Lymph node fibroblastic reticular cells directly present peripheral tissue antigen under steady-state and inflammatory conditions.

Abstract

Lymph node stromal cells (LNSCs) can induce potent, antigen-specific T cell tolerance under steady-state conditions. Although expression of various peripheral tissue-restricted antigens (PTAs) and presentation to naive CD8+ T cells has been demonstrated, the stromal subsets responsible have not been identified. We report that fibroblastic reticular cells (FRCs), which reside in the T cell zone of the LN, ectopically express and directly present a model PTA to naive T cells, inducing their proliferation. However, we found that no single LNSC subset was responsible for PTA expression; rather, each subset had its own characteristic antigen display. Studies to date have concentrated on PTA presentation under steady-state conditions; however, because LNs are frequently inflammatory sites, we assessed whether inflammation altered stromal cell-T cell interactions. Strikingly, FRCs showed reduced stimulation of T cells after Toll-like receptor 3 ligation. We also characterize an LNSC subset expressing the highest levels of autoimmune regulator, which responds potently to bystander inflammation by up-regulating PTA expression. Collectively, these data show that diverse stromal cell types have evolved to constitutively express PTAs, and that exposure to viral products alters the interaction between T cells and LNSCs.

Full text

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The Rockefeller University Press $30.00
J. Exp. Med. Vol. 207 No. 4 689-697
www.jem.org/cgi/doi/10.1084/jem.20092642
689
Brief Definitive Report
Autoreactive T cells are ubiquitous to the nor-
mal lymphocyte repertoire, presumably to max-
imize potential immune responses to pathogens.
In healthy individuals, peripheral tolerance
mechanisms keep these cells in check to pre-
vent autoimmunity.
The role of nonhematopoietic LN stromal
cells (LNSCs) in peripheral tolerance is an
emerging, quickly evolving field of study. Vari-
ous groups have shown that LNSCs shape the
T cell repertoire under noninflammatory condi-
tions. In the steady state, they express a range of
clinically relevant peripheral tissue–restricted
antigens (PTAs; Lee et al., 2007; Nichols et al.,
2007; Magnusson et al., 2008) and transcription
factors (Gardner et al., 2008; Yip et al., 2009),
and are highly effective at tolerizing autoreactive
T cells (Lee et al., 2007; Nichols et al., 2007;
Gardner et al., 2008; Magnusson et al., 2008).
Reactive CD8+ T cells are activated, induced to
proliferate, and lost from the peripheral T cell
pool (Lee et al., 2007; Nichols et al., 2007;
Gardner et al., 2008; Magnusson et al., 2008).
Although bone marrow chimeras show that
tolerance requires nonhematopoietic cells in
these systems (Lee et al., 2007; Nichols et al.,
2007; Gardner et al., 2008; Magnusson et al.,
2008), the LN stromal niche is heterogeneous
and poorly studied. As such, identification of
the tolerizing cell type is difficult, requiring
mice with a genetic trace for stromal lineages,
or the ability to isolate these rare cells with
high efficiency and purity.
The primary hypothesis regarding the iden-
tity of a tolerogenic LNSC suggests analogy to
medullary thymic epithelial cells (mTECs), which
express a wealth of PTAs (Derbinski et al., 2001;
CORRESPONDENCE
Shannon Turley:
Shannon_Turley@
dfci.harvard.edu
Abbreviations used: AFP, 
fetoprotein; Aire, autoimmune
regulator; BEC, blood endo-
thelial cell; DN, double negative;
ECM, extracellular matrix;
eTAC, extrathymic Aire-
expressing cell; FRC, fibroblas-
tic reticular cell; iFABP,
intestinal fatty acid–binding
protein; LEC, lymphatic endo-
thelial cell; LNSC, LN stromal
cell; mTEC, medullary thymic
epithelial cell; PLP, proteolipid
protein; PTA, peripheral tissue–
restricted antigen; QPCR,
quantitative PCR; TLR, Toll-like
receptor; tOVA, truncated OVA;
Tyr, tyrosinase.
