retinoic acid-induced gut tropism improves the protective capacity of treg in acute but not in...

Retinoic acid-induced gut tropism improves theprotective capacity of Treg in acute but not in chronic gutinflammation

Astrid Menning1, Christoph Loddenkemper2, Astrid M. Westendorf3,

Balint Szilagyi1, Jan Buer3, Christiane Siewert1, Alf Hamann1 and

Jochen Huehn1,4

1 Experimental Rheumatology, Charite University Medicine, Berlin, Germany2 Institute of Pathology, Charite University Medicine Berlin, Germany3 Institute of Medical Microbiology, University of Duisburg-Essen, Germany4 Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany

Treg are endowed with immunosuppressive activities and have been proposed as

promising targets for the therapy of autoimmune diseases. As the suppressive capacity of

Treg depends on their migration into the affected tissues, we tested here whether

modulation of Treg homing would enhance their capacity to suppress inflammation in

mouse models of inflammatory bowel disease. Retinoic acid (RA) was used to induce the

gut-specific homing receptor a4b7 efficiently and, to some extent, the chemokine receptor

CCR9 on in vitro expanded Treg. Upon transfer, RA-treated Treg were indeed more potent

suppressors in an acute, small intestinal inflammation model, compared with Treg

stimulated without RA. By contrast, the efficacy of Treg to resolve an established, chronic

inflammation of the colon in the transfer colitis model was not affected by RA-treatment.

In the latter model, a rapid loss of RA-induced a4b7 expression and de novo induction of

a4b7 on previously negative cells was observed on transferred Treg, which implies that Treg

acquire gut-seeking properties in vivo under inflammatory and/or lymphopenic condi-

tions. Together, our data show that the induction of appropriate homing properties prior to

transfer increases the protective potential of adoptively transferred Treg in acute, but not

in chronic, inflammatory disorders of the gut.

Key words: a4b7. CCR9 . Colitis . Foxp3 . Migration

Supporting Information available online

Introduction

A central dilemma of the immune system is to simultaneously

mount protective immune responses while maintaining tolerance to

‘‘self’’- or innocuous antigens. In particular, mucosal surfaces are

exposed to a vast variety of both pathogenic agents and environ-

mental antigens derived from air, food or commensal microorgan-

isms. To ensure unresponsiveness towards non-pathological agents,

a strict control over immune responses is necessary at these sites

and Treg have been shown to be operative in this process [1].

Dysregulated immune responses in the gut and a loss of

tolerance to intestinal microflora are thought to cause inflam-

matory bowel diseases (IBD) such as Crohn’s disease or ulcerative

colitis [1, 2]. Several animal models for IBD have highlighted theCorrespondence: Prof. Jochen Huehne-mail: [email protected]

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2010. 40: 2539–2548 DOI 10.1002/eji.200939938 Immunomodulation 2539

necessity of different Treg populations, such as thymus-derived

naturally occurring Treg (nTreg) [3], IL-10-producing Tr1 cells

[4] or TGF-b-induced Treg [5, 6] for preventing the development

of colitis. Importantly, recent studies have demonstrated that a

single transfer of nTreg into mice suffering from colitis is suffi-

cient to resolve an established inflammatory disorder [7, 8],

indicative of the curative potential of Treg.

In this context, active resolution of an established colitis is

supposed to take place in the intestinal lamina propria (LP)

[7, 8]. In general, Treg entry into the inflamed tissue is thought to

be indispensable for an efficient suppression in vivo [9–14]. For

instance, in mouse models for skin-inflammation, we have

previously shown that adoptively transferred Treg require the

expression of selectin ligands to migrate to the inflamed tissue

and to exert their suppressive function during the effector phase

of an inflammatory response [9]. Conversely, suppression of the

initiation of an immune response required the CCR7-dependent

localization of Treg in LN [15]. These findings imply that organ-

specific targeting of Treg by the modulation of their homing

profile might increase the in vivo suppressive efficacy of Treg

devised for therapeutic application.

Tissue-specific migration is achieved by specific expression

patterns of adhesion molecules [16]. The integrin a4b7 is the

main homing receptor mediating lymphocyte entry into the

intestine and associated lymphoid tissues [17, 18] based on the

interaction with musosal addressin cell adhesion molecule-1,

which is expressed by vascular endothelium of the gut. Further-

more, CCR9-expressing T cells are preferentially attracted to sites

of CCL25 production in parts of the small intestine [19–21];

however T-cell entry to this site does not ultimately depend on

CCR9 [22]. Expression of these gut-specific homing receptors is

supposed to be imprinted during naıve T-cell activation in MLN

and Peyer’s Patches (PP) [23] and the vitamin A metabolite

retinoic acid (RA), presumably derived from local DC [24, 25]

and stromal cells [26, 27], has lately been identified as a central

factor in this process [28]. Similar to conventional T cells, our

recent data demonstrated that RA potently induces high expres-

sion of a4b7 and low, but significant, levels of CCR9 on murine

naıve-like Treg and that those cells preferentially migrate into the

inflamed gut of mice suffering from colitis [29].

