or 1 3 105WT SP CD4+CD252T cells. As
a negative control, RAG-22/2mice were transferred with splenic
WT CD4+CD252T cells (3 3 105cells per mouse) and CD4+
CD25+Tregs (3 3 105cells per mouse; Supplemental Fig. 4A).
However, no differences were found in clinical and histologic
colitis scores or the absolute number of LP CD3+CD4+T cells
between IL-7Ra2/2and WT transferred groups, irrespective of
lower or higher number of donor T cells (Supplemental Fig. 4B–
E). Although we also checked the expression of MHC class II on
CD11b2CD11c+classical dendritic cells and CD11b+CD11c+
myeloid dendritic cells in this experiment (Supplemental Fig. 4F),
no differences were detected between any groups. Diminished
expression of MHC class II on dendritic cells in RAG-22/2mice,
which is caused by elevated level of IL-7 with lymphopenia, may
recover after transferred CD4+T cells consume IL-7.
This study has demonstrated that the high expression of IL-7Ra
on colitic CD4+T cells, but not on non-T cells, is essential for the
development and persistence of colitis. This finding is supported
by the findings that 1) the MFI of IL-7Ra expression of LP CD4+
T cells is significantly higher than that of other non-CD4+T cells
in colitic conditions, 2) the MFI of IL-7Ra expression of colitic
LP CD4+T cells is significantly higher than that of normal LP
CD4+T cells, 3) RAG-22/2mice transferred with IL-7Ra2/2
CD4+CD252T cells do not develop colitis, and 4) IL-7Ra2/23
RAG-22/2mice transferred with WT CD4+CD252T cells de-
velop colitis similar to that in transferred IL-7Ra+/+3 RAG-22/2
mice. Collectively, IL-7Ra expression on colitic CD4+T, but not
on other cells, is essential for the development and persistence of
It was originally reported that IL-7Ra is highly expressed
on lymphocytes such as T cells (16). Consistent with this report,
we have previously reported that the IL-7/IL-7R signaling path-
way is critical for the maintenance of IL-7Rahighcolitogenic
CD4+memory T cells (18, 20). Furthermore, we showed that
treatment with neutralizing anti–IL-7Ra mAb ameliorated ongo-
ing chronic colitis (18). More recently, several reports have proved
the importance of the IL-7/IL-7R signal in nonlymphocytes.
Guimond et al. (31) have reported that IL-7Ra is expressed on
some types of dendritic cells, and that in the lymphopenic envi-
ronment the IL-7/IL-7R signal of dendritic cells leads to de-
pression of its MHC class II molecule, which results in the
suppression of the proliferation of CD4+T cells. Other recent
reports that IL-7Ra is broadly expressed on NK cells, dendritic
cells, and macrophages in normal conditions (16, 17), suggesting
the need for us to further investigate the importance of the IL-7/
IL-7R signaling pathway in non-T cells for the development and
persistence of chronic colitis. Although IL-7Ra expression on
The Journal of Immunology2629
some LP populations, such as NK cells, granulocytes, macro-
phages, and CD11b+CD11c+myeloid dendritic cells, in colitic
mice was significantly downregulated compared with that in
normal mice, the expression level of IL-7Ra on colitic CD4+
T cells was conversely high, with the result that colitogenic
memory CD4+T cells sustain the highest expression of IL-7Ra in
IL-7Ra2/2mice are originally lymphopenic, because of the
loss of IL-7/IL-7R signaling pathway in lymphocytes, which is
a critical factor for their development in the thymus and their
maintenance in the periphery. Comparison of the surface pheno-
types of SP CD4+T cells in IL-7Ra2/2and WT mice by flow
cytometric analysis revealed no significant differences in the ex-
pression of CD69, CD25, and Foxp3 (Fig. 2A). Manifestation of
an antiapoptosis molecule Bcl-2 of CD4+T cells from IL-7Ra2/2
mice was lower than that of CD4+T cells from WT mice, which
corresponds to the previous reports that the IL-7/IL-7R signal
maintains T cells, upregulating the antiapoptosis molecule. Nev-
ertheless, we detected a substantial number of CD44lowCD62L+
naive CD4+T cells resident in the SPs of IL-7Ra2/2mice, al-
though their relative number in IL-7Ra2/2mice was significant-
ly lower than that in WT mice. Because of the scarcity of naive
CD4+T cells in IL-7Ra2/2mice, it was possible that the failure
of some part of naive T cells to develop might occur in the thy-
mus, which would lead to the loss of some TCR repertoires
needed for the onset of colitis. Thus, we compared the TCR Vb
repertoires of SP CD4+T cells in IL-7Ra2/2mice to those in WT
mice. However, except in the ratio of Vb8.3, no evidence was
found of skewed development in TCR Vb repertoires between
age-matched IL-7Ra2/2and WT mice.