Lymph node fibroblastic reticular cells
directly present peripheral tissue antigen
under steady-state and inflammatory conditions
Anne L. Fletcher,1 Veronika Lukacs-Kornek,1 Erika D. Reynoso,1,3
Sophie E. Pinner,1 Angelique Bellemare-Pelletier,1 Mark S. Curry,2
Ai-Ris Collier,1 Richard L. Boyd,5 and Shannon J. Turley1,4
1Department of Cancer Immunology and AIDS and 2Flow Cytometry Core Facility, Dana-Farber Cancer Institute,
Boston, MA 02115
3Division of Medical Sciences and 4Department of Pathology, Harvard Medical School, Boston, MA 02115
5Monash Immunology and Stem Cell Laboratories, Monash University, Clayton 3800, Australia
Lymph node stromal cells (LNSCs) can induce potent, antigen-specific T cell tolerance
under steady-state conditions. Although expression of various peripheral tissue–restricted
antigens (PTAs) and presentation to naive CD8+ T cells has been demonstrated, the stromal
subsets responsible have not been identified. We report that fibroblastic reticular cells
(FRCs), which reside in the T cell zone of the LN, ectopically express and directly present a
model PTA to naive T cells, inducing their proliferation. However, we found that no single
LNSC subset was responsible for PTA expression; rather, each subset had its own character-
istic antigen display. Studies to date have concentrated on PTA presentation under steady-
state conditions; however, because LNs are frequently inflammatory sites, we assessed
whether inflammation altered stromal cell–T cell interactions. Strikingly, FRCs showed
reduced stimulation of T cells after Toll-like receptor 3 ligation. We also characterize an
LNSC subset expressing the highest levels of autoimmune regulator, which responds
potently to bystander inflammation by up-regulating PTA expression. Collectively, these
data show that diverse stromal cell types have evolved to constitutively express PTAs, and
that exposure to viral products alters the interaction between T cells and LNSCs.
© 2010 Fletcher et al. This article is distributed under the terms of an Attribu-
tion–Noncommercial–Share Alike–No Mirror Sites license for the first six months
after the publication date (see http://www.rupress.org/terms). After six months
it is available under a Creative Commons License (Attribution–Noncommercial–
Share Alike 3.0 Unported license, as described at http://creativecommons.org/
licenses/by-nc-sa/3.0/).
The Journal of Experimental Medicine

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FRCs present self-antigen to naive T cells | Fletcher et al.
and 47 integrins expressed by lymphocytes), CD140a
(common to mesenchymal fibroblasts), and CD44 (a matrix
metalloproteinase dock, heparin sulfate proteoglycan, and
hyaluronate receptor often expressed by fibroblastic cells;
Fig. 1 B). LECs and BECs shared a similar phenotype, with
Anderson et al., 2002) and tolerize the developing T cell
repertoire. However, although Lee et al. (2007) reported ex-
pression of an intestinal PTA by a gp38+ LNSC, Gardner
et al. (2008) identified a tolerogenic gp38 stromal cell type.
Each subset shared markers with mTECs.
In this report, we show that fibroblastic reticular cells
(FRCs) endogenously express PTAs and directly stimulate
naive antigen-specific CD8+ T cells. We also report that
lymphatic endothelial cells (LECs) are the only LNSC to
express the melanocyte-associated enzyme tyrosinase (Tyr),
suggesting an important contribution to peripheral toler-
ance, because LN expression of this PTA is crucial for delet-
ing Tyr-specific T cells from the normal repertoire (Nichols
et al., 2007). We further report that LNSC subsets respond
to signaling through Toll-like receptor 3 (TLR3), with
FRCs showing a reduced capacity to stimulate T cells. We
also characterize a hitherto unstudied stromal subset, which
showed unique up-regulation of PTAs and autoimmune reg-
ulator (Aire) in response to inflammation. These results
carry novel implications for peripheral tolerance theory,
showing that cells of highly diverse lineage, phenotype, and
function can express PTAs and shape the T cell repertoire.
RESULTS AND DISCUSSION
The LN stromal compartment consists of discrete subsets
The LN stromal niche supports leukocyte entry, exit, migra-
tion, survival, and activation (Gretz et al., 1996; Katakai et al.,
2004; Bajénoff et al., 2006; Link et al., 2007). Multiple op-
portunities therefore exist for tolerogenic interactions be-
tween T cells and stroma. With many studies emphasizing the
biological, pathological, and therapeutic implications of a resi-
dent cell type that naturally deletes T cells in an antigen-
specific manner (Lee et al., 2007; Nichols et al., 2007; Gardner
et al., 2008; Magnusson et al., 2008; Reynoso et al., 2009;
Yip et al., 2009), identification of the cells responsible is in-
creasingly crucial. Limiting factors include the rarity of LNSCs,
the collagen-rich structure of the LN, requiring enzymatic
digestion, and sparse phenotypic and functional data.