Based on these findings, it is tempting to speculate that the

transfer of in vitro expanded, RA-treated gut-specific Treg into

IBD patients could be an efficient approach to cure the disease.

Accordingly, we tested whether RA-induced gut-specific Treg

migration would increase the efficacy of transferred Treg to

suppress gut-specific inflammations in mice.

The results of this study show that pre-treatment of Treg with

RA leads to enhanced suppression of a rapid, acute inflammation

in the small intestine. However, the RA-treatment failed to

improve Treg-mediated resolution of a chronic colon-specific

inflammation. In the latter situation, instability of RA-induced

a4b7 expression on transferred Treg and a de novo induction of

gut homing receptors was observed, a process that was inde-

pendent of prior RA-conditioning. Our data suggest that the

therapeutic potential of Treg to cure chronic IBD is not increased

by the present techniques to modulate the migratory behavior of

in vitro expanded Treg, in part due to a significant flexibility in

the expression of homing receptors.

Results

RA-treatment of Treg enhances suppression of anacute small intestinal inflammation

In vitro expansion of ex vivo isolated Treg will be indispensable to

gain high Treg numbers for therapeutic applications. To test

options for optimizing the homing of such cells into the gut, we

stimulated murine ex vivo isolated CD62LhighCD251CD41 nTreg

in vitro with anti CD3/CD28 antibody either under neutral or

polarizing conditions with RA [29]. On average, in vitro

stimulation yielded fourfold expansion of Treg numbers (data

not shown). Importantly, expression of the Treg-specific trans-

cription factor Foxp3 was maintained to more than 90%

(Fig. 1A) resulting in equal in vitro suppressive activity of non-

polarized (neutral) and RA-treated Treg [29]. As shown in

Fig. 1A, RA-treated cells, but not control cells, expressed high

levels of a4b7, while only a small fraction expressed CCR9 upon

treatment with RA. P-selectin ligand (P-lig) and CD62L were

expressed at lower levels by RA-treated Treg than by neutral Treg

(Fig. 1A). In a preceding study, we have already demonstrated

that, in line with their phenotype, RA-treated Treg preferentially

home to the gut, PP and MLN compared with neutral Treg [29].

Based on these results, we tested the capacity of such cells to

suppress an intestinal inflammation in a model that is sensitive to

the transfer of Treg [30]. In this model, the inflammatory

response is initiated by transfer of HA-specific T-cell receptor

transgenic CD81 T cells from the CL4-strain into mice expressing

the HA protein under control of the villin-promotor selectively in

enterocytes of the small intestine (VILLIN-HA) [30]. In vitro

expanded, polyclonal Treg were co-transferred with naıve HA-

specific CD81 T cells into VILLIN-HA mice and 5 days later, small

intestinal tissue was analyzed by histology. In vitro expanded

Treg were found to be capable of reducing the inflammatory

score compared with mice receiving CD81 T cells alone and this

suppression was significantly enhanced by RA-treatment of Treg

prior to transfer (Fig. 1B). Treg-mediated suppression in the

small intestine was reflected by decreased inflammatory

cell infiltrates and reduced epithelial and mucosal damage

(Fig. 1C).

To verify that the reduced disease activity relies on a prefer-

ential migration of RA-treated Treg into the inflamed intestinal

tissue we performed in vivo homing assays with radioactively

labeled in vitro expanded Treg that were transferred into VILLIN-

HA mice suffering from an established intestinal inflammation. As

shown in Fig. 1D, migration of RA-treated Treg into small

intestines and PP was significantly enhanced when compared

with the migration of Treg cultured without RA. These experi-

ments demonstrate that RA-induced expression of gut-specific

homing receptors on Treg correlates with an increased capacity of

Eur. J. Immunol. 2010. 40: 2539–2548Astrid Menning et al.2540


Treg to enter the inflamed intestine and to suppress the

development of a local inflammation in this model of acute

disease.

RA-treated Treg are not superior suppressors inchronic colitis

However, in humans IBD rather occurs as a chronic, relapsing

disease. Therefore, we compared RA-treated and neutral Treg for

their capacity to cure an established colitis in the slower, but

chronic CD41 T-cell transfer model [7, 8].

Colitis was induced by transferring CD45RBhighCD41 naıve

T cells into RAG1�/� mice, followed by adoptive transfer of

in vitro expanded Treg into groups of mice with comparable

clinical signs of colitis (diarrhea, weight loss, hunching) about

3 wk later. The progression of colitis was monitored by measuring

the weight of recipients for additional 3 wk, and histology was

applied to assess the severity of inflammation in the colon about

6 wk after the initial naıve T-cell transfer (Fig. 2A). The weight

loss observed in mice, which had received naıve T cells alone, was

clearly reversed by the transfer of Treg; however, neutral and RA-

treated Treg did not differ in their therapeutic capacity

(Fig. 2B and Supporting Information Fig. 1). In line with the body

weight data, all mice treated with either neutral or RA-

treated Treg showed strongly reduced histological scores

compared with control mice receiving naıve T cells alone (Fig. 2C

and D). Colon samples taken on the day of Treg transfer

confirmed the ongoing inflammation at the start point of ther-

apeutic intervention. Thus, both neutral and RA-treated Treg

have the potential to almost completely resolve an established

inflammation.

