To Investigate the Necessity of STRA6 Upregulation in T Cells during T Cell Immune Responses
et al. (2013) To Investigate the Necessity of STRA6 Upregulation in T Cells during T Cell Immune
Responses. PLoS ONE 8(12): e82808. doi:10.1371/journal.pone.0082808
To Investigate the Necessity of STRA6 Upregulation in T Cells during T Cell Immune Responses
Rafik Terra 0
Xuehai Wang 0
Yan Hu 0
Tania Charpentier 0
Alain Lamarre 0
Ming Zhong 0
Hui Sun 0
Jianning Mao 0
Shijie Qi 0
Hongyu Luo 0
Jiangping Wu 0
George Kassiotis, MRC National Institute for Medical Research, United Kingdom
0 1 Laboratoire d'immunologie, Centre de recherche, Centre hospitalier de l'Universite de Montre al (CRCHUM) - Ho pital Notre-Dame , Montre al, Que bec , Canada , 2 Service de ne phrologie, Centre de recherche, Centre hospitalier de l'Universite de Montre al (CRCHUM) - H opital Notre-Dame , Montre al, Que bec , Canada , 3 Institut national de la recherche scientifique (INRS) - Institut Armand-Frappier , Laval, Que bec , Canada , 4 Department of Physiology, Jules Stein Eye Institute, David Geffen School of Medicine, University of California Los Angeles , Los Angeles, California , United States of America
Our earlier study revealed that STRA6 (stimulated by retinoic acid gene 6) was up-regulated within 3 h of TCR stimulation. STRA6 is the high-affinity receptor for plasma retinol-binding protein (RBP) and mediates cellular vitamin A uptake. We generated STRA6 knockout (KO) mice to assess whether such up-regulation was critical for T-cell activation, differentiation and function. STRA6 KO mice under vitamin A sufficient conditions were fertile without apparent anomalies upon visual inspection. The size, cellularity and lymphocyte subpopulations of STRA6 KO thymus and spleen were comparable to those of their wild type (WT) controls. KO and WT T cells were similar in terms of TCR-stimulated proliferation in vitro and homeostatic expansion in vivo. Naive KO CD4 cells differentiated in vitro into Th1, Th2, Th17 as well as regulatory T cells in an analogous manner as their WT counterparts. In vivo experiments revealed that anti-viral immune responses to lymphocytic choriomeningitis virus in KO mice were comparable to those of WT controls. We also demonstrated that STRA6 KO and WT mice had similar glucose tolerance. Total vitamin A levels are dramatically lower in the eyes of KO mice as compared to those of WT mice, but the levels in other organs were not significantly affected after STRA6 deletion under vitamin A sufficient conditions, indicating that the eye is the mouse organ most sensitive to the loss of STRA6. Our results demonstrate that 1) in vitamin A sufficiency, the deletion of STRA6 in T cells does no affect the T-cell immune responses sofar tested, including those depend on STAT5 signaling; 2) STRA6-independent vitamin A uptake compensated the lack of STRA6 in lymphoid organs under vitamin A sufficient conditions in mice; 3) STRA6 is critical for vitamin A uptake in the eyes even in vitamin A sufficiency.
Funding: This work was supported by grants from the Canadian Institutes of Health Research to J.W. (MOP69089 and MOP123389), H.L. (MOP97829) and A.L.
(MOP89797). It was also made possible by grants from the Heart and Stroke Foundation of Quebec, the Natural Sciences and Engineering Research Council of
Canada (203906-2012), Juvenile Diabetes Research Foundation (17-2013-440) and Fonds de Recherche Quebec-Sante/National Sciences Foundation of China to
J.W. (AG-6), and the Jean-Louis Levesque Foundation to J.W. and A.L. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
During T-cell immune responses, naive T cells are activated by
stimuli through TCR in the company of co-stimulation signals,
and undergo multiple rounds of proliferation before entering the
differentiation phase, after which they become effector T cells.
The expression of many molecules is modulated during activation
and differentiation stages, with some of them playing pivotal
regulatory roles, while others exert support and house-keeping
functions to cope with increased metabolic demands. We
undertook unbiased exploration with DNA microarray analysis
of molecules up- or down-regulated in T cells within the first 16 h
after stimulation by anti-CD3 with a view to identifying those that
are critical in the early T-cell activation stage. A group of
molecules with the highest levels of altered expression in activated
T cells was chosen, with resting T cells as reference, and verified
by Northern blotting analysis. STRA6 (stimulated by retinoic acid
gene 6) is among those that have been validated. We generated
STRA6 gene knockout (KO) mice to assess the significance of its
up-regulation in T-cell activation and, consequently, T-cell
At the outset of our investigation in 2004, no function was
ascribed to STRA6, a 74-kDa protein with multiple
transmembrane domains that was first identified in retinoic acid-stimulated
P19 embryonic carcinoma cells upon retinoic acid stimulation .
