The Kidney-Renal Lymph Node-System
Contributes to Cross-Tolerance against
Innocuous Circulating Antigen
This information is current as
of June 18, 2017.
Veronika Lukacs-Kornek, Sven Burgdorf, Linda Diehl,
Sabine Specht, Miroslaw Kornek and Christian Kurts
J Immunol 2008; 180:706-715; ;
doi: 10.4049/jimmunol.180.2.706
http://www.jimmunol.org/content/180/2/706
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References
The Journal of Immunology
The Kidney-Renal Lymph Node-System Contributes to
Cross-Tolerance against Innocuous Circulating Antigen1
Veronika Lukacs-Kornek,* Sven Burgdorf,* Linda Diehl,* Sabine Specht,† Miroslaw Kornek,*
and Christian Kurts2*
D
endritic cell (DC)3 capture Ags in nonlymphatic organs
and transport them to draining lymph node (LN) for presentation to naive T cells (1– 4). DC migration can be
triggered by microbial stimuli and requires the chemokine receptor
CCR7 (5–7). Activation of cytotoxic CD8⫹ T cells is facilitated by
specialized CD8␣⫹ DC capable of presenting endocytosed Ag to
CD8⫹ T cells (cross-presentation) (8, 9). Recent findings showing
that migratory DC transferred viral Ag to CD8␣⫹ DC resident in
cutaneous or pulmonary LNs (10 –12) have challenged the traditional view that migratory DC directly activate CD8⫹ T cells in
draining LNs.
In the absence of infection, low-level CCR7-dependent migration of immature DC has been observed, for example, in the skin,
gut, and lungs (4, 6, 7, 13–16). Such “steady-state” migratory DC,
which presumably carry peripheral tissue autoantigens, are believed to directly tolerize autoreactive T cells in draining LNs (4,
13). Although tolerance of CD8⫹ T cells could be induced by
LN-resident non-DC-expressing autoantigen themselves (17), tolerance against exogenous autoantigens (cross-tolerance) has been
shown to be induced by DC presenting, for example, pancreatic,
*Institute of Molecular Medicine and Experimental Immunology and †Institute of
Medical Microbiology, Immunology, and Parasitology, Friedrich-Wilhelms-Universität, Bonn, Germany
Received for publication April 27, 2007. Accepted for publication October 27, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Sonderforschungsbereich 704 and by the Klinische
Forschergruppe 155 of the Deutsche Forschungsgemeinschaft.
2
Address correspondence and reprint requests to Dr. Christian Kurts, Institute of
Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-Universität,
53105 Bonn, Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; rLN, renal
LN; kDC, kidney DC; MHC II, MHC class II; aOVA, Alexa647-labeled OVA; 7AAD,
7-aminoactinomycin D; cLN, cutaneous LN; MFI, mean fluorescence intensity; NX,
nephrectomy; MR, mannose receptor; FSC-w, forward scatter pulse width.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
renal, and intestinal Ag (9, 18 –20). Cross-tolerizing DC in pancreatic LNs expressed CD8␣ (21), whereas those in the mesenteric
LN were CD8␣⫺ (20) and those in renal LNs (rLNs) could be
either (22). However, in these sites, steady-state DC migration has
not been reported. Thus, it remains to be clarified whether crosstolerance is induced by migratory DC that carried Ag into draining
LNs, or by LN-resident DC that received organ-derived Ag by
other means, for example, from other, migratory DC, as reported in
cutaneous or pulmonary herpes virus infection (10 –12).
Cross-tolerance may be required also against certain nonself
proteins, such as innocuous self-serum or food proteins or orally
administered Ag. Systemic injection of soluble Ag devoid of inflammatory stimuli has been widely used to study such T cell tolerance (23–25). Cross-tolerance against blood-borne soluble Ag is
thought to be induced in the spleen, because cross-presentation of
such Ag is particularly efficient in this organ (26 –28), but not in
LNs (29). Experimental evidence also suggested a role of the liver
in tolerogenic presentation of soluble Ag (30).