To exclude that differences in the suppressive efficacy of

neutral versus RA-treated Treg were only detectable at levels of

suboptimal suppression, we also applied lower Treg numbers in

this experiment. Although suppression was reduced, histological

scores did not significantly differ between mice receiving neutral

or RA-treated Treg populations, even when 5�105 Treg (neutral

Treg 1.070.9; RA-treated Treg 1.370.8) or only 2� 105 Treg

(neutral Treg 1.671.0; RA-treated Treg 1.871.3) were trans-

ferred. Together, these data demonstrate that the induction of

gut-specific homing properties on Treg by RA-treatment in vitro

does not improve their ability to cure an established chronic

colitis.

ATregs Neutral

B

17% 0% 58% 12% 95%GMFI 151

Gut-specific Tregs RA

CCR9 CD62L P-selectin ligand Foxp3

C Dhealthy control HA-specific CD8

40

NeutralRA

T N l T RA

30

+ Tregs Neutral + Tregs RA

10

20p=0.1 p=0.02 p=0.03 p=0.1

* *

30 ***

20

15

25 *

hist

olog

ical

sco

re

0

10

5

HAWT HA-VillinWT

+ HA-specific CD8

+TregsNeutral

+TregsRA

% o

f rec

over

ed r

adio

activ

ity

0

98% 18% 29% 9% 95%GMFI 2533

Figure 1. RA-treatment of Treg enhances suppression of an acute small intestinal inflammation. (A) Ex vivo isolated CD62LhighCD251CD41 Tregwere stimulated with plate-bound anti-CD3/28-antibody and IL-2 with or without RA (100 nM). On day 5 or 6, cells were harvested and analyzed forhoming receptor – and Foxp3 expression by FACS. (B) VILLIN-HA mice received ex vivo isolated naıve CL4-transgenic CD81 T cells (2.5� 106) aloneor together with in vitro expanded Treg in a ratio of 1:1. After 5 days, the severity of disease was assessed by histological examination ofhematoxilin/eosin-stained small intestinal tissue samples (n 5 7–10, pooled data from two independent experiments). (C) Hematoxilin and eosinstainings of representative duodenum samples are shown (magnification � 10). (D) In vitro modulated Treg were radioactively labeled with 51Cr andinjected i.v. into VILLIN-HA mice 4 days after disease induction by transfer of CL4-transgenic CD81 T cells. Recipients were sacrificed 24 h later anddistribution of radioactivity was measured in indicated organs using a g-counter. Percentage of total recovered radioactivity is shown (n 5 6, pooleddata from two independent experiments). Graphs present mean1SD (�pr0.05, ��pr0.01, Student’s t-test). GMFI 5 Geometric mean of fluorescenceintensity.

Eur. J. Immunol. 2010. 40: 2539–2548 Immunomodulation 2541


Homing receptor expression by transferred Tregassimilates under inflammatory andlymphoproliferative conditions

In order to understand why RA-treated Treg did not show a

superior suppressive activity in the chronic disease model, we

analyzed expression of a4b7, CCR9 and Foxp3 by Thy1.11CD41

Treg re-isolated from recipient mice. At the occurrence of first

clinical signs of colitis, the recipient mice received in vitro

expanded neutral or RA-treated Thy1.11CD41 Treg (Fig. 3A)

labeled with the proliferation tracer dye far-red fluorescent cell

tracer dye (SE = succinimidyl ester). Five days later, cells from

spleen and MLN were analyzed for their homing receptor- and

Foxp3-profile as well as for proliferation. Surprisingly, both

neutral and RA-treated Treg expressed similar levels of a4b7 and

CCR9 (Fig. 3B and C). Compared with high a4b7 levels on RA-

treated Treg before transfer (Fig. 3A), expression was clearly

reduced on recovered Thy1.11CD41 T cells (Fig. 3B), implying

that RA-induced a4b7 expression rapidly vanishes in vivo and

approaches levels reached by neutrally cultured cells de novo

acquiring a4b7 expression in vivo. Density plots shown in Fig. 3E

suggest that de novo expression of a4b7 on previously negative

T cells is mainly observed in proliferating cells. Proliferation of

neutral and RA-treated Treg was similar at that time point as

indicated by similar DDAO-SE fluorescence intensity (Fig. 3E and

data not shown). Numbers of Thy1.11CD41 cells in the colonic

LP were not sufficient to allow analysis at this early time point

(Fig. 3C).

Similar expression levels of a4b7 and CCR9 were also detected

3 wk post Treg transfer on recovered neutral and RA-treated

Thy1.11CD41 T cells from spleen, MLN and the colonic LP

(Fig. 3C). Even in the colonic LP and in MLN, recovered Treg only

expressed intermediate levels of a4b7, while at the same time

substantial amounts of P-lig were detected, a homing receptor

that targets T cells into inflamed tissues (e.g. MLN: neutral Treg

45.674.4%; RA-treated Treg 42.972.8%).