By flow cytometry, nonhematopoietic LNSCs form four
subsets based on expression of gp38 and CD31 (Fig. 1 A;
Link et al., 2007). FRCs (gp38+CD31) are the major stro-
mal subset in skin-draining LNs, and provide survival factors
and directional support for DCs and lymphocytes, which
crawl along FRC meshwork to reach intra-LN niches
(Katakai et al., 2004; Bajénoff et al., 2006; Link et al., 2007).
T cell entry to and exit from LNs is regulated by blood en-
dothelial cells (BECs; CD31+ gp38) and LECs (gp38+CD31+).
FRCs, BECs, and LECs comprise 80–85% of nonhemato-
poietic LN cellular content. The double-negative (DN) pop-
ulation expresses neither gp38 nor CD31 (Fig. 1 A) and has
not previously been characterized. In our hands, follicular
DCs, which are confined to B cell zones, comprised <1% of
CD45 stroma. Because of their rarity, these cells were not
further characterized in this study.
In assessing the LNSC phenotype, we found that FRCs
showed strong expression of VCAM-1 (a ligand for VLA-4
Figure 1. LNSC subsets are phenotypically distinct. (A) Flow cytom-
etric strategy used to identify and sort CD45 LNSC subsets according
to gp38 and CD31 expression. The percentage of CD45 stroma (left)
and LNSC subsets (right) is shown. (B) LNSC subsets were analyzed
using flow cytometry. Shading indicates isotype control. Data represent
five to six mice from three experiments. (C) TLR expression in sorted
LNSC subsets assessed by RT-PCR. Images represent three experiments.
BM-DC, bone marrow–derived DC; SSC, side scatter.

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FRCs directly present a model peripheral tissue antigen
to naive T cells
We have previously shown that an unknown component of
nonhematopoietic stroma deletes autoreactive T cells in the
intestinal fatty acid–binding protein (iFABP)–truncated OVA
(tOVA) mouse model, where LNSCs express tOVA as a
model PTA under the control of the tissue-specific promoter
for iFABP (Lee et al., 2007). Robust proliferation of naive
OT-I T cells is induced within 2 d after their transfer into
iFABP-tOVA but not C57BL/6 hosts (Fig. 2 A), even when
hematopoietic cells cannot present antigen (Lee et al., 2007).
However, the nature of these initial experiments did not
permit identification of the stromal subset responsible. By
sorting FRC, LEC, BEC, and DN subsets to high purity
and examining mRNA expression, we were able to identify
FRCs as the stromal cell type predominantly responsible for
tOVA expression in the iFABP-tOVA model (Fig. 2 B). Low
transcript was evident in LEC, BEC, and DN subsets at high
PCR cycle number (>45 cycles), which was not the result of
contamination, because other FRC-restricted transcripts
identified (see Fig. 3) could not be amplified from these sub-
sets. Expression in FRCs was 40-fold higher than LECs, 73-
fold higher than BECs, and 14-fold higher than DNs
(unpublished data).
In vivo, naive OT-I T cells responding to tOVA presented
by LNSCs in iFABP-tOVA mice strongly proliferate and are
subsequently deleted, inducing tolerance (Lee et al., 2007).
We conducted an in vitro stimulation assay and found that
FRCs from iFABP-tOVA mice, but not C57BL/6 mice,
induced division of OT-I T cells (Fig. 2 C). Up to 85% of the
OT-I T cells up-regulated CD25, an early activation marker
(Fig. 2 D). FRCs therefore present tOVA at levels high enough
to functionally interact with T cells, inducing proliferation as
seen before their deletion in vivo (Lee et al., 2007). This is the
first demonstration that FRCs can act as APCs in LNs.
Interestingly, T cells stimulated by FRCs showed lower ex-
pression of CD25 compared with those stimulated by peptide-
pulsed splenocytes (Fig. 2 C). A reduction in IL-2R may
indicate a reduced capacity to respond to IL-2, reflecting the
functional juxtaposition resulting from these interactions
(activation versus eventual deletion). This, as well as any
other difference in stimulated T cells, warrants further study.