In 2007, Kawaguchi et al. used an unbiased technique to identify
STRA6 as a specific cell-surface receptor for plasma retinol
binding protein (RBP) and showed that STRA6 mediates cellular
vitamin A uptake from holo-RBP (RBP/vitamin A complex) in
bovine retinal pigment epithelium cells . STRA6-mediated
vitamin A uptake from holo-RBP is coupled to intracellular
proteins as confirmed by several independent studies , and its
mechanism in coupling to specific intracellular proteins has been
elucidated . Pasutto et al.  observed that mutations in
STRA6 correlated with many eye, heart, diaphragm and lung
malformations as well as mental retardation in Matthew-Wood
syndrome in humans, corroborating its reported roles in vitamin A
uptake by cells as vitamin A is vital in organogenesis. Recent
reports indicate that single nucleotide polymorphisms or mutations
in STRA6 gene are correlated with the congenital eye
malformations microphthalmia, anophthalmia and coloboma [7,8] as well
as Matthew-Wood syndrome . Genetic null mutation of
STRA6 in mice results in significant retinoid reduction in the
retinal pigment epithelium and neurosensory retina, diminished
visual responses and eye morphology, although the last-mentioned
defect is not as serious as in patients with STRA6 mutations .
There is a report suggesting that STRA6 is not only a vitamin A
transporter but can also function as a cytokine receptor. Upon
binding with holo-RBP, STRA6 is phosphorylated at tyrosine
residue 643, which, in turn, recruits and triggers JAK2 and
STAT5 activation .
The ascribed roles of STRA6 in vitamin A transport and the
STAT5 signalling pathway are certainly relevant to T-cell
activation and function. Retinoids are known to modulate Th1
(T helper 1), Th2, Th17 and reglulatory T (Treg) cell development
and function . At the molecular level, it has been
demonstrated that retinoic acid opens up the FoxP3 promoter
tertiary structure for activated FoxP3 transcription . RARa
can interact with STAT5a and b , which are critical molecules
in the signaling pathway of a key T activation cytokine IL-2 .
Vitamin A is absorbed from dietary nutrients. There are several
possible modes of vitamin A transport to cells in different organs.
Vitamin A in the diet can be transported to liver cells and other
cell types in the form of chylomicron-bound retinyl ester [21,22].
The liver is the primary storage site for vitamin A in the form of
all-trans-retinyl ester, which can be reverted to vitamin A . As
alluded to above, vitamin A associates with RBP in blood, and
such complexes can deliver vitamin A to cells via the RBP receptor
STRA6 . Transthyretin can associate with vitamin A-bound
RBP, and such coupling serve to prevent renal filtration of the
holo-RBP , Recently, Alapatt et al.  discovered a second
RBP receptor, a STRA6 homologue called RBPR2. RBPR2 is
expressed in the liver, intestines, fatty tissues, and spleen. Like
STRA6, RBPR2 is fully capable of binding to RBP and
transporting vitamin A into cells. As vitamin A is hydrophobic,
it should also be able to diffuse through cell membranes without
any specific receptors.
The relative contribution of STRA6 to vitamin A cellular
import in lymphoid organs has not been evaluated and is a
secondary goal of our study.
In this study, we demonstrated that STRA6 KO mice were vital
and fertile, manifesting no apparent anomalies in their lymphoid
organs and T cell-dependent immune responses under vitamin A
sufficient conditions. Intracellular vitamin A concentrations in
lymphoid organs, such as the thymus and spleen of the KO mice
were comparable to those of WT controls, although the vitamin A
content in cells from KO eyes was significantly lower that that
from the WT eyes. The implications of these data are discussed.
Materials and Methods
STRA6 mRNA in cells and tissues from KO, heterozygous and
WT mice was measured by RT-qPCR. Total RNA was extracted
with TRIzolH (Invitrogen, Carlsbad, CA, USA) and then
reversetranscribed with Superscript IITM reverse-transcriptase
(Invitrogen). The forward and reverse primers were 59-AGG CAT CTG
AGA ATG GAA GCC AGA-39 and 59-AGC AGA ACC AGG
AAC GAC AGT GAA-39, respectively. A 184-bp product was
detected with the following amplification program: 95uC615 min,
1 cycle; 94uC615 s, 55uC630 s, 72uC630 s, 35 cycles. b-actin
mRNA levels were measured as internal controls; the forward and
reverse primers were 59-TGGTACCACAGGCATTGTGAT-39
and 59-TGATGTCACGCACGATTTCC CT-39, respectively,
with the same amplification program as for STRA6 mRNA. The
data were expressed as ratios of STRA6 and b-actin signals.
Generation of STRA6 KO mice
A PCR fragment amplified with the STRA6 cDNA sequence
served as a probe to isolate genomic BAC DNA clone 7O8 from
the RPCI-22 129/sv mouse BAC genomic library. The targeting
vector was constructed by recombination  and routine cloning
methods using an 11-kb STRA6 genomic fragment from clone
7O8 as the starting meterial. A 2.7-kb MunI-XbaI genomic
fragment containing exon 2 was replaced by a 1.1-kb Neo cassette
from pMC1Neo-Poly A flanked by 2 diagnostic restriction sites,
XbaI and ScaI, as illustrated in Figure 1A. The final targeting
fragment was excised from its cloning vector backbone by Not I
digestion and electroporated into R1 embryonic stem (ES) cells for
G418 selection . The targeted ES cell clones were injected into
C57BL/6 blastocysts. Chimeric male mice were mated with
C57BL/6 females to establish mutated STRA6 allele germline
Southern blotting with probes corresponding to the 59 and 39
sequences outside the targeting region, as illustrated in Figure 1A
(red squares), were used to screen for gene-targeted ES cells and
eventually to confirm gene deletion in mouse tail DNA. With the
59 probe, the targeted allele should present a 7.8-kb XbaI band,
and the WT allele, a 5.2-kb XbaI band. With the 39 probe, the
targeted allele should present a 6.9-kb ScaI band, and the WT
allele, a 4.3-kb ScaI band (Fig. 1A).