In accordance with recently described positive effects of RA on

Foxp3 expression [31, 32], analysis of Foxp3 expression 3 wk post

Treg transfer revealed higher Foxp3 frequencies among

RA-treated Thy1.11CD41 T cells in spleen, MLN and the colonic

A

CB

hist

olog

ical

sco

re

0

1

2

3

4

5

kw 6kw 30d

i.v. transfer of Thy1.2+ naive T cells

i.v. transfer ofThy1.1+ Tregs

- histology of colon tissue- phenotype of Thy1.1+ Tregs

% o

f ini

tial w

eigh

t

80

90

100

110

120

naive T cells - + + + naive T cells - + + ++

first clinicalsigns of colitis

established colitisand weight loss in

control mice

healthyRAG1-/- mice

****

****

D

TregsNeutral RA

--- + + +

+ ++ + TregsNeutral RA

--- + + +

+ ++ +-+

~d40~d20

sllec T on sllec T evian6 wk

+ Tregs Neutral6 wk

+ Tregs RA6 wk

naive T cells 3 wk

Figure 2. RA-treated Treg are not superior suppressors in chronic colitis. (A) Design of the experiment: 3� 105 CD45RBhighCD41 FACS-sorted naıveT cells were transferred i.v. into RAG1�/� recipients. At first clinical signs of colitis (�d20), in vitro modulated Thy1.11 Treg were transferred intothese mice. (B) Disease severity was monitored by measuring body weight. (C) Recipients were sacrificed (�d40 5�d20 after Treg transfer) andparts of the colon were fixed, stained with hematoxilin/eosin and scored in a blinded fashion. (D) Hematoxilin and eosin stainings ofrepresentative colon samples are shown (magnification x10). Graphs present mean 1 SD of pooled data from three independent experiments(n 5 14–16, ��pr0.01, Student’s t-test).



LP compared with their neutrally cultured counterparts (Fig. 3C).

These differences were most evident in MLN (neutral Treg

54.577.1% versus RA-treated Treg 71.973.4%; p 5 0.04) and in

the LP (neutral Treg 39.272.7% versus RA, 50.874.9%;

p 5 0.05).

Nevertheless, in both groups Foxp3 expression in MLN and the

colonic LP was decreased compared with the spleen, suggesting

that Treg proliferate locally, which is apparently accompanied by a

loss of Foxp3 or an outgrowth of Foxp3- cells (Fig. 3C). Concerning

the overall recovery of transferred Thy1.11 cells, we observed

comparable numbers and percentages of both populations in MLN

and spleen, but significantly higher numbers of neutral Treg in the

colonic LP compared with RA-treated cells (total cell numbers:

neutral Treg 4.771.0� 104 versus RA-treated Treg 2.170.3� 104;

p 5 0.017; percentages: neutral Treg 5.971.1% versus RA-treated

Treg 2.870.5%; p 5 0.016) (Fig. 3D).

These experiments demonstrate that the present conditions of

RA treatment do not ensure long-lasting, high levels of a4b7

expression in transferred Treg under inflammatory and lympho-

penic conditions, which allow intense proliferation of the cells.

Instead, RA-treatment seems to prevent the outgrowth of non-

Treg cells in the transferred cell population, which leads to an

overall lower recovery of transferred cells but to an increased and

stabilized Foxp3 expression.

Maintenance of a4b7 expression on RA-treated Tregunder homeostatic conditions

To assess the possibility that the rapid change in homing receptor

expression is caused by the lymphoproliferative and inflamma-

tory environment in RAG�/� hosts that were reconstituted with

BA

ED

Neutral Neutral

Tregs before transfer recovered Tregs (d5) LPL MLN spleenC

1500

GMFI 490 GMFI 3040 GMFI 1241 GMFI 1026

GM

FI α

4β7

NeutralRA

NeutralRA

0

500

1000

n.d.

α4β7 α4β750

203040

CCR9 CCR9

% C

CR

9+

100

75

010

*p 0 05

*p=0.04

n.d.

Foxp3

92.6% 91.2% 92.5% 92.1%

Foxp3%

Fox

p3+

d5 d20 d5 d20 d5 d200

25

50

75 p=0.05

n.d.

Neutral RA

10

4

6

8*

p=0.016recovered Tregs (d5)

α 4β 7

DDAO SE

% T

hy1.

1+

0

2

0.6

0.8

*

DDAO-SE cell

num

ber

Thy

1.1+

x 1

06

0

0.2

0.4 *p=0.017

0.047 0.021

3 wk

2.0% 13.3% 18.7% 10.5%

RA RA

Figure 3. Homing receptor expression by neutral and RA-treated Treg assimilates under in vivo conditions. Experiments were performed asdescribed in Figure 1. (A) Prior to transfer, in vitro expanded Treg were FACS-analyzed for the expression of a4b7, CCR9 and Foxp3. (B) Five days postTreg transfer, cells were isolated from colon, spleen and MLN and were characterized by FACS. Representative histogram plots of recovered CD41