The kidney is a site of extensive turnover of circulating soluble
protein Ags. Ags below albumin molecular size (68 kDa) can pass
the glomerular filter and reach the tubular lumen (31). To prevent
their loss with the urine, they are reabsorbed by tubular epithelial
cells (32) and released in degraded form into peritubular capillaries. Recently, it was shown that filterable dextran molecules are
not only taken up by tubular epithelial cells, but also by kidney
DCs (33). The kidney contains remarkably abundant numbers
of DC (34), whose function is largely unresolved. Murine kidney
DCs (kDCs) were characterized by CD11c, MHC class II (MHC
II), and CD11b expression and an immature phenotype, and elicited weak T cell responses in vitro (34). In vivo, they suppressed
pathogenic CD4⫹ T cells in experimental crescentic glomerulonephritis and attenuated disease (35). Their location in the tubulointerstitium may allow capture of pathogens that ascend through the
tubular system or of circulating Ags that entered this system via
glomerular filtration (36). In the present study, we demonstrate
selective enrichment of circulating filterable Ag in the kidney. This
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Soluble Ags devoid of inflammatory stimuli, derived for example from self-serum or food proteins, induce T cell tolerance,
predominantly in the spleen. In this study, we describe an additional role of the kidney-renal LN (rLN) system in tolerogenic
presentation of circulating soluble Ags. Protein below albumin molecular mass constitutively passed the kidney glomerular filter
and was concentrated in the tubular compartment. Enriched filterable Ag was endocytosed by kidney dendritic cells (kDCs).
Simultaneously, it was transported cell independently within 2 min to DCs resident in rLNs. These DC phenotypically differed
from kDCs carrying filterable Ag, and used a distinct mechanism, mannose receptor-mediated endocytosis, to internalize Ag. They
activated specific CD8ⴙ T cells, which subsequently proliferated without producing effector cytokines or developing cytotoxic
activity, showed a curtailed lifespan and signs of apoptosis. Such T cell tolerization was independent of steady-state migratory
kDC, because it occurred also when nephrectomy was performed soon after Ag injection. These findings demonstrate that the
kidney dispatches concentrated blood-borne Ags to the rLNs, where they are captured by resident DCs, resulting in CD8ⴙ T cell
cross-tolerance. This mechanism may contribute to avoiding immunity against innocuous circulating protein Ags below albumin
size. The Journal of Immunology, 2008, 180: 706 –715.
The Journal of Immunology
707
phenomenon allowed studying transport of kidney-derived Ag to
rLNs, and consequences for T cell activation. We provide evidence
for a complementary mechanism of cross-tolerance induction that
is unique to the kidney and independent of steady-state DC
migration.
rompun (aniMedica). The peritoneal cavity was opened by a lateral cut, the
kidney was lifted and hilus vessels were ligated. The kidney was then
removed, leaving rLN, adrenal gland in situ. Renal blood vessels and the
ureter were cauterized and the body cavity was closed.
Materials and Methods
The FLICA apoptosis detection kit (Axxora) was used to detect caspase 3/7
activity according to the manufacturer’s guidelines. As the FAM reagent
was fluorescent at the same wavelength as CFSE, OT-I cells were labeled
by resuspension in PBS containing 4 ␮M Far-red (Molecular Probes) at
37°C for 15 min. To determine caspase 3/7 activity, single-cell suspensions
from mice injected with Far-red labeled OT I cells at a concentration of
2 ⫻ 105 cells/200 ␮l were incubated with 5 ␮l of FAM reagent at 37°C for
45 min. Late apoptotic/dead cells were labeled with 7AAD.
Reagents and mice
Abs and flow cytometry analysis
Cells were stained using the following fluorochrome-labeled Abs: antiCD8, V␣2-TCR, V␤-5 TCR, CD11c, CD44-biotin, CD62L-biotin,
CD25-biotin (BD Pharmingen), B7-DC, B7-H1, and CD90.2 (Thy1.2)
(eBioscience). FcRs were blocked with rat anti-FcR from 24G2 hybridoma
supernatant; dead cells were excluded by Hoechst-33342 dye or 7-aminoactinomycin (7AAD). Flow cytometry was performed on a LSR II using
FACS Diva software (BD Biosciences) and analyzed using FlowJo software (Tristar Software).