As expected, RAG22/2mice transferred with SP IL-7Ra2/2
CD4+CD252T cells did not develop colitis, in sharp contrast to
colitic RAG22/2mice transferred with WT CD4+CD252T cells.
Nevertheless, flow cytometric analysis revealed that SP and LP
CD4+T cells from RAG22/2mice transferred with IL-7Ra2/2
CD4+CD252T cells differentiated to CD44highCD62L2TEMcells
as well as those from colitic RAG22/2mice transferred with WT
CD4+CD252T cells. This result suggests that IL-7R deficiency in
CD4+T cells causes the disorder of cell proliferation or mainte-
nance rather than the impaired development of memory CD4+
T cells, in accordance with the downmodulated Bcl-2 expression
of IL-7Ra2/2CD4+T cells. As shown in Fig. 3H, production of
Th1 cytokines from recovered LP CD4+T cells of the IL-7Ra2/2
CD252group was significantly lower than that of the WT CD252
group. However, IL-7Ra2/2CD4+T cells could express Th1 and
Th17 cytokines to an extent similar to that in WT CD4+T cells in
the colitic condition (Fig. 4). Therefore, we conclude that disorder
of IL-7Ra2/2CD4+T cells to proliferate and survive is the main
mechanism underlying their inability to induce colitis, whereas
their reduced inflammatory cytokine production is a secondary
effect. Furthermore, we also analyzed other common g-receptor–
associated receptor IL-15Rb to determine whether it was upreg-
ulated to compensate for the lack of IL-7Ra. However, no dif-
T cells developed chronic colitis. A, RAG-22/2mice and IL-7Ra2/23
RAG-22/2mice were transferred with splenic WT CD4+CD252T cells
(3 3 105cells per mouse). As a negative control, RAG-22/2mice were
transferred with splenic WT CD4+CD252T cells (3 3 105cells per mouse)
and CD4+CD25+Tregs (1 3 105cells per mouse). B, Disease activity index
during 8 wk after transfer. *p , 0.05. C, Gross appearance of the colon, SP,
and mesenteric lymph nodes from IL-7Ra2/23 RAG-22/2mice trans-
ferred with CD4+CD252T cells (top), RAG-22/2mice transferred with
CD4+CD252T cells (middle), and RAG-22/2mice transferred with CD4+
CD252T cells and CD4+CD25+Tregs. D, Clinical scores were determined
at 8 wk after the transfer as described in Materials and Methods. Data are
indicated as the mean 6 SEM of seven mice in each group. *p , 0.001. E,
Histologic examination of the colon from IL-7Ra2/23 RAG-22/2mice
transferred with CD4+CD252T cells (left), RAG-22/2mice transferred
with CD4+CD252T cells (middle), and RAG-22/2mice transferred with
CD4+CD252T cells and CD4+CD25+Tregs (right). Original magnification
340 (upper) and 3100 (lower). F, Histologic scoring 8 wk after transfer.
IL-7Ra2/23 RAG-22/2transferred with WT CD4+CD252
Data are indicated as the mean 6 SEM of seven mice in each group. *p ,
0.05. G, LP CD3+CD4+T cells were isolated at 11 wk after transfer, and the
numberwasdetermined byflowcytometry. Data are indicatedasthe mean6
CD4+T cells. LP CD4+T cells were isolated at 11 wk after transfer and
stimulated with anti-CD3 and anti-CD28 mAbs for 48 h. IFN-g and TNF-a
concentrations in culture supernatants were measured by ELISA. Data are
indicated as the mean 6 SD of seven mice in each group. *p , 0.05.