Thy1.11 cells from MLN are presented. (C) Graphs show the geometric mean of fluorescence intensity (GMFI) of a4b7 as well as frequencies of CCR9and Foxp3 of CD41Thy1.11 cells recovered from analyzed organs five days and 3 wk post Treg transfer. (D) Numbers and percentages of recoveredCD41Thy1.11 cells from indicated organs are presented. (E) Proliferation of DDAO-SE labeled Thy1.11CD41 cells from MLN five days after transfer isreflected by representative density plots. Shown is the mean7SD of pooled data from two (five days time point, n 5 7–8) or three (3 wk time point,n 5 14–16, �pr0.05, Student’s t-test) independent experiments. n.d., not detectable; LPL, lamina propria lymphocytes.



naıve CD41 T cells, we transferred in vitro expanded Thy1.11

Treg into fully immunocompetent, healthy C57Bl/6 mice. Five

days after transfer cells from lymphoid organs were analyzed

with respect to their homing receptor and Foxp3 expression. In

contrast to the adoptive transfer into mice suffering from colitis,

RA-treated Treg maintained a4b7 expression under these homeo-

static conditions (Fig. 4). Nevertheless, a minor fraction of RA-

treated Treg down-regulated a4b7 expression, and these cells

were particularly enriched in peripheral LN (pLN). Notably,

neutral Treg did not up-regulate a4b7 expression under these

conditions, suggesting that inflammatory stimuli and/or a

lymphopenic environment are required for this process. We also

noted that RA-treated Treg contained slightly but in pLN and PP

even significantly higher Foxp3 frequencies than neutral Treg

(Fig. 4B and data not shown), which underlines our previous

finding that RA could be helpful to improve the stability of Foxp3

in transferred Treg cells.

Accordingly, RA-treatment is sufficient to generate gut-seek-

ing Treg under non-inflammatory conditions and to stabilize

Foxp3 expression, at least within a short time period of 5 days.

Discussion

Until today, conventional therapies of IBD in humans mainly rely

on the application of immunosuppressive drugs being accom-

panied by a general, unspecific down-regulation of the whole

immune system [33]. Numerous studies in mice [3–8, 30] have

suggested that the transfer of autologous, in vitro expanded Treg

into IBD patients might be an interesting approach for the

treatment of IBD.

In order to improve this strategy, we attempted to achieve a

site-specific homing of Treg required for an efficient suppression

of a local inflammation [9, 15]. With respect to IBD, an induction

of mucosa-seeking behavior would be desirable. Stimulation of

CD41 T cells in the presence of RA has been shown to induce

expression of a4b7 and, to a lesser extent, CCR9 [28]. In contrast

to a4b7, which is indispensable for T-cell migration to the gut-

associated lymphoid tissue, CCR9 supports entry into the small

intestine, but is not essential in this process [22]. In our

preceding study, we have demonstrated that also Treg up-regu-

late gut-specific homing receptors in response to RA-treatment

and that these cells preferentially home to intestinal tissues in an

a4b7-dependent manner in the dextran sodium sulfate-induced

colitis model [29].

Our present data show that RA-treated Treg are indeed

superior suppressors of an acute and rapid CD81 T-cell-mediated

colitis compared with Treg stimulated under neutral conditions.

Since we also show that RA-treated Treg display enhanced

migratory preferences for the small intestine in this model, it can

be assumed that the stronger therapeutic potential of RA-treated

Treg, at least in part, results from the modulation of homing

properties by RA. By contrast, although in vitro expanded Treg

were able to efficiently cure a chronic inflammation in the

transfer colitis model, RA-induced gut-homing potential did not

increase the capacity of Treg to dampen the colonic inflammation

under these conditions.

The different suppressive capacities observed in both models

of intestinal inflammation are supposed to reflect the localization

of transferred neutral and RA-treated Treg in diseased mice,

although it cannot be excluded that they relate to difference in

the affected tissues (small intestine versus colon). In particular,

RA-induced CCR9 expression could selectively mediate the

retention of RA-treated Treg in the small intestine of diseased

VILLIN-HA mice, an effect that has no relevance for Treg accu-

mulation in the inflamed colons of RAG�/� mice since the CCR9

ligand CCL25 is not expressed in the colon [21]. It also has to be

considered that other homing receptors could contribute to

NeutralRA

** **

Neutral RA BA

*9%GMFI 164

63%GMFI 526

400

p=0.002 p=0.002100

80

MLN

MLNpLN

pLNMLN

p=0.029% 63%

500

100

200

300

GM

FIα

4β 7

20

40

60Fox

p3

pLN3%

GMFI 12040%

GMFI 329

0 0% F

oxp3

+ o

f Thy

1.1+

CD

4+

α4β

7

Figure 4. Maintencance of a4b7 expression on RA-treated Treg under homeostatic conditions. In vitro expanded Thy1.11 Treg were adoptivelytransferred into C57Bl/6 recipients. Five days later mice were sacrificed and homing receptor expression on cells isolated from indicated organswas analyzed by flow cytometry. (A) Representative FACS plots of recovered CD41Thy1.11 cells from MLN and pLN are presented. (B) Graphs showthe geometric mean of fluorescence intensity (GMFI) of a4b7 as well as frequencies of Foxp3 among CD41Thy1.11 cells recovered from indicatedorgans. Shown is the mean7SD of pooled data from two independent experiments (n 5 6, �pr0.05, ��pr0.01, Student’s t-test).



lymphocyte migration into inflamed colons, for instance, P-lig

[34] or, as is known for IgA-secreting plasma cells, CCR10 [35].