Intracellular cytokine staining
Forty-eight hours after priming, single-cell suspensions were prepared and
restimulated with SIINFEKL for 4 h in the presence of GolgiPlug (BD
Biosciences). Cells were fixed for 15 min at 4°C with 2% paraformaldehyde in PBS, then washed and incubated for 20 min in PBS 0.1% BSA,
0.5% saponin, followed by an additional 30 min of incubation with fluorochrome-conjugated Ab IL-2 PE and IFN-␥ allophycocyanin (BD Biosciences) in PBS 0.1% BSA, 0.5% saponin, and unconjugated rat IgG.
Purification of OT-I cells
OT-I cells were isolated from OT-I.Rag⫺/⫺ mice as described (9). Briefly,
spleen and LN cells were treated with erythrolysis buffer (146 mM NH4Cl,
10 mM NaHCO3, 2 mM EDTA) to remove RBC. Cells then were labeled
with CFSE (Invitrogen Life Technologies) as described (18) by resuspension at 10 –20 ⫻ 106 cells/ml in PBS 0.1% BSA (GERBU), followed by
addition of CFSE to a final concentration of 5 ␮M for 10 min at 37°C.
Purity of OT-I cells was usually ⬃75– 85% of viable lymphocytes.
Isolation of DC from mice
DC were isolated from kidney, spleen, and LN by digestion with collagenase
(Roche Diagnostic) and DNase I (Sigma-Aldrich) as described (34). Tubular
fragments from digested kidneys were removed by sedimentation. For determining expression of DC subtype markers such as CD8␣, only forward scatter
pulse width (FSC-w) single events were analyzed as shown in Fig. 2F.
In vivo cytotoxicity assay
In vivo cytotoxicity assays were performed as described (12). In brief,
spleen cells were either pulsed with SIINFEKL (1 ␮g/ml, 45 min at 37°C)
and labeled with 1 ␮M CFSE (CFSEhigh cells), or were not pulsed with
peptide and labeled with 0.1 ␮M CFSE (CFSElow cells). A total of 1 ⫻ 107
of both target cell types were injected i.v. After 4 h, the survival of target
cells in spleen and LN was analyzed by flow cytometry. Specific lysis was
calculated using the formula: percent-specific cytotoxicity ⫽ 100 ⫺
(100 ⫻ (CFSEhigh/CFSElow)primed/(CFSEhigh/CFSElow)control).
Nephrectomy (NX)
NX was conducted as described (9). Briefly, mice were anesthetized with
0.1 mg/g body weight ketamine (Pharmacia) and 0.01 mg/g body weight
Statistical analysis
Results are expressed as mean ⫾ SD; n indicates the number of animals per
group. Comparisons were drawn using a two-tailed Student t test (Prism 4;
Graphpad Software).
Results
kDCs preferentially endocytose filterable Ag
Molecules below albumin molecular size (68 kDa) constitutively
pass the kidney glomerular filter and are reabsorbed by tubular
epithelial cells (37, 38). kDCs have recently been shown to take up
filterable 40-kDa and nonfilterable 500-kDa dextran molecules
(33). We speculated that the concentration of the glomerular filtrate in the tubular system might cause enrichment of filterable
dextran in kDCs, but not in DCs in other sites such as the spleen
or the cutaneous LNs (cLNs). To test this hypothesis, we investigated the simultaneous uptake of filterable and nonfilterable dextrans by coinjecting Cascade-Blue-labeled 10-kDa and FITC-labeled 500-kDa dextran i.v. into C57BL/6 mice. Indeed, 1 h after
injection, kDCs carried more filterable than nonfilterable dextran,
whereas DC from cLNs had internalized both markers with similar
efficiency (Fig. 1A). Splenic DC took up high amounts of large
dextran, but reproducibly little small dextran for unknown reasons
(Fig. 1A).