PCR was adopted for routine genotyping of the targeted
allele(s). The following PCR conditions were applied: 4 min at
94uC, followed by 35 cycles of 30 s at 94uC, 30 s at 60uC, and 30 s
at 72uC, with final incubation at 72uC for 10 min. The KO
forward primer 59- GCG TCA CCT TAA TAT GCG AAG
TG39 and reverse primer 59-CAA GAA GTC CGT GGC TGA GTC
TA-39 detected a 400-bp fragment from the targeted allele. The
WT forward primer 59-TCT CCC AGG TCT GGT TTG AG-39
and reverse primer 59-TTA GGG CAA CAC CCT ACT GG-39
detected a 197-bp fragment from the WT allele.
The KO mice were backcrossed to the C57BL/6 background
for 8 generations and then used for experimentation. All mice
were housed under specific pathogen-free conditions and fed with
mouse chow (Teklad Global 2018, Teklan Diets, Madison, WI)
containing 15 IU/g Vitamin A. The mice had access to water and
chow ad libitum. The mouse organs and tissues were retrieved after
the mice were euthanatized by i.p. injection of 100 ml of Euthanyl
(pentobarbital sodium, 120 mg/ml containing 1% lidocaine). The
same method of euthanasia was used for unwanted heterozygous
mice or extra unused mice. The studies were approved by the
Institutional Animal Protection Committees of the CRCHUM
Single cell suspensions from the thymus, spleen and lymph
nodes were prepared and stained immediately or after culture with
antibodies (Abs) against CD4, CD8, CD25, CD19, B220, CD69
and STRA6. In some experiments, intracellular proteins, such as
FoxP3, IFN-c, IL-4, IL-17, and TNF-a, were detected after the
cells were pre-stained with Abs against cell surface antigens fixed
with BD Cytofix/CytopermTM solution (BD Biosciences, San
Figure 1. STRA6 mRNA expression in organs and activated T cells. STRA6 mRNA in organs (A) and activated total spleen T cells (B) was
measured by RT-qPCR. WT spleen total T cells were cultured in wells coated with solid-phase anti-CD3 mAb and anti-CD28 mAb (0.5 mg/ml and
4 mg/ml, respectively, for coating) for the durations indicated. The cells were then harvested and their STRA6 mRNA levels measured by RT-qPCR.
Samples were in triplicate for RT-qPCR, and means 6 SD of ratios versus b-actin signals are reported. Experiments were conducted twice, and
representative data are illustrated.
Diego, CA) and then stained with monoclonal Abs (mAb) against
intracellular antigens. The Abs deployed for flow cytometry are
listed in Table 1. Flow cytometry analysis of the stained cells are
described in our previous publications .
Flow cytometry was also employed to assess lymphocytic
choriomeningitis virus (LCMV)-specific T cells. Synthetic peptides
gp3341: KAVYNFATC (LCMV-GP, H-2Db); np396404:
FQPQNGQFI (LCMV-NP, H-2Db); gp276286:
SGVENPGGYCL (LCMV-GP, H-2Db); and gp6180:
GLNGPDIYKGVYQFKSVEFD (LCMV-GP, I-Ab) were purchased from
Sigma-Genosys (Oakville, Ontario, Canada). PE-gp3341,
PE-np396404 and PE-gp276286 H-2Db tetrameric complexes were
synthesized in-house and used at 1/100 dilution as previously
described . These MHC-tetramers were used to detect
LCMV-specific CD8+ T cells on day 8 post LCMV infection.
Briefly, splenocytes were first stained with PE-gp3341, PE-np396
404 or PE-gp276286 tetramers for 30 minutes at 37uC, followed by
staining with FITC-rat anti-mouse CD8a and APC-rat anti-mouse
CD62L mAbs at 4uC for another 20 minutes. 7-AAD was used for
exclusion of dead cells. After washing, cells were fixed in 0.5%
paraformaldehyde and samples were analyzed by flow cytometry.
One million splenocytes from LCMV-infected mice were seeded
in single wells of 96-well round-bottomed plates. They were
maintained in 5% RPMI-1640 supplemented with 100 units/ml
interleukin-2, 10 mg/ml brefeldin A, 10 mM gp3341 or gp6180
peptide. After 5 h of incubation at 37uC, the cells were stained
with PE-conjugated rat anti-mouse CD8a or CD4 mAbs and
7-AAD. They were then fixed, permeabilized and stained with
APC-labeled rat anti-mouse TNF-a and FITC-labeled rat
antimouse IFN-c mAbs. IFN-c and TNF-a-secreting T cells were
counted by flow cytometry .