However, we did not detect differences in the expression of either

of these molecules in neutral or RA-treated Treg (Fig. 1A and

data not shown).

It seems rather unlikely that the different type of pathogenic

effector cell, CD81 versus CD41 T cells, plays a critical role for the

different effects of RA treatment on the suppressive capacity of

the Treg in the two models of intestinal inflammation used in the

present study, since Treg have repeatedly been reported to

control both CD81 as well as CD41 T-cell responses [36] and are

obviously protective in either model used here.

In contrast to our result that RA does not increase the

suppressive potential of Treg in the chronic T-cell transfer

colitis model, Mucida et al. recently have shown that TGF-b-

induced, in vitro generated Treg more efficiently prevented the

induction of colitis in this model when stimulated in the presence

of RA [31]. However, RA has been demonstrated to improve the

otherwise poor stability of TGF-b-induced Treg [31, 32],

or to counteract the inhibitory effect of certain memory T-cell-

derived cytokines on TGF-b-mediated Treg conversion [37].

Therefore, it cannot be excluded that the latter effects, rather

than RA-induced gut-homing receptor expression, were critical

under the reported conditions [31]. In line with the published

positive effects of RA on Foxp3 expression [31, 32], we found

slightly higher Foxp3 frequencies among RA-treated Treg recov-

ered after transfer, indicating that Foxp3-expression is influenced

by RA-signaling not only in TGF-b-induced Treg but also in

nTreg. Therefore, by preventing the outgrowth of Foxp3- cells,

RA-treatment resulted in an increased Foxp3 stability, but slightly

decreased total cell numbers of transferred Thy1.11 cells

(Fig. 3D).

Our central finding that RA-treatment did not increase the

capacity of transferred Treg to cure a chronic colitis was asso-

ciated with similar expression levels of a4b7, CCR9 (Fig. 3C) and

CD62L (data not shown) on neutral and RA-treated Treg isolated

from recipient mice 3 wk after transfer. Notably, compared with

high a4b7 levels on in vitro expanded cells, a4b7 expression was

clearly reduced on recovered RA-treated Treg that were analyzed

5 days and 3 wk post Treg transfer, implying that the high a4b7

expression on RA-treated Treg decreases in vivo. It has previously

been shown for CD81 T cells that a4b7 becomes partially down-

regulated upon entry into the small intestine, while expression of

aEb7, mediating the retention of cells in the intestinal epithelium

[38], increases simultaneously. This mechanism might hold true

for Treg in the colon, as well.

Apart from down-regulation of a4b7, we observed a quick de

novo induction of a4b7 on neutral Treg in vivo, which was

predominantly found for proliferating cells. Higher levels of a4b7

were also found to be associated with proliferation in RA-treated

Treg, suggesting that its expression is in general dynamically

regulated in the context of a stimulatory environment in vivo. As

a consequence of these two independent processes – loss of

unstable RA-induced expression and de novo induction in vivo –

the expression of a4b7 by neutral and RA-treated Treg rapidly

equalizes in vivo. Similarly, an assimilation of expression levels

was observed for the chemokine receptor CCR9.

In contrast to the observed instability of RA-induced mucosal

homing receptors under lymphopenic and inflammatory condi-

tions, a considerable fraction of RA-treated Treg maintained their

high a4b7 expression when transferred into healthy, immuno-

competent mice, while no up-regulation was observed on neutral

Treg. These findings suggest that under homeostatic conditions

a4b7 expression induced by RA-treatment in vitro is stable to a

certain extent. This result is consistent with further studies from

our group demonstrating stable a4b7 expression on effector/

memory CD41 T cells (Szilagyi et al, in preparation) and long-

term expression of P-lig on Th1 cells [39]. However, to which

extent the observed instability of RA-induced mucosal homing

receptors on transferred Treg in the present study is due to

lymphopenia-induced proliferation in RAG�/� hosts or to the

concomitant inflammatory milieu could not be addressed by the

use of the adoptive transfer colitis model and would require

additional investigations using inflammatory disease models in

lymphocompetent animals.

In conclusion, our findings are of significant importance for

therapeutic strategies since novel options to treat chronic

inflammatory and autoimmune diseases with reagents targeting

homing pathways (e.g. anti-a4-antibody natalizumab) have not

yet been carefully analyzed with respect to potential effects on

the localization of suppressive cell populations.

Materials and methods

Mice

BALB/c, C57BL/6, congenic Thy1.1 C57BL/6 and RAG1�/� mice

were bred at the BfR (Bundesinstitut fuer Risikobewertung,

Berlin, Germany) and were used at 6–12 wk of age. CL4-TCR

transgenic – and VILLIN-HA mice on BALB/c background have

been described elsewhere [30]. All animal experiments were

performed under specific pathogen-free conditions and in

accordance with institutional, state and federal guidelines.