Each LNSC subset has a unique PTA expression signature
A prominent hypothesis (Lee et al., 2007; Nichols et al., 2007;
Gardner et al., 2008; Yip et al., 2009) suggests that a stromal
subset analogous to thymic mTECs could bear the responsi-
bility of PTA expression and tolerance induction in LNs. We
assessed expression of representative PTAs in sorted LNSC
subsets (Fig. 3). To our surprise, no single subset was respon-
sible for PTA expression. Even antigens primarily expressed
in melanocytes showed differential expression in LNSCs:
although FRCs were the only cells expressing Mlana, only
LECs expressed Tyr. Both proteins are current immunother-
apy targets for melanoma (Trefzer et al., 2006). This finding
had clinical relevance, because Tyr-reactive T cells escape
low levels of VCAM-1 and a CD44-low subset. A distinct
subset of LECs and BECs was stained by MTS16 (Fig. 1 B;
Godfrey et al., 1990). In the thymus, MTS16 stains extra-
cellular matrix (ECM) with similar reactivity to ER-TR7, an
antibody that also stains FRCs; however, FRCs did not stain
with MTS16, suggesting qualitative differences in ECM pro-
duced by thymus and LN fibroblastic cells. BECs uniquely
expressed high levels of Sca-1 (Fig. 1 B), associated with self-
renewal and ECM remodeling (Bonyadi et al., 2003; Kafadar
et al., 2009). DN cells resembled FRCs in the level of CD44
and Sca-1 expression but lacked other fibroblastic markers
such as CD140a (Fig. 1 B).
We did not detect epithelial cell adhesion molecule in
LNSCs in C57BL/6 mice either by flow cytometry (Fig. 1 B)
or quantitative PCR (QPCR; not depicted). Although
Gardner et al. (2008) identified an epithelial cell adhesion
molecule+ Aire+ cell type in LNs (termed extrathymic Aire-
expressing cells [eTACs]), their low frequency compared
with other stroma, high motility, and the fact that a genetic
tracer is required to identify them makes them difficult to
compare with classical nonmotile stroma. In addition, the
Adig transgenic mice were bred on a nonobese diabetic
mouse background, and these eTACs may differ in pheno-
type or frequency from those in other strains. In accordance
with Hubert et al. (2008), we did not observe Aire protein
expression (unpublished data). We were thus unable to iden-
tify eTACs in our preparations; these cells are likely too rare
in nonautoimmune mice or express protein at levels too low
to identify without a genetic reporter system.
Because virally infected FRCs have been linked to re-
duced CD8+ T cell immunopathology (Mueller et al., 2007),
we investigated expression of viral-associated TLRs in LNSC
subsets. TLRs are critical components of the innate immune
system that enable cells to detect highly conserved moieties
associated with pathogen exposure. All classes of professional
APCs express TLRs; however, in an unexplained paradox,
there is precedence for TLR expression in stromal cell types
associated with a microenvironment that is tolerogenic or
poorly immunogenic. This includes intestinal epithelium,
bone marrow–derived mesenchymal stromal cells, nonparen-
chymal liver cells, and ocular epithelial cells (Cario et al.,
2000; Ueta et al., 2004; Wu et al., 2007; Liotta et al., 2008).
Given substantial evidence that LNSCs are also primarily
tolerogenic and may be immunosuppressive, even when virally
infected (Mueller et al., 2007), we investigated TLR expres-
sion in LNSC subsets.
In the steady state, FRCs, LECs, and BECs expressed
TLR3 (Fig. 1 C), which detects double-stranded RNA pro-
duced either as primary genetic material or as a replicative
intermediate in most viral infections (Weber et al., 2006).
DN cells uniquely lacked expression of all viral-associated
TLRs. No stromal subsets expressed TLR7 or 8, which
detect single-stranded RNA, using bone marrow–derived
DCs as a positive control. Expression of TLR3 suggests that
FRCs, LECs, and BECs are capable of directly responding
to viral infection.

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FRCs present self-antigen to naive T cells | Fletcher et al.
Conversely, the Aire-like transcriptional modulator Deaf-1
was equally expressed in all skin-draining LNSCs (Fig. 3,
A and B). Deaf-1 drives PTA expression in pancreatic LNs
(Yip et al., 2009), and its reduction is associated with type I
diabetes in humans and mice. Comparison of expression in
primary tissue (Fig. 3 C) showed that FRCs expressed Mlana at
levels equivalent to a melanocyte-enriched skin preparation,
whereas PLP was 200-fold higher in the spinal cord compared
with FRCs, and AFP, which is expressed by the liver at a low
basal level after birth but increases after liver carcinoma or
viral hepatitis, was expressed almost 100-fold lower in paren-
chyma-enriched adult mouse liver compared with FRCs.
Activation through TLR3 reduces FRC-mediated
T cell stimulation
A prevailing view of tolerance states that DCs are primar-
ily tolerogenic in the steady state but become profoundly
negative selection in the thymus; tolerance to this self-antigen
relies on its expression by a nonhematopoietic cell in LNs
(Nichols et al., 2007), and we identify that cell type as a LEC.