T-cell proliferation in vitro and in vivo after being
transferred to sub-lethally-irradiated mice
Spleen cells were loaded with carboxyfluorescein succinimidyl
ester (CFSE; 5 mM for 5 mins), and then cultured in the presence
APC-rat anti-mouse CD25 (clone PC61)
FITC-rat anti-mouse CD25 (clone 7D4)
PE-rat anti-mouse CD4 (clones GK1.5 and H129.19)
PerCP-rat anti-mouse CD4 (clone RM4-5)
biotin-rat anti mouse CD8b (clone 53-5.8)
APC-Cy7- anti-mouse B220 (clone RA3-6B2)
PE- or APC-hamster anti-mouse CD3e(clone 145-2C11)
biotin- or FITC-rat anti-mouse CD44 (clone 1M7)
FITC- or PE-rat anti-mouse CD8a (clone 53-6.7)
APC-rat anti-mouse CD8a (clone H57-597)
PE- and APC-rat anti-mouse IFN-c
PE- and APC-rat anti-mouse IL4 mAbs
PerCP-streptavidin and 7-Amino-actinomycin D (7-AAD)
APC-rat anti-mouse TNF-a (clone MP6-XT22)
FITC-rat anti-mouse IFN-c (clone XMG1.2)
APC-rat anti-mouse IL17A (clone eBio17B7)
APC-rat anti mouse/rat Foxp3 (clone FJK-16s) mAbs
intracellular antigen fixation buffer
106 permeabilization buffer
APC-CyTM PE-rat anti-mouse CD25 (clone PC61)
Goat anti-mouse STRA6 Ab
eBioscience (San Diego, CA)
Cedarlane Laboratories Ltd (Burlington, Ontario, Canada)
Everest Biotech (Upper Heyford, Oxfordshire, UK)
of soluble hamster against mouse CD3 mAb (clone 2C11;
0.5 mg/ml) [27,28,32,33]. After 3 days, CFSE fluorescence of
the CD4 and CD8 subpopulations was analyzed by flow cytometry
for TCR-stimulated proliferation. T-cell homeostatic expansion
was evaluated by i.v. injection of 56106 CFSE-loaded spleen cells
into C57BL/6 recipients 5 h after sub-lethal irradiation (650 Rad).
On day 5, the CFSE fluorescence of CD4 and CD8 cells from the
spleen and LN was studied by flow cytometry.
In vitro Th1, Th2, Th17 and Treg cell polarization
In vitro Th and Treg cell differentiation was conducted as follows
[27,34]. Nave CD4 T cells (CD4+CD62L+CD44low) were isolated
from KO or WT mouse spleens with MagCellect Mouse Nave
CD4+ T cell Isolation kits (R & D Systems). T cell-depleted WT
spleen cells were irradiated at 3000 Rad and used as feeder cells.
The nave CD4 cells (0.16106/well) were mixed with the feeder
cells (0.56106/well) and cultured in 96-well plates in the presence
of soluble anti-CD3e mAb (clone 145-2C11, 2 mg/ml; BD
Biosciences). Cultures were supplemented with recombinant
mouse IL-12 (10 ng/ml; R & D Systems) and anti-IL-4 mAb
(10 mg/ml; R & D Systems) for the Th1 condition; recombinant
mouse IL-4 (20 ng/ml; R & D Systems), and anti-IL-12 mAb
(10 mg/ml; BD Biosciences) and anti-IFN-c mAb (10 mg/ml; R &
D Systems) for the Th2 condition; recombinant mouse IL-6
(20 ng/ml; R & D Systems), recombinant human TGF-b1
(5 ng/ml; R & D Systems) and anti-IL-4 and anti-IFN-c mAbs
(10 mg/ml for each; R & D Systems) for the Th17 condition;
recombinant human TGF-b1 (5 ng/ml; R & D Systems), and
antiIL-4 and anti-IFN-c mAb (10 mg/ml; R & D Systems) for the Treg
LCMV clone 13 was obtained from Dr. R.M. Zinkernagel
(University of Zurich, Zurich, Switzerland). Viral stock was
propagated in vitro, and viral titers were quantified by
focusforming assay . Mice were infected by the i.v. route with
26106 focus-forming units of LCMV clone 13. They were
sacrificed 8 days post-infection, and their spleens were harvested
for primary immune response analysis.