2630COLITOGENIC IL-7Ra–EXPRESSING CD4+T CELLS
ference was found in the expression of IL-15Rb on SP or LP
CD4+T cells from each group. These results suggest that IL-7Ra
expression on colitogenic CD4+T cells is essential for the de-
velopment and persistence of colitis.
Next, we used IL-7Ra2/23 RAG22/2mice to access the
importance of the IL-7/IL-7Ra signaling pathway in non-T cells.
At the start of this project, we hypothesized that IL-7Ra2/23
RAG-22/2mice transferred with CD4+CD252T cells would
develop more severe colitis than the control transferred RAG-22/2
recipient mice by considering two points. First, we thought that
the availability of IL-7 for colitogenic CD4+T cells might in-
crease in IL-7Ra2/23 RAG22/2mice as a result of the loss of
IL-7 consumption by IL-7Ra–lacking non-T cells. Actually, IL-7
concentration in serum from IL-7Ra2/2mice is reported to be
higher than that from WT mice (31). Thus, it was possible that the
persistence of colitogenic memory CD4+T cells is affected by
those cells in the form of IL-7 competition. Second, we had to
consider the presence of newly identified RORgt+IL-22–pro-
ducing NK cells (so called NK-22 cells) (27–29, 32, 33) for the
development of chronic colitis, because it has been shown that
these NK-22 cells constitutively express IL-7Ra. Importantly, it
has been reported recently that IL-22 is protective in murine DSS-
induced colitis model using IL-222/23 RAG-22/2mice (33),
leading to speculation that these NK-22 cells reside in intestinal
LP of RAG-22/2mice and may be regulated by the IL-7/IL-7R
signaling pathway. Unexpectedly, we could not detect any sig-
nificant differences regarding the severity of colitis between
RAG-22/2and IL-7Ra2/23 RAG-22/2recipient mice. This find-
ing was also confirmed by the experiment using a smaller num-
ber of CD4+CD252T cells as donor cells. Instead, we found that
the expression of IL-7Ra on colitic LP CD4+T cells was sig-
nificantly higher than that on normal LP CD4+T cells (Fig. 1),
suggesting a mechanism for exclusive use of IL-7 by highly IL-
7Ra–expressing colitic CD4+T cells.
Previously, we showed that IL-7Ra expression on LP CD4+
T cells in CD4+CD45RBhighT cell-transferred RAG-22/2mice is
downmodulated at the early effector phase of colitogenic CD4+
T cell differentiation (1–2 wk after transfer) and is again upreg-
ulated at the memory phase when colitis is established (.4 wk
after transfer) (22). Thus, it is possible that the competition for
IL-7 between colitogenic CD4+T cells and other non-T cells
occurs during such an early phase of colitis development. Other-
wise, IL-7 competition between T cells versus non-T cells may
occur at more acute immune responses, such as acute bacterial
infections, which is mainly regulated by IL-7Ra–downmodulating
effector T cells (10).
Finally, it is important to discuss the therapeutic strategies for
the treatment of IBD. Because IL-7 is the most important cytokine
forthe maintenance of homeostasis ofall the resting memory CD4+
T cells, it seems to be unsafe to adopt the blockade of IL-7/IL7R
signaling pathway for the treatment of IBD. As shown in this
study, however, it should be emphasized that the highest expres-
sion of IL-7Ra is found in colitogenic memory LP CD4+T cells
as compared with non-CD4+T cell compartments and normal
CD4+T cells. In such a situation, it is possible that a neutralizing
or depleting anti–IL-7Ra mAb would preferentially target col-
itogenic memory CD4+T cells with the highest expression of IL-
7Ra. Consistent with this notion, a recent report has shown that
targeted depletion of pathogenic Th1 and Th17 cells, which ex-
press high levels of lymphotoxin-a, inhibits autoimmune diseases
(34). In addition, it may be necessary to develop a molecular
targeting therapy against the IL-7Ra molecule that is more spe-
cific for the target organ, rather than a systemic therapy, using
effective drug delivery to inflamed mucosa of IBD.
Collectively, we have shown that IL-7Ra expression on CD4+
T cells is essential for the development of colitis in this model.
This finding suggests that IL-7Ra on colitogenic memory LP
CD4+T cells is one of the important targets in IL-7/IL-7R signal
The authors have no financial conflicts of interest.
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2632COLITOGENIC IL-7Ra–EXPRESSING CD4+T CELLS