Antibodies, staining and sorting reagents

The following antibodies were produced in our laboratory: anti

FcgR II/III (2.4G2), anti CD62L (Mel14), anti CD3 (145.2C11)

and anti CD28 (37.51). The recombinant P-selectin-human IgG

fusion proteins were kindly provided by D. Vestweber (Muenster,

Germany). Cy5-labeled anti human IgG antibodies were obtained

from Jackson Immuno Research. The following antibodies and

secondary reagents were purchased from BD PharMingen: anti

CD4 (RM4-5), anti CD25 (PC6.1), anti CD45RB (16A), anti

Thy1.1 (OX-7), anti Thy1.2 (53-2.1) anti a4b7 (DATK32) and

appropriate isotype controls. PE conjugated anti CCR9 (242503)

was obtained from R&D Systems. The FITC-, PE- and PECy5-anti-



mouse Foxp3 staining kit as well as Pacific Blue conjugated anti

CD4 (RM4-5) were purchased from eBioscience. All microbeads

were obtained from Miltenyi Biotec. The cell tracker dye DDAO-

SE (CellTraceTM

Far Red DDAO-SE) was obtained from Molecular

Probes.

Flow cytometry

Cytometry was performed using LSRII Flow Cytometer (BD

Biosciences) and FlowJo software. Intracellular Foxp3

staining was performed with the FITC-, PE- or PECy5- anti-

mouse Foxp3 staining set according to the manufacturer’s

instructions.

Cell Sorting

For in vitro expansion of naıve Treg, pooled erythrocyte-depleted

spleen and LN cells from BALB/c, C57Bl/6 or Thy1.1 congenic

C57Bl/6 mice were stained with anti CD62L FITC, anti CD25 APC

and anti CD4 PerCP. Labeled cells were enriched using anti APC

microbeads and the AutoMACS magnetic separation system

(Miltenyi Biotec). Subsequently, CD62LhighCD251CD41 T-cell

subsets were separated by FACS (Aria, BD Biosciences).

For T-cell reconstitution of RAG1�/� mice, lymphocytes from

C57Bl/6 or from Thy1.1-congenic C57Bl/6 mice were labeled

with anti CD4 FITC, anti CD25 APC and anti CD45RB PE and

enriched for CD4 using anti FITC microbeads and the AutoMACS

magnetic separation system. FACS (Aria or Diva, BD Biosciences)

was used to separate CD45RBhighCD25lowCD41 T cells.

All sorted subsets were at least 90% pure on re-analysis.

Foxp3 expression of all Treg populations was tested after sorting

and was consistently 495%.

In vitro expansion and homing receptor modulation ofTreg

Sorted Thy1.11 or Thy1.21 Treg were stimulated on anti CD3

(1–4 mg/mL) and anti CD28 (4–8 mg/mL, depending on the

antibody batch)-coated plates, in the presence of IL-2 (40 ng/mL,

R&D systems) either under neutral (no further supplements) or

under polarizing conditions with RA (100 nM, Sigma). On day 3

of the culture, Treg were removed from the stimulus and rested

for another 2–3 days before analysis of homing receptor

expression by FACS. Cell culture was done in RPMI 1640 (Gibco)

supplemented with 10% FCS (Sigma) [29].

T-cell reconstitution of RAG1�/�mice and cure of colitis

Colitis was induced in RAG1�/� mice by i.v. injection of 3�105

sorted Thy1.11 or Thy1.21 CD45RBhighCD25lowCD41 T cells.

Mice developed clinical signs of colitis between 3–4 wk post

transfer, which became manifest in weight loss, loose feces and

hunched movement.

Mice displaying clinical signs of colitis were matched into

groups according to similar disease activity and received 1�106,

0.5�106 or 0.2� 106 Thy1.11 Treg (either neutral or RA-trea-

ted) by adoptive transfer or no treatment. On the day of Treg

transfer, some animals were sacrificed to assess the severity of

colitis.

To study the stability of RA-induced a4b7 expression in vivo,

Treg were labeled with 2.5mM DDAO-SE (Molecular Probes) in

PBS for 20 s with subsequent washing of the cells in RPMI 1640

containing 10% FCS. Weight of mice was monitored twice a week

and mice showing 420% weight loss were sacrificed.

Treg transfer into immunocompetent mice

To assess the stability of a4b7 expression on cultivated Treg under

homeostatic conditions 3� 106–5�106 in vitro expanded Thy

1.11 Treg were adoptively transferred into healthy C57Bl/6

(Thy1.2) recipient mice. Five days later, mice were sacrificed and

homing receptor expression on cells isolated from spleen, pLN,

MLN and PP was analyzed by flow cytometry.

Examination of homing receptor and Foxp3 expressionon transferred Treg

All mice were sacrificed 3 wk (on average on day 20) or 5 days

after Treg transfer. Cells from spleen, MLN and the colonic LP

were stained with anti-CD4 Pacific Blue, anti Thy1.1 FITC or

PerCP, anti a4b7 PE or anti CCR9 PE, anti CD62L Cy5 or P-

selectin-human IgG with subsequent anti human IgG Cy5

staining. After surface staining, cells were fixed and stained with

anti Foxp3 PECy5 or anti Foxp3 FITC. As control, cells from all

samples were pooled and stained with appropriate isotype

control antibodies. As additional control, we included MLN

CD41 T cells from healthy C57Bl/6 mice to standardize the

sensitivity of our stainings.