Glucose tolerance tests
The KO and WT mice were fasted for 16 h and injected i.v.
with D-glucose (2 mg/g body weight) in PBS. Blood samples from
the tail vein were taken at 5, 15, 30, 60, and 90 min after the
injection for glucose measurements with a glucose meter (Bayer,
Measurement of serum and intracellular vitamin A and
retinyl ester concentrations by high-pressure liquid
Serum and tissue samples, collected in a dark, cold room, were
stored at 280uC until their analysis. Retinoids were extracted by
homogenizing tissues in a butanol-acetonitrile mixture (1:1) with a
tissue/solvent ratio of 200 mg/700 ml, in Eppendorf tubes on ice
by 5 30-s pulses with 1-min intervals. K2HPO4 solution (6.89M)
was added to the tubes in proportion to the homogenized mixture
(20 ml for 900 ml homogenized mixture). For retinoid extraction
from sera, 200-ml butanol-acetonitrile mixture (1:1) was added to
200-ml serum, and the mixture was vortexed for 1 min; 20 ml
K2HPO4 solution (6.89M) was then added to the mixture before
30-s vortexing. The tissue and serum samples thus prepared were
centrifuged for 20 min at 14,000 g at 4uC. Cleared supernatants
were passed through Spin-X filters (0.45 mm pore size; Costar,
Batavia, Illinois, USA) at 14,000 g for 10 min at 4uC. For retinyl
ester measurement, the samples prepared as aforementioned
before the step of filtration were vacuum-dried and re-dissolved in
100% methanol, followed by centrifugation at 14,000 g for
10 min. The supernatants were then analyzed by HPLC.
Vitamin A in extracts was quantified by HPLC in an AKTA
Purifier (Model UPC10; GE Healthcare, Baie dUrfe, Quebec,
Canada) and reverse-phase column (m-RPC C2/C18 ST 4.6/100;
GE Healthcare). Samples (100 ml) were eluted with a linear
gradient from 100% eluent A (acetontrile:water = 65:35) to 100%
eluent B (acetontrile:water = 90:10) in 5-column volumes at a flow
rate of 1 ml/min. Both eluates contained 10 mM ammonium
acetate. Vitamin A was detected at 313 nm wave-length. Its
characteristic retention volume was identified with pure Vitamin A
from Sigma (Oakville, ON, Canada) as a standard. Areas under
the curves were computed by UNICORN5.11 software (GE
Healthcare). The sensitivity of the assay was 250 ng.
Retinyl ester in extracts was quantified by HPLC in an Eclipse
XDB-C18 reverse-phase column (4.66150 mm, 5 mm, Agilent,
Santa Clara, CA). Samples (200 ml) were eluted with a linear
gradient from 100% methanol to 100% ethyl acetate in 5 column
volumes at a flow rate of 1 ml/min. Retinyl ester was detected at
324 nm wavelength. Its characteristic retention volume was
identified with retinyl palmitate (Sigma) as standard. Areas under
the curves were computed by Agilent LC software. Sensitivity of
the assay was 1.5 ng.
Students t tests were employed to analyze statistical differences
between WT and KO mice for their lymphoid organ weight and
cellularity, and for their retinoid contents (retinol and retinyl
esters) in different organs. ANOVA was used to compare the
glucose tolerance between WT and KO mice.
STRA6 expression in different organs and activated
STRA6 mRNA expression was assessed by RT-qPCR. Among
the organs and tissues examined, the thymus had the highest
expression level, followed by the heart and kidneys (Fig. 2A).
STRA6 expression in the spleen was moderate. The skeleton
muscles and liver had barely detectable STRA6 mRNA. The
STRA6 mRNA expression levels in the lung, liver, spleen and
kidney assessed by our RT/qPCR was consistent with Northern
results reported previously by Bouillet . High STRA6
expression in the thymus suggested that it might have some
critical functions in T-cell development and T-cell function. As
depicted in Figure 2B, STRA6 expression in resting spleen T cells
(0 h) was modest, consistent with values of the whole spleen. The
expression was augmented with in 3 h after T-cell activation by
TCR cross-linking, and reached a peak at 48 h. This result
corroborates our initial DNA microarray data, through which
STRA6 was found upregulated during T-cell activation.
Generation of STRA6 KO mice
To evaluate the roles of STRA6 in the immune system in
general and T cell-mediated immune responses in particular, we
produced STRA6 KO mice. The targeting strategy is illustrated in
Figure 1A. Germline transmission was confirmed by Southern
blotting of tail DNA (Fig. 1B). With the 59 end probe, the WT
allele after XbaI digestion presented a 7.8-kb band, and the KO
allele, a 5.2-kb band (Fig. 1B, upper panel). With the 39 end probe,
the WT allele after ScaI digestion presented a 6.9-kb band, and
the KO allele, a 4.3-kb band (Fig. 1B, lower panel). WT (mice 3, 4,
and 5) and heterozygous mice (mice 1, 2 and 6) were thus
identified. Mouse 1 was backcrossed to the C57BL/6 background
for 8 generations, and then used in the experiments described
To ascertain whether STRA6 gene deletion results in its lack of
expression, we measured STRA6 mRNA in spleen cells by
RT-qPCR. STRA6 mRNA was detectable in WT but not in KO
spleen cells (Fig. 1C). The lack of STRA6 expression in KO cells at
the protein level was confirmed by flow cytometry, as STRA6 was
detectable in WT but not KO thymocyte surface (Fig. 1D).
