Immunity, Vol. 21, 527–538, October, 2004, Copyright 2004 by Cell Press
Retinoic Acid Imprints Gut-Homing Specificity
on T Cells
and development of Th1 and Th2 (Iwata et al., 1992,
2003; Yang et al., 1993; Racke et al. 1995; Hoag et al.,
2002; Stephensen et al., 2002). RAs bind to two families
of nuclear receptors, the RA receptor (RAR) isotypes (?,
?, and ?) and the retinoid X receptor (RXR) isotypes (?,
?, and ?) (Kastner et al., 1995; Mangelsdorf and Evans,
1995). 9-cis-RA binds to both RAR and RXR, whereas
the major physiologic RA, all-trans-RA, binds to RAR
and may bind to RXR at high concentrations. These
receptors function as ligand-inducible transcription fac-
tors mainly as RXR/RAR heterodimers.
Since vitamin A is critical especially for the gut immu-
nity, we considered a possibility that vitamin A might
play a role in the specific homing of T cells to the gut.
Naive T cells can migrate into any secondary lymphoid
organs but usually cannot migrate into nonlymphoid tis-
sues before they are activated with antigen (Butcher et
al., 1999; von Andrian and Mackay, 2000; Masopust et
al., 2001). Antigen-experienced effector/memory T cells
migrate preferentially to tissues that are associated with
tered specific antigen (Guy-Grand et al., 1978; Kantele
ential homing of T cells to the gut, expression of the
gut-homing receptors, namely the integrin ?4?7 and the
chemokine receptor CCR9, is essential (Hamann et al.,
1994; Kantele et al., 1999; Svensson et al., 2002). The
?4?7 integrin ligand, mucosal addressin cell adhesion
molecule-1 (MAdCAM-1), mediates recruitment of T
cells into normal and inflamed intestinal endothelium
(Butcher et al., 1999). The CCR9 ligand TECK is ex-
pressed by epithelial cells in the small intestine, espe-
cially those in the crypt region most closely associated
with MAdCAM-1?vessels (Kunkel et al., 2000; Wurbel
on T cells by antigenic stimulation with dendritic cells
(DC) from Peyer’s patches (PP) or by stimulation with
DC from mesenteric lymph nodes (MLN) in the presence
of antigen and adjuvant or anti-CD3 antibody (Mora et
al., 2003; Johansson-Lindbom et al., 2003; Stagg et al.,
2002). However, the mechanism of the imprinting is un-
known. In this study, we found that physiological con-
centrations of RA, but not retinol or retinal, enhanced
the expression of the gut-homing receptors ?4?7 and
CCR9 in T cells upon activation and that RA imprints
T cells with the gut-homing specificity. In vitamin
A-deficient mice, ?4?7?memory/activated T cells were
markedly reduced in secondary lymphoid organs, and
T cells were depleted from the intestinal lamina propria
(LP), suggesting that the RA effect is physiological. RA
production by DCs from the gut-associated lymphoid
organs and binding of RA to RAR in T cells appeared
to be involved in the imprinting of T cells with the gut-
Makoto Iwata,1,* Asami Hirakiyama,1Yuko Eshima,1
Hiroyuki Kagechika,2Chieko Kato,1
and Si-Young Song1
1Mitsubishi Kagaku Institute of Life Sciences
11 Minamiooya, Machida-shi
2Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku
For a preferential homing of T cells to the gut, expres-
sion of the integrin ?4?7 and the chemokine receptor
CCR9 is essential and is induced by antigenic stimula-
tion with dendritic cells from the gut-associated lym-
metabolite, retinoic acid, enhances the expression of
them with the gut tropism. Dendritic cells from the
gut-associated lymphoid organs produced retinoic
acid from retinol. The enhanced ?4?7 expression on
T cells by antigenic stimulation with these dendritic
cells was suppressed by the retinal dehydrogenase
inhibitor citral and the retinoic acid receptor antago-
nist LE135. Accordingly, vitamin A deficiency caused
a reduction in ?4?7?memory/activated T cells in
lymphoid organs and a depletion of T cells from the
intestinal lamina propria. These findings revealed a
novel role for retinoic acid in the imprinting of gut-
homing specificity on T cells.
Persistent diarrhea with infectious diseases is a major
cause of the infant mortality in developing areas in the
world. It has been demonstrated that vitamin A supple-
mentation significantly reduces the mortality (Sommer
processes including vision, reproduction, epithelial cell
differentiation, bone development, and immunity (Kast-
ner et al., 1995; Napoli, 1999). Vitamin A deficiency
causes IFN-? overproduction and impaired antibody re-
sponses. There is a helper T cell imbalance with excess
Th1 and insufficient Th2 function (Cantorna et al., 1994).