We decided to investigate whether this led to immunological
consequences. To this end, we replaced the 10-kDa dextran with
the model Ag, OVA, in an aOVA form, and coinjected it together
with 500-kDa dextran as marker for nonfilterable molecules, which
could be acquired only from the blood. Splenic DC endocytosed
both molecules with high efficiency exceeding the uptake by cLN
DCs, but the ratio was similar (Fig. 1B). In contrast, kDCs efficiently endocytosed aOVA, but little FITC-dextran (Fig. 1B),
again indicating superior uptake of filterable molecules and inferior uptake of blood-borne molecules. Analysis of control mice not
injected with fluorescent tracers confirmed absence of autofluorescent DCs (Fig. 1B). Uptake of aOVA was very rapid and could be
detected already after 30 s (Fig. 1, C and D). It was even larger in
kidney cells deficient for the common leukocyte marker CD45
(Fig. 1, C and D). Histology identified these cells as tubular epithelial cells (data not shown), consistent with their established
function of reabsorbing filterable protein (32, 39).
kDCs acquired i.v.-injected aOVA very rapidly. Already 30 s
after injection, ⬃12% of the kDC had accumulated some aOVA
(Fig. 1C). After 10 min, the mean fluorescence intensity (MFI) had
risen ⬎5-fold (Fig. 1, C and D), and Ag was detectable within
nearly half of the kDCs (Fig. 1C), equivalent to 30 –50 ⫻ 103 DCs
per kidney. All these DCs showed the CD11b⫹ myeloid phenotype, whereas Gr-1⫹ plasmacytoid and CD8␣⫹ DCs were absent
(Fig. 1E). Endocytosis of aOVA by kDC did not result in DC
maturation as evidenced by lack of CD80 or MHC II up-regulation
(Fig. 1F).
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All reagents were obtained from Sigma-Aldrich, if not specified otherwise.
A total of 500-kDa FITC-dextran and 10-kDa Cascade-Blue dextran were
obtained from Molecular Probes. Alexa647-labeled OVA (aOVA) was prepared using a labeling kit (Molecular Probes) according the manufacturer’s
guidelines. Dextrans diluted in PBS were injected i.v. in a total volume of 400
␮l. C57BL/6, OT-I.RAG⫺/⫺, Thy1.1, and Rag⫺/⫺ mice were bred under specific pathogen-free conditions at the central animal facility of the University
Clinic of Bonn (House for Experimental Therapy). MR⫺/⫺ mice were obtained from M. Nussenzweig (New York, NY); CCR7⫺/⫺ mice were obtained
from M. Lipp (Berlin, Germany). For all experiments, female mice between 8
and 12 wk of age were used in accordance with local animal experimentation
guidelines. For priming mice with soluble protein, OVA (grade V) was dissolved in PBS and run over Sephadex G-25 (PD10 column; Amersham) to
remove peptide contaminations. A total of 10 ␮g of soluble OVA per gram of
body weight in a total volume of 400 ␮l was injected i.v. For priming under
immunogenic conditions, an emulsion of 200 ␮g of OVA in 100 ␮l of 0.9%
NaCl and 100 ␮l of CFA was injected s.c into the flank.
Apoptosis detection
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CROSS-TOLERANCE BY LN-RESIDENT DC
Rapid cell-independent transport of filterable Ag to rLN DCs
Efficient cross-presentation of filterable Ag in the rLN
To investigate whether filterable Ag was presented to T cells, we
injected mice with OVA and CFSE-labeled transgenic OVA-specific CD8⫹ T cells (OT-I cells), which have been widely used for
detecting cross-presentation of OVA (18). Indeed, OT-I cells proliferated more vigorously in rLNs than in cLNs (Fig. 4, A and B).