Normal lymphoid organs and lymphocyte
subpopulations in STRA6 KO mice
STRA6 KO mice were viable and fertile with no apparent
anomalies upon visual inspection. Weight and cellularity of the
KO thymus and spleen were comparable to those of WT mice
(Fig. 3A). T-cell (CD4+ plus CD8+ versus non T-cell (CD42CD82)
subpopulations, and CD4 versus CD8 T-cell subpopulations in the
spleen and LN of WT and KO mice showed no consistent
differences (Fig. 3B). The percentages of B cells (CD19+B220+) in
the spleen and lymph nodes in WT and KO mice were also similar
(Fig. 3C). In KO thymi, the percentages of CD4 single-positive
and CD8 single-positive, CD4CD8 double-positive cells and
CD4+/FoxP3+ Treg cells were comparable to those in WT thymi
(Fig. 3D). The comparable percentages of Treg cells in the thymi of
WT and KO mice were confirmed by the measurement of FoxP3+
cells among CD4+CD25+ thymocytes (Fig. 3E).
In the periphery, the percentages of FoxP3+ Treg cells among
CD4 cells in the spleen (Fig. 3F) and lymph nodes (Fig. 3G) from
WT and KO mice were also similar.
These results show that STRA6 KO mice have normal
lymphoid organ and T-cell development.
Normal activation, proliferation and differentiation of
STRA6 KO T cells
KO and WT T cells were stimulated by solid-phase anti-CD3
mAb for 16 h. The activation markers CD25 and CD69 in CD4
and CD8 T cells were quantified by flow cytometry. KO and WT
T cells showed similar up-regulation of these markers (Fig. 4A). To
assess T-cell proliferation, KO and WT T cells in total spleen cells
were loaded with CFSE and stimulated by soluble anti-CD3 mAb.
After 3 days, their proliferation was assessed by flow cytometry.
CD4 and CD8 KO T cells proliferated like their WT counterparts,
as shown in Figure 4B. To measure T-cell homeostatic expansion,
spleen T cells were loaded with CFSE and then injected into
sublethally-irradiated syngeneic recipients. The proliferation of these
transferred KO CD4 and CD8 cells in recipient spleens and LN
during the 5 days after the injection was measured based on their
CFSE content according to flow cytometry. As shown in
Figure 4C, the cells from WT and KO mice proliferated similarly
in vivo. Therefore, KO T-cell proliferation, whether caused by
TCR stimulation in vitro or homeostatic expansion in vivo, was
When KO and WT nave CD4 cells were cultured under Th1,
Th2, Th17 and Treg conditions, they achieved comparable Th1,
Th2, Th17 and Treg cell percentages (Fig. 5), indicating normal
differentiation of nave KO CD4 cells into these subpopulations.
The effect of STRA6 deletion in anti-LCMV immune
responses in vivo
The function of STRA6 KO T cells in vivo was evaluated in the
LCMV infection model. As illustrated in Figure 6A, the number of
total splenocytes, and CD4 and CD8 cells on day 8 post-infection
(8 dpi) presented no significant differences in WT and KO mice
(Fig. 6A). The absolute numbers (Fig. 6B) and relative percentages
(Fig. 6C) of LCMV-specific tetramer-positive (gp33+, np396+ and
gp276+) CD8 cells in virus-infected mice were all increased in
comparison to uninfected control C57BL/6 mice (data not
shown), but there were no significant differences between KO
and WT mice with regard to these parameters. The absolute
numbers and relative percentages of LCMV-specific
TNF-aproducing CD4 (gp61) and CD8 cells (gp33) (Figs. 6D and 6E),
and LCMV-specific IFN-c-producing CD4 and CD8 cells (Figs. 6F
and 6G) in KO mice were comparable to those in WT controls.
These results indicate that the STRA6 deletion had no discernable
effect on anti-LCMV immune responses.
Normal glucose tolerance in STRA6 KO mice
One report suggests that STRA6 stimulation by RBP induces
the expression of SOCS3, which inhibits insulin signaling . We
assessed the glucose tolerance of KO mice, and found that, KO
and WT mice showed no significant difference in glucose tolerance
(Fig. 7), suggesting that in the absence of STRA6, the insulin
signaling of the KO mice on a normal diet is not enhanced.
Organ retinyl ester and retinol levels in STRA6 KO mice
As vitamin A has been reported to play an important role in
immune regulation , a lack of immunological phenotype
so-far tested in the KO mice prompted us to examine vitamin A
contents of lymphoid organs as well as several other organs
including the eyes. Vitamin A is stored in organs predominantly in
the form of retinyl ester, which is a lipid and can reach high
concentrations. A minor stored form is retinol bound to CRBP,
and the retinol content in the cells is limited by the availability of
CRBP. Retinyl esters and retinol and can be quickly converted to
each other inside the cells. We thus measured the contents of both
retinyl esters and retinol in these organs.
As shown in Figure 8A, the WT and KO spleen and thymus
had no significant difference in their retinyl ester contents, nor did
the brains and kidneys. The retinyl ester contents in WT eyes were
much higher than that of other organs, and the contents in the KO
eyes were significantly lower compared to those of the WT
counterparts. WT or KO blood had no detectable retinyl ester
(data not shown). Retinyl ester is known to be high in the blood
only right after a meal enriched in vitamin A.