The visual function of vitamin A depends on its metabo-
lite 11-cis-retinal, but most of the other vitamin A func-
tions, including immunological functions, depends on
retinoic acid (RA), principally all-trans-RA and 9-cis-RA
(Kastner et al., 1995). RA is formed mainly through the
intracellular oxidative metabolism of retinol via retinal
(Duester, 2000). RA directly and indirectly regulates
T cell activities including activation-induced apoptosis
Enhanced Expression of the Gut-Homing Receptor
?4?7 Integrin by RA
We first determined the effect of RA on the expression
of gut-homing receptors on T cells in the absence of
Figure 1. RA and an RAR Agonist Enhance the Expression of the Gut-Homing Receptor ?4?7 Integrin on CD4?T Cells upon Activation
Naive CD4?T cells were stimulated for 2 days with antibodies to CD3 and CD28 and expanded for 2 days under the unskewed condition (A
and B) or the Th1- or Th2-skewed condition (A). (A) The cells cultured with or without 10 nM all-trans-RA were analyzed for ?4?7, CD62L,
E-lig, and P-lig expression by flow cytometry. (B) Effects of various doses of all-trans-RA, all-trans-retinal, all-trans-retinol, 9-cis-RA, and
effects of 100 nM of the RAR? (and ?) agonist Am80 and the RXR pan-agonist HX600 on the expression of ?4?7 and E-lig. The results are
expressed as ?MFI ? SD in triplicate cultures. Effects of selected concentrations of retinoids were examined for the statistical significance
by performing separate experiments (n ? 5 per group). Asterisks indicate that the effect of retinoid at the indicated concentration is statistically
of at least three independent experiments.
other cell types in vitro. Naive CD4-expressing (CD4?)
tamination with antigen-experienced T cells. The naive
T cells were stimulated with plate bound antibodies to
CD3 and CD28 and were expanded with IL-2 (unskewed
condition). The addition of 10 nM all-trans-RA, which is
the major physiologic RA, enhanced the expression of
the gut-homing receptor ?4?7 integrin on these cells
and suppressed that of E-selectin ligands (E-lig) (Figure
1A). The effect of RA was significant when it was added
within 2 days from the start of the culture (data not
shown). RA was also effective under the skewed condi-
tions to induce Th1 or Th2 (“Th1 condition” or “Th2
and Th2 was low, but RA markedly enhanced it (Figure
on Th1 was suppressed by RA, and Th2 cells that were
induced with or without RA expressed low levels of E-lig
and P-lig (Figure 1A). The expression of CD62L (L-selec-
memory T cells to secondary lymphoid organs (Butcher
et al., 1999; Sallusto et al., 1999; Weninger et al., 2001),
was moderately suppressed. RA also enhanced ?4?7
Retinoic Acid Imprints Gut-Homing Specificity
1 ?M ovalbumin peptide P323-339 in 1 ml of the medium containing
10% FCS and 2.5 ?Ci of [3H]-all-trans-retinol. The suspension was
cultured for 18 hr, and the cells were washed three times with ice-
cold PBS. The cells were then lysed in 0.5 ml of Tris-HCl buffer (50
mM Tris-HCl, 25 mM NaCl, 2 mM EDTA, 1 mM DTT [pH 7.5]) con-
taining 100 ?M citral and mixed with 0.5 ml of ethanol containing 1
nmole of each unlabeled retinoid (all-trans-retinol, all-trans-retinal,
and all-trans-RA) andbutylated hydroxytoluene (50 ?g/ml).The mix-
ture was adjusted to pH 3.0 by adding 10 ?l of 1 M HCl. Retinoids
were extracted, dried, and dissolved in 40 ?l acetonitrile. 25 ?l of
the extract was fractionated with the HPLC system as described
above. A half of each fraction (1 ml) was added into a piece of
ReadyCap (Beckman,Fullerton, CA),dried, andits radioactivitywas
counted. Internal retinoid standards were detected by measuring
ultraviolet absorption at 350 nm.
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We wish to thank S. Kamijo and his coworkers for their help with
animal care and vitamin A-modified diet, Dr. K. Ishiguro for helpful
advice for HPLC analysis, and K. Hirose and Y. Ono for secretarial
assistance. We also thank Dr. H. Takayama for warm encour-
Received: April 8, 2004
Revised: June 30, 2004
Accepted: August 18, 2004
Published: October 19, 2004
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