After injection of 10 ␮g of OVA/g body weight, the division index
was three times higher in rLN than in cLN (Fig. 4, A and B). Also
higher aOVA doses were presented more efficiently in rLNs, whereas
doses lower than 5 ␮g/g body weight did not cause significant T cell
activation in any LN (Fig. 4, A and B). OT-I cell proliferation in the
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FIGURE 1. Selective uptake of filterable Ag by kDC. A, C57BL/6 mice
were i.v. injected with Cascade-Blue-labeled dextran (10 kDa) and 500-kDa
FITC-labeled dextran at 2:1 molar ratio. After 1 h, CD11c⫹ DCs from the
cLN, the kidney, and the spleen were analyzed for fluorescence uptake. B,
C57BL/6 mice were i.v. injected with 8 ␮g/g body weight of aOVA and of
500-kDa FITC-dextran. After 20 min, CD11c⫹ DCs from the cLN, the kidney,
and the spleen were analyzed for fluorescence uptake. Uninjected mice served
as control for background fluorescence. C, Mice were i.v. injected with 8 ␮g/g
body weight aOVA. After 30 s or 10 min, fluorescence uptake by CD45⫺
intrinsic kidney cells, and CD11c⫹ kDC was determined. Numbers in the
lower right dot plots give proportions of DCs containing aOVA ⫾ SD (n ⫽ 3
mice). D, Statistical analysis of the MFI of aOVA uptake in C. MFI background of cells from uninjected animals was subtracted (n ⫽ 3 mice). E,
Expression of CD11b, CD8␣, and Gr-1 by CD11c⫹ kDC. F, Expression of
CD80 and MHC II by CD11c⫹ kDC before (dashed line) and 1 h after aOVA
injection (tinted). Isotype controls are shown as bold lines. Shown are results
typical of three individual experiments.
Kidney autoantigens have been reported to be presented to CD8⫹
T cells in the rLN (9, 18). To test whether also filterable Ag
reached this LN, we injected mice i.v. with aOVA and determined
its uptake by rLN cells. A cLN that did not drain the kidney was
used as a control to assess the amount of aOVA taken up from the
blood. Indeed, more cells captured aOVA in the rLNs than in cLNs
(Fig. 2, A and B), and higher Ag uptake was detected (Fig. 2, A and
B). Importantly, aOVA accumulation was evident already 90 s
after aOVA injection and was further elevated after 5 min (Fig. 2,
A and B). Left NX before aOVA injection abrogated Ag accumulation in the left rLN, while that in the right rLN remained unaffected (Fig. 2C). Thus, aOVA accumulation in rLNs depended on
its enrichment in the kidney, and was not due to particularities
of rLNs.
Most rLN cells that contained filterable (kidney-derived) aOVA
were CD11c⫹CD11b⫹ cells, and thus most likely DCs, while
CD11c⫺CD11b⫹ macrophages represented a minority (Fig. 2, A
and D). Determining the absolute numbers of aOVA⫹ DCs
showed 20-fold less DCs in one rLN than in one kidney (Fig. 2E).
Interestingly, in rLNs aOVA was detected mostly in CD11clow
cells (Fig. 2, A and D), whereas aOVA⫹ DCs in the kidney were
predominantly CD11chigh (Fig. 1C). The rLN differed from the
kidney also by containing some CD8␣⫹ DCs that had captured
aOVA (Fig. 2F).
These phenotypic differences and the rapid kinetics of aOVA
accumulation suggested cell-independent Ag transport into rLNs.
To test this possibility, we took advantage of our recent discovery
that endocytosis of soluble OVA by DCs in lymphatic tissues was
facilitated by the mannose receptor (MR) (29). Indeed, this receptor was used by rLN DCs for uptake of aOVA, because no uptake
was seen in MR⫺/⫺ mice (Fig. 3A). In contrast, kDCs internalized
aOVA in a MR-independent fashion and at somewhat less efficiency (Fig. 3A). The absence of kidney DCs containing high
amounts of MR-endocytosed aOVA argued against this organ as
origin of DCs carrying such Ag in rLNs.
To confirm this interpretation, we used CCR7⫺/⫺ mice, in
which DC trafficking from nonlymphoid organs to their draining
LN is incapacitated (6, 7). Despite this defect, rLN DCs of
CCR7⫺/⫺ mice showed strong uptake of aOVA 5 min after injection, while again little uptake was noted in the cLN (Fig. 3B).
These findings verified that rapid Ag transport to the rLN must
have occurred cell independently.