The retinol levels in these WT and KO organs were of the same
pattern as retinyl ester, although at much lower levels (Fig. 8B;
note the scale difference). The KO spleen, thymus, kidney and
brain had no significant difference in retinol contents compared to
their WT counterparts. Unlike retinyl ester, the retinol was
detectable in the sera, but was of similar levels in WT and KO
sera. The eyes contained the highest levels of retinol. KO eyes
presented significantly lower levels of retinol than the WT
These data indicate that under a vitamin A sufficient condition,
lymphoid organs still take up vitamin A without STRA6 in mice.
This explains the lack of immunological phenotype in the KO
mice. However, even under such a vitamin A sufficient condition,
the eyes still heavily depend on SRAT6 for vitamin A uptake, as
they are the organ with the highest vitamin A demand.
STRA6 is a receptor of holo-RBP (i.e., vitamin A-bound RBP)
for cellular vitamin A uptake. STRA6 is up-regulated after T-cell
activation. In this study, we generated STRA6 KO mice to assess
whether such up-regulation was essential for T cell-mediated
immune responses and STRA6s role in vitamin A uptake. Under
a vitamin A sufficient condition, STRA6 KO mice developed
normally and were fertile. Their T cells presented no signs of
abnormality in terms of development, activation marker
upregulation, proliferation, and Th and Treg cell differentiation. KO
mice also had normal anti-LCMV immune responses. There was
no significant difference in intracellular vitamin A content, in the
forms of both retinyl ester and retinol, in lymphoid organs from
WT and KO mice. However, even under the vitamin A sufficient
condition, the KO eyes contained significantly lower amounts of
retinyl ester and retinol, indicating a critical role of vitamin A
uptake in this organ.
A caveat of whole organ retinoid analysis is that contribution of
retinoid in the blood can affect the total retinoid levels. This is
especially true if the organ is rich in blood, which contains
RBPbound vitamin A. Despite this caveat, whole organ retinoid
analysis can be used as an approximation of cellular retinoid
uptake. This is especially true as several of the organs we tested
Figure 4. Normal activation and proliferation of KO T cells. A. Normal activation marker CD69 and CD25 expression on KO T cells. Total spleen
cells were stimulated overnight by soluble anti-CD3 mAb (0.5 mg/ml). CD69 and CD25 expression on CD4 (left panel) and CD8 (right panel) T cells was
measured by 3-color flow cytometry. B. Normal proliferation of KO CD4 and CD8 cells upon TCR activation in vitro. Total spleen cells were loaded with
CFSE and then stimulated with soluble anti-CD3 mAb (0.5 mg/ml). The cells were harvested after 72 h, and stained for CD4 and CD8; CFSE levels in
these cells were analyzed by 3-color flow cytometry. C. KO CD4 and CD8 cells present normal homeostatic expansion in vivo. Five million CFSE-loaded
spleen cells were injected i.v. into sub-lethally irradiated (650 Rad) C57BL/6 recipients 5 h after the irradiation. On days 6, the CFSE fluorescence of
CD4 and CD8 cells from the spleen and LN was analyzed by flow cytometry. All experiments in this figure were conducted twice or more, and
representative histograms are shown.
(i.e., the thymus, brain and eyes) are not blood rich. Moreover,
sera had no detectable retinyl ester (data not shown); so the retinyl
ester levels of the organs tested will not be upward influenced by
the blood retinyl ester levels. The reduced vitamin A contents in
the eyes of STRA6 KO mice is not unexpected, as the eyes have
the highest concentrations of vitamin A among all the organs
(Fig. 8) due to its heavy reliance on vitamin A for vision, and
probably need all the capacities of vitamin A transport including
the pathway of RBP/STRA6 to achieve this high vitamin A
content, even in vitamin A sufficiency. Consistent to our findings,
RBP KO mice have normal vitamin A levels in most of their
organs, but a reduced one in the eyes .
Vitamin A and its metabolites retinoic acids are clearly
required in immune responses [1318,36]. It is reported that in
hepatocytes, holo-RBP triggers STRA6, leading to the activation
of JAK2/STAT5 signaling pathway, which is also essential in the
activation and function of immune cells. STRA6 is up-regulated
within 24 h of T-cell activation (Fig. 1B). Is such up-regulation, or
more fundamentally, the existence of STRA6, essential for T
cellmediated immune responses? We demonstrated that in STRA6
KO mice, a lack of STRA6 did not affect T-cell activation/
proliferation/differentiation in vitro and anti-viral immune
responses in vivo under a vitamin A sufficient condition. These
observations suggest following possible and not necessarily
mutually exclusive explanations: 1) STRA6
up-regulation/existence only becomes important for T-cell functions during vitamin
A deficiency, when all capacities of vitamin A import to immune
cells are required; 2) STRA6 homologue RBPR2 can compensate
for STRA6 function in the immune cells, as this homologue is
expressed in the spleen; also, retinyl esters bound to lipoproteins
secreted by the small intestine can deliver vitamin A to peripheral
organs under vitamin A sufficient conditions; 3) STRA6 plays a
minimal role in modulating the JAK2/STAT5 signalling pathway
in immune cells, and its upregulation after T cell activation has
nothing to do with JAK2/STAT5 signaling; 4) We cannot exclude
the possibility that STRA6 deletion might still affect certain T
cellmediated immune responses to some extent, but they have not
been assessed in our experiments, or their magnitude was too small
to be discerned by current assays; 5) Such up-regulation might be a
parallel and irrelevant event during T-cell activation.