It will be of the utmost clinical significance to determine
whether PTA expression in LECs occurs by mechanisms spe-
cific to LN endothelium or is a property of lymphatic endo-
thelium in general. Retinal S antigen was primarily expressed
in BECs, although FRCs showed consistent, low transcript,
while Rrad, a pancreas-associated PTA controlled by Aire in
the spleen (Gardner et al., 2008), was expressed in all LNSC
subsets.  Fetoprotein (AFP) and proteolipid protein (PLP),
respectively associated with the liver and central nervous sys-
tem, were similarly expressed in all LNSC subsets. Interest-
ingly, QPCR showed that expression of Aire was predominant
(60-fold higher) in DNs (Fig. 3 B), with expression in
FRCs, LECs, and BECs at the limits of detection. In two out
of four sets of template, Aire was not detected in sorted BECs.
Figure 2. FRCs ectopically express and present OVA to OT-I T cells. (A) CFSE-labeled OT-I T cells were transferred into B6 or iFABP-tOVA hosts.
T cell division, measured as the percentage of CFSE dilution, was assessed by flow cytometry. Graphs represent 12 mice from four experiments. (B) OVA
transcription was assessed by RT-PCR. -Actin was used to demonstrate cDNA integrity. Images are representative of three experiments. (C) Proliferation
of CFSE-labeled OT-I T cells (shown as the percentage divided) was assessed after culture alone, with SIINFEKL-pulsed splenocytes, or with FRCs purified
from C57BL/6 or iFABP-tOVA mice. Plots represent three experiments. (D) The cell division index (left) and proportion of divided cells (right) were calcu-
lated from three experiments. Graphs depict means + SD. SkLN, skin-draining LN.

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(Fig. 4 A). After PolyI:C injection, FRCs strongly up-regulated
MHC class I and showed some increase in CD80 and CD86,
whereas LECs up-regulated only CD80, and BECs showed
no alteration of co-stimulatory receptors (Fig. 4 A). DN cells
showed a modest increase in CD86 expression. However, the
strongest response across all LNSC subsets occurred with
PD-L1 (Fig. 4 A), the strongest prognostic indicator of
deletion after interactions between T cells and LNSCs in
experimental models of inflammation and autoimmunity
(Mueller et al., 2007; Reynoso et al., 2009). Indeed, blocking
PD-1 signal is sufficient to break the robust T cell tolerance nor-
mally induced after interaction between OT-I T cells and OVA-
expressing LNSCs, causing autoimmunity (Reynoso et al., 2009).
Increased expression of CD86 and PD-L1 on DN cells
showed that these phenotypic alterations could occur indi-
rectly (because DN cells are TLR3), probably through
inflammatory mediators from TLR3+ cells. To test the net
outcome of up-regulated MHC and co-stimulatory mole-
cules balanced against a large increase in PD-L1, we tested
the capacity of PolyI:C-treated iFABP-tOVA FRCs to stim-
ulate OT-I T cells in vitro. Strikingly, FRCs were less capa-
ble of stimulating OT-I T cell division, whereas peptide-pulsed
immunostimulatory after exposure to “danger” signals such
as tissue damage or microbial products. Although LNSC sub-
sets similarly appear tolerogenic in the steady state, their
function in an inflammatory milieu has not been formally
tested. Expression of TLR3 in FRCs, LECs, and BECs sug-
gests the capacity to respond directly to viral threat.
We injected mice with PolyI:C, which signals through
TLR3. The reaction of LNSCs to viral infection is of partic-
ular interest, given that Mueller et al. (2007) showed that
infection of FRCs correlated with viral persistence. It was
unclear whether immunosuppression was intrinsic to FRCs
under inflammatory conditions or whether the virus was
subverting FRC function.
Because inflammation induces an escalatory effect within
LNs, we tested stromal phenotype after PolyI:C injection at a
relatively early time point (16 h) just sufficient for translation of
co-stimulatory receptors, based on the response of TLR3+ DCs.
At this time point, DCs were in the process of up-regulating
MHC class I, CD40, CD80, CD86, and PD-L1 (Fig. 4 A).
Control (PBS-injected) mice showed steady-state expres-
sion of CD40 and PD-L1 in all LNSC subsets, and CD80 was
low in all subsets except LECs. CD86 was not expressed
Figure 3. LNSC subsets show distinct PTA expression patterns. (A) RT-PCR was performed for a panel of PTAs. Images represent two to four
experiments. (B) QPCR was performed for selected PTAs and transcriptional regulators. (C) QPCR was used to examine PTA expression in primary tissue
compared with FRCs (normalized to 1, relative to GAPDH). All QPCR data show expression calculated relative to GAPDH and are depicted as fold change
relative to FRCs, normalized to 1. All graphs depict means + SD from three to four experiments. ND, not detected; Ret S, retinal S antigen.