Isolation of lamina propria lymphocytes from thecolon

Colons were cut longitudinally, flushed with PBS and agitated at

371C in RPMI 1640 supplemented with 5% FCS for 30 min.

Subsequently, intraepithelial lymphocytes were removed from

colons by shaking them twice for 20 s in cold PBS/2% FCS. Colons

were cut into pieces of 5 mm and stirred at 371C for 50 min in

RPMI/5% FCS containing Collagenase VIII (Sigma, 200U/mL) and

Collagenase D (Roche, 0.1 U/mL). Supernatants containing lamina

propria lymphocytes were washed with cold PBS/2% FCS. Cells

were resuspended in a 40% Percoll solution (Biochrom) and were

layered onto a 70% Percoll solution. Centrifugation was carried out

at RT for 30 min at 2000 rpm without brake. The interface



containing lamina propria lymphocytes was removed, washed in

PBS/2%FCS and used for FACS analysis.

Histological examination of colitis

For histology of reconstituted RAG1�/� mice, colon tissue

samples were fixed in 10% phosphate-buffered formalin. Paraf-

fin-embedded sections were stained with hematoxilin and eosin.

The degree of inflammation in the colon was graded in a blinded

fashion from 0 to 5 as previously described [4]. Briefly, a grade of

0 corresponds to no obvious signs of inflammation; grade 1

involves minimal scattered mucosal inflammatory cell infiltrates,

with or without minimal epithelial hyperplasia; grade 2

represents mild scattered to diffuse inflammatory cell infiltrates,

sometimes extending into the submucosa and associated with

erosions, with minimal to mild epithelial hyperplasia and

minimal to mild mucin depletion from goblet cells; grade 3 was

given when mild to moderate inflammatory cell infiltrates that

were sometimes transmural, often associated with ulceration,

with moderate epithelial hyperplasia and mucin depletion were

detected; grade 4 corresponds to marked inflammatory cell

infiltrates that were often transmural and associated with

ulceration, with marked epithelial hyperplasia and mucin

depletion; and grade 5 represents marked transmural inflamma-

tion with severe ulceration and loss of intestinal glands.

Antigen-specific acute small intestinal inflammationin VILLIN-HA mice

CL4-TCR transgenic CD81 T cells were isolated as described

elsewhere [30]. Briefly, splenocytes were depleted of non-naıve

CD81 T cells using the MACS CD8 T-cell isolation kit (Miltenyi).

2.5� 106 CL4-TCR transgenic CD81 T cells, alone or mixed with

neutral or RA-treated Treg from BALB/c mice in a ratio of 1:1, were

transferred i.v. into VILLIN-HA recipients. Mice were sacrificed 5

days later. Duodenum, jejunum and ileum were fixed separately in

10% phosphate-buffered formalin. Paraffin-embedded sections were

stained with hematoxilin and eosin and were histologically analyzed

in a blinded fashion as described elsewhere [30].

Homing of adoptively transferred Treg

In vitro expanded Treg were labeled with 51Cr (Sodium

Chromate, Perkin Elmer): 2�107 cells/mL in complete RPMI

medium, 20 mCi/mL for 1 h at 371C, followed by 1 h of incubation

at 371C in fresh medium and removal of dead cells on Nycodenz

(17.1% isotonic Nycodenz; Nyegaard). Labeled cells were

injected i.v. into VILLIN-HA mice 4 days after disease induction

by CL4-TCR transgenic CD81 T-cell transfer. Mice were sacrifized

24 h after transfer of labeled cells and the distribution of

radioactivity in different organs, serum and the remaining body

was measured in a g-counter (Wallac).

Statistical analysis

Data were presented as mean7SD. Significance was determined

with Student’s t test since values were found to be normally

distributed by the Kolmogorov Smirnov test. Differences were

considered statistically significant with pr0.05 and highly

significant with pr0.01.

Acknowledgements: The authors thank Katharina Raba and

Thoralf Kaiser for their excellent FACS sorting and Simone

Spieckermann for assistance in histology. This work was

supported by the Collaborative Research Center programs

SFB633, SFB650 and SFB621 of the German Research Foundation

(DFG).

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: IBD: inflammatory bowel disease � LP: lamina propria �nTreg: naturally occurring Treg � P-lig: P-selectin ligand � pLN:

peripheral lymph node � PP: Peyer’s Patch � RA: retinoic acid

Full correspondence: Prof. Jochen Huehn, Experimental Immunology,

Helmholtz Centre for Infection Research, Braunschweig, Germany

Fax: 149-30-53161813399

e-mail: [email protected]

Additional correspondence: Prof. Alf Hamann

e-mail: [email protected]

Received: 31/8/2009

Revised: 30/5/2010

Accepted: 29/6/2010

Accepted article online 9/7/2010



retinoic acid-induced gut tropism improves the protective capacity of treg in acute but not in...

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