There is little systemic documentation on vitamin A sufficiency
status in wild mammals in todays world. However, it is
welldocumented that vitamin A deficiency is prevalent in African and
Southeast Asian populations, particularly affecting children and
pregnant women, according to the World Health Organization
, and such deficiency predisposes them to infectious diseases
. It is conceivable that, during evolution, mammals might have
experienced vitamin A deficiencies in certain periods or regions in
the world. Better cellular vitamin A transport will confer an
evolutionary advantage to these animals with regard to but not
restricted by their ability to cope with infectious diseases. If
STAR6 is universally critical in all cell types for vitamin A uptake,
its function should be revealed in immune responses in vitamin A
deficiency. Experiments addressing this possibility are in progress.
STRA6 point mutations are found in some patients, with
microphthalmia, anophthalmia, coloboma  and
MatthewWood syndrome [referring to combinations of microphthalmia/
anophthalmia, cardiac malformations, pulmonary dysgenesis, and
diaphragmatic hernia; ref. 9]. In a study of 2 unrelated
consanguineous families with malformation syndromes sharing
anophthalmia and distinct eyebrows as common signs,
homozygous mutations were identified in STRA6 . Our STRA6 KO
mice and those generated by Ruiz et al.  did not have dramatic
phenotypes, such as a total absence of eyes, as seen in humans with
Why cannot STRA6 KO in mice reproduce human disease
phenotypes caused by STRA6 mutations? A simple explanation is
that this is due to species differences. It is not unprecedented that
gene mutations in mice and humans have very different
phenotypes. For example, partial or complete loss of ABCA4
functions cause many blinding diseases in humans including
retinitis pigmentosa, cone-rod dystrophy and Stargdardt macular
dystrophy, but ABCA4 KO in mice does not cause blindness
unless combined with a deletion of other genes such as RDH8
. On the other hand, disease loci of microphthalmia and
anophthalmia have been mapped to multiple chromosomes [42
45]. Patients with Matthew-Wood syndrome or malformation
syndromes have quite large phenotype variations in terms of organ
affliction and disease severity. Such observations suggest that these
syndromes are not monogenic, and STRA6 mutation alone is not
sufficient to evoke all such phenotypes. It could explain why no
serious ophthalmic  or other organ malformations are
apparent in STRA6 null mutation mice. If STRA6s major
function is cellular vitamin A uptake, and human organ
malformation syndromes are mainly caused by a lack of available
intracellular vitamin A, it would support the notion that STRA6
only plays a minor role in cellular vitamin A uptake in vitamin A
sufficiency, especially in organs other than the eyes. Unless other
routes of cellular vitamin A uptake such as those mediated by
RBPR2 or by retinyl esters bound to lipoproteins are
simultaneously compromised, vitamin A in the cells of most, if not all,
organs vitamin A contents will remain in the normal range, and
the organs will develop and function normally in vitamin A
Figure 7. Glucose tolerance of KO and WT mice. WT (n = 5) and KO (n = 7) mice were fasted for 16 h, and then injected i.p. with D-glucose
(2 mg/g body weight). Blood glucose was measured at different time points from the time of injection until 120 min. Means 6 SD of glucose levels
(mg/dL) are reported. No statistical significant difference is observed between the KO and WT groups (ANOVA).
sufficiency. However, significant phenotype might be revealed in
vitamin A deficiency. This hypothesis is supported by results from
RBP KO mice. These KO mice are fertile and have no organ
abnormality other than the vision phenotype , as is the
case of STRA6 KO mice when fed with a vitamin A sufficient diet.
However, they manifest severe systemic phenotype of embryonic
lethality under a vitamin A deficient condition [49,50].
Consistently, in mouse embryo culture where is no retinyl ester pathway,
RBP knockdown also causes severe developmental defects .
In summary, we conclude that, under normal dietary
conditions, mouse lymphoid organ development, T-cell activation and
differentiation, including Treg cell development, and anti-LCMV
Figure 8. Intracellular retinoid contents in lymphoid and other organs of STRA6 KO mice were comparable to those of WT mice.
Retinoid (retinyl ester, A; retinol, B) contents (nmol/gram tissue or nmol/ml serum) of the eyes, brain, kidney, spleen, thymus, spleen, thymus and sera
from KO and WT mice were measured by HPLC. The mouse numbers (n) per group are indicated. The results are expressed as means+SD. The
p-values are indicated when significant (Students t test).
responses, could proceed normally in the absence of STRA6 under
vitamin A sufficient conditions.
Conceived and designed the experiments: RT XW YH HL JW. Performed
the experiments: RT XW YH TC MZ JM SQ. Analyzed the data: RT
XW YH AL HS JW. Contributed reagents/materials/analysis tools: SQ
HL AL HS JW. Wrote the paper: RT XW YH AL HS JW.
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