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FRCs present self-antigen to naive T cells | Fletcher et al.
Although the outcome for a T cell recognizing a non–
down-regulated PTA on a PolyI:C-treated FRC is unknown,
at least one other study suggests that FRCs are tolerogenic
after direct exposure to virus (Mueller et al. 2007).
The functional impact on T cells is the subject of further
study, but it is apparent that the tolerogenic capacity of stroma
is altered upon viral exposure. A mechanism similar to activa-
tion of tolerogenic DCs may occur, where administering TLR
ligands alters the ability of DCs to stimulate T cells without
compromising tolerance (Hamilton-Williams et al., 2005).
OT-I T cells activated by TLR-stimulated DCs could not
hematopoietic cells were not affected (Fig. 4, B and C). How-
ever, DCs and FRCs showed similar phenotypic changes in
PD-L1, MHC class I, and co-stimulatory molecules after
PolyI:C treatment (Fig. 4 A). These did not, therefore,
account for the altered FRC–T cell interaction after PolyI:C
exposure. Instead, we found that OVA expression was re-
duced sevenfold in FRCs sorted from PolyI:C-treated mice
(Fig. 4 D), suggesting a possible mechanism for the reduced
antigen-specific T cell stimulation observed. This was not the
result of broadly reduced gene transcription, because FRCs
did not down-regulate expression of all PTAs (Fig. 4 E).
Figure 4. Activation through TLR3 alters LNSC phenotype and reduces FRC-mediated T cell stimulation. (A) LNSC expression of co-stimulatory
and inhibitory receptors was assessed by flow cytometry. Plots represent six to seven mice from two to three experiments. Shading indicates isotype control;
thin and thick lines depict data from PBS- or PolyI:C-treated mice, respectively. (B) Proliferation of CFSE-labeled OT-I T cells (shown as percentage divided)
with and without PolyI:C. Cells were cultured without APCs, with SIINFEKL-pulsed splenocytes, or with FRCs purified from iFABP-tOVA mice. Plots represent
three experiments. (C) OT I cell division was calculated across FRC/T cell dilutions. Graph depicts mean + SD from three experiments. (D) OVA expression
levels in FRCs were assessed by QPCR after PolyI:C or PBS treatment of iFABP-tOVA mice. Graph depicts mean + SD from three experiments. (E) QPCR was
performed for selected PTAs. Graph depicts mean + SD from two to three experiments. For all QPCR data, expression in PolyI:C-treated mice was calculated
relative to GAPDH and depicted as fold change relative to expression in the appropriate subset from PBS-injected mice, normalized to 1. ND, not detected.

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mouse line generated by L. Lefrançois (University of Connecticut,
Farmington, CT; Vezys et al., 2000). C57BL/6 OT-I TCR transgenic
Rag/ mice were obtained from Taconic. All mice were specific pathogen
free and cared for in accordance with institutional and National Institutes
of Health guidelines. Experimental procedures were conducted with the
approval of the Research Animal Care subcommittee at the Dana-Farber
Cancer Institute.
Stromal cell isolation and staining for flow cytometry. Skin-draining
LNs (inguinal, axillary, and brachial) for each mouse were digested separately
using 1 ml of enzyme mix containing 0.2 mg/ml Collagenase P (Roche),
0.1 mg/ml DNase I (Invitrogen), and 0.8 mg/ml Dispase (Roche) at 37°C
for 15 min. Tissues were agitated, media were collected, and the digestion
mix was replaced. This proceeded for 50–60 min, when LNs were com-
pletely digested. As previously described (Fletcher et al., 2009), 5 × 106 cells
were stained, acquired on a FACSCalibur or FACSAria (BD), and analyzed
using FlowJo software (Tree Star, Inc.).
Stromal cell enrichment and sorting. LNs from 6–10 mice per group
were pooled for digestion, as detailed in the previous section, using 5 ml of
enzyme mix. Cells were counted and filtered as described, and enriched for
CD45 stroma as previously described (Fletcher et al., 2009). Stained cells
were sorted using a FACSAria fitted with a 100-µm tip at a pressure of 20 psi.
The purity of sorted stromal populations routinely exceeded 96%. Tail skin
was digested for 90 min using enzyme mix, with fractions collected at 15-min
intervals. Highly pigmented fractions enriched in melanocytes were retained
for QPCR analysis. Spinal cord was digested to a single-cell suspension within
15 min; liver was digested and enriched for CD45 stroma similarly to LNs.
Treatment with PolyI:C. Mice were injected i.v. with either 100 µg
PolyI:C (GE Healthcare) in PBS or PBS alone, and sacrificed for analysis
after 16–18 h.
Analysis of T cell proliferation. For in vivo analysis, 4 × 106 CFSE-
labeled OT-I T cells were transferred i.v. into C57BL/6 or iFABP-tOVA
mice, and skin-draining LNs were analyzed 42 h later. For in vitro analysis,
skin-draining LNs were enzymatically digested. CD45+ and CD31+ cells
were depleted to high purity using MACS separation (Miltenyi Biotec). Re-
maining FRCs were cultured with CFSE-labeled OT-I T cells in culture
media (MEM; 10% FCS, 10 U/ml IL-2, 1% penicillin/streptomycin) with
2.5 µg/ml PolyI:C where indicated in the figures. As a positive control,
splenocytes were incubated with 1 mg/ml SIINFEKL (AnaSpec). OT-I
T cells were identified by TCR clonotype, and proliferation was measured
using CFSE dilution. The division index was calculated as the mean number
of divisions among cells that had divided at least once.
PCR. Preparation of sorted cells and cDNA synthesis were performed as
previously described (Fletcher et al., 2009). The concentration of cDNA
was adjusted to 250 nM for all subsets. Cycling conditions for PTA analysis
were 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 54.5°C
for 45 s, and 72°C for 45 s. For TLR analysis, PCR conditions were
94°C for 5 min, followed by 40 cycles of 94°C for 60 s, 55°C for 60 s,
and 72°C for 1 min. QPCR was performed with 200 nM of validated
primers in 25-µl reactions using the Platinum SYBR Green One-Step
qPCR kit (Invitrogen) and the following program: 50°C for 2 min, 95°C
for 10 min, and 40 cycles of amplification at 95°C for 15 s and 60°C for
60 s. Relative levels of target mRNA were compared with GAPDH using
the 2Ct method, where the control cell type or treatment group was
normalized to 1. Primers were obtained from Integrated DNA Technolo-
gies. Sequences were as follows: TLR3, (For) 5-TTGTCTTCTGCAC-
GAACCTG-3 and (Rev) 5-CGCAACGCAAGGATTTTATT-3;
TLR7, (For) 5-TTCCGATACGATGAATATGCACG-3 and (Rev)
5-TGAGTTTGTCCAGAAGCCGTAAT-3; TLR8, (For) 5-GGCAC-
AACTCCCTTGTGATT-3 and (Rev) 5-CATTTGGGTGCTGTTGT-
TTG-3; B-actin, (For) 5-TGGAATCCTGTGGCATCCATGAAAC-3
induce autoimmunity unless present at very high frequency
and with CD4+ T cell help (Hamilton-Williams et al., 2005).
Although tolerance in the iFABP-tOVA model is pri-
marily deletional, residual OT-I T cells retain the ability to
induce intestinal disease if cross-primed during an unrelated
inflammatory process (Vezys et al., 2000; Vezys and Lefrançois,
2002). Up-regulation of PD-L1 may decrease the chance of
this occurring. Cross-priming in this model has also been
linked to CD40 activation, and it is striking that, of all the
co-stimulatory molecules tested, LNSCs uniformly lack any
change in CD40 expression in response to inflammation
(Vezys and Lefrançois, 2002).
Collectively, these data paint a far more intricate picture
of LNSC-mediated tolerance than previously appreciated.
We show that LN FRCs express PTAs and can act as effec-
tive APCs for naive CD8+ T cells, and that FRCs possess
reduced capacity to stimulate T cells after TLR3 ligation.
FRCs, LECs, and BECs all express virus-responsive TLR3,
and LECs are the only LNSC subset to express Tyr, an anti-
gen for which LNSC-based tolerance is crucial (Nichols et al.,
2007). Tolerance to other PTAs, particularly retinal S anti-
gen, which is strongly expressed by BECs, is yet to be dem-
onstrated, but our results indicate that multiple stromal cell
types are likely to contribute to peripheral tolerance.
The function, location, and lineage of the DN subset is
unknown, but the evidence suggests that these cells are im-
portant components of the stromal niche, expressing the
highest levels of Aire but lacking TLR3 expressed by DCs
and other LNSC subsets, and showing a strong, unique re-
sponse to inflammation by up-regulating PTA expression
(Fig. 4 E). These cells will form the subject of further study.
It is striking that LNSCs should show reduced ability to
stimulate T cells after contact with microbial products that
signal a potential threat. There are two plausible reasons for
this. The first is to prevent low-affinity autoreactive T cells
from acquiring effector function during an immune response.
The second is more crucial: to preserve LN structure and
function during an immune response by preventing activation
of CD8+ T cells specific to viral antigens presented by stromal
cells. Indeed, Mueller et al. (2007) reported that a lympho-
cytic choriomeningitis virus strain directly infecting FRCs
was associated with reduced T cell–mediated damage to the
structure of the LN. Clearly the tolerogenic capacity of these
cells is not limited to endogenous PTAs. This makes them an
attractive target for autoimmune and transplantation tolerance
therapy. Finally, the expression of PTAs across stromal subsets
of diverse primary function and lineage suggests that tolerance
induction is not a specialist function in LNs, supporting and
extending a recent parallel hypothesis that PTA expression
may be a general function of all epithelium (Dooley et al.,
2009). In this report, we show that in LNs, PTA expression is
a responsibility shared between all parenchymal cell types.
MATERIALS AND METHODS
Mice. Male C57BL/6 mice aged 4–6 wk were obtained from the Jackson
Laboratory; iFABP-tOVA mice were bred in house from the 232-4 transgenic

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and (Rev) 5-TAAAACGCAGCTCAGTAACAGTCCG-3; OVA, (For)
5-CACAAGCAATGCCTTTCAGA-3 and (Rev) 5-GAATGGATGGT-
CAGCCCTAA-3 or (For) 5-GCAAACCTGTGCAGATGATG-3 and
(Rev) 5-CACCAACATGCTCATTGTCC-3; Mlana, (For) 5-CTGCT-
GAAGAGGCCGCAGGG-3 and (Rev) 5-GGAGCGTTGGGAACCA-
CGGG-3; Tyr, (For) 5-ATTGATTTTGCCCATGAAGC-3 and (Rev)
5-GGCAAATCCTTCCAGTGTGT-3; retinal S antigen, (For) 5-TGA-
CTACCTACCCTGTTCAG-3 and (Rev) 5-TTCACTGGATGTGA-
GCTCTC-3; Rrad, (For) 5-GGGAACAGGATGGGGGCTGC-3 and
(Rev) 5-TGGCGCGGAAGGCCATCTTG-3; AFP, (For) 5-GCTGT-
GGTGAGGGAATGGCCG-3 and (Rev) 5-CCGCCAGCTGCTCCTC-
TGTC-3; PLP, (For) 5-CAGGGGGCCAGAAGGGGAGG-3 and (Rev)
5-GCAGCACCCACAAACGCAGC-3; Aire, (For) 5-AGGCTCCCA-
CCTGAAGACTAA-3 and (Rev) 5-CACGGTCACAGCTCTCTGG-3;
Deaf-1, (For) 5-ACTCTGAGTGGCCCTGTCAG-3 and (Rev) 5-TGT-
CAAAGGTCAGTGCTCC-3; and GAPDH, (For) 5-AACTTTGGCA-
TTGTGGAAGG-3 and (Rev) 5-ACACATTGGGGGTAGGAACA-3.
Antibodies. Primary antibodies were as follows: CD45 (clone 30-F11),
CD106 (clone 429), Sca-1 (clone D7), and PD-L1 (clone MIH5; BD); anti-
CD31 (clone MEC13.3), MHC class I (clone 28-8-6), CD40 (clone
HM40.3), CD80 (clone I6-10A1), CD44 (clone IM7), and CD8 (clone
53-6.7; BioLegend); and anti-CD140a (APA5) and CD25 (clone PC61.5;
eBioscience). Anti-CD86 (clone RMMP-2) and V2 TCR (clone B20.1)
were purchased from Invitrogen, and anti-CD326 (clone G8.8) was pur-
chased from Santa Cruz Biotechnology, Inc. Clone MTS16 was created and
grown in house. Anti-gp38 was purified in house from clone 8.1.1
obtained from the Developmental Studies Hybridoma Bank.
Statistical analysis. Statistical analysis was performed using Prism 4 for
Macintosh (GraphPad Software, Inc.). Data were compared using an un-
paired two-tailed t test with 95% confidence intervals.
This work was supported by National Institutes of Health grants R01 DK074500
and P01 AI045757 (to S.J. Turley), and a National Health Medical Research Council
Postdoctoral Biomedical Training Fellowship (to A.L. Fletcher).
The authors have no conflicting financial interests.
Submitted: 10 December 2009
Accepted: 23 February 2010
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