In addition, we noted that DCs with high CD11c⫹ expression
were fewer in the rLNs of CCR7⫺/⫺ mice. These DCs had captured only little amounts of aOVA within this short time (Fig. 3B),
and therefore did not appear to belong to the subset of rLN DCs
that capture filterable Ag. However, it is possible that these cells
represented steady-state migratory DCs that continuously carry
Ags at a low rate to the rLN, reminiscent of the situation in the
lung draining LNs (15).
The Journal of Immunology
709
spleen was always higher than in rLNs (Fig. 4, A and B), consistent
with the efficient OVA uptake by splenic DCs (Fig. 1A).
We next investigated whether the proliferation of OT-I cells in
rLNs had been induced by filterable Ag rapidly conveyed there in
a cell-independent fashion, or by Ag shuttled there by steady-state
migratory DCs at later time points, as suggested by Fig. 3B. To
distinguish between these two possibilities, we examined proliferation of OT-I cells in mice unilaterally nephrectomized after injection of OVA. NX was conducted 90 min after Ag injection,
because this time had been reported to suffice for clearing the murine plasma of a single bolus injection of filterable molecules (40).
OT-I cell proliferation was similar in the ipsilateral and contralateral rLN of nephrectomized mice, and in sham-operated animals
(Fig. 4C), indicating that rapidly transported Ag was sufficient to
drive T cell proliferation. Hypothetical DC-mediated steady-state
Ag transport at later time points was not required.
Filterable Ag activates T cells in a tolerogenic fashion
We next investigated the phenotype of OT-I cells activated by
filterable Ag. The activation marker CD44 was increased, but
CD62L only moderately decreased when compared with naive
OT-I cells (Fig. 5A). However, these activation signs were
much less pronounced than those displayed by OT-I cells proliferating in the draining cLN of mice s.c. injected with OVA in
CFA (OVA/CFA), which served as control for immunogenic
priming (Fig. 5A). In particular, immunogenic priming resulted
in strong up-regulation of the high-affinity IL-2R ␣-chain,
CD25, whereas OT-I cells in the rLN showed levels hardly
above those of naive cells (Fig. 5A). This CD44⫹CD25⫺CD62Lint
expression pattern was consistent with previous studies on the phenotype of tolerized T cells (9, 18, 19, 30, 41– 43). The few OT-I
cells proliferating in response to soluble OVA in nondraining
cLNs also showed this phenotype, albeit less pronounced (Fig. 4,
A and B).
Consistent with this surface phenotype, OT-I cells activated by
filterable Ag produced little IFN-␥ and hardly any IL-2 upon restimulation with the antigenic OVA peptide, whereas OT-I cells
responding to OVA/CFA produced both cytokines well (Fig. 5, B
and C). OT-I cells produced low cytokine amounts also when mice
were unilaterally nephrectomized 90 min after OVA injection (Fig.
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FIGURE 2. Rapid accumulation of filterable Ag in rLN DCs. A, C57BL/6 mice were injected i.v. with 8 ␮g/g body weight aOVA. After 90 s or 5 min,
cells from rLNs and cLNs were stained for CD11c. Dot plots show typical aOVA content and CD11c expression in total viable 7AAD⫺ LN cells. B,
Statistical analysis of A, giving MFI of CD11c⫹ DCs 90 s and 5 min after aOVA injection (n ⫽ 5 mice). C, A total of 8 ␮g/g body weight aOVA was
i.v. injected 1 day after surgical removal of the left kidney. aOVA content in CD11c⫹ DCs was detected in the left and right rLN and in the cLN (n ⫽
6 mice, rLNs from two animals were pooled for analysis). D, Proportions of CD11clow, CD11chigh DCs and of CD11c⫺CD11b⫹ cells within the aOVAcontaining rLN cells from A. E, Absolute numbers of CD11c⫹ aOVA⫹ DCs in one kidney and in the left rLN (n ⫽ 3 mice). F, Viable CD11c⫹ DCs in
rLNs were analyzed for CD8␣ expression and aOVA uptake. Doublet events were excluded by a FSC-w gate. The number gives mean ⫾ SD of CD11c⫹
positive for both parameters (n ⫽ 3 mice). Without doublet exclusion, this value was 2.8 ⫾ 0.5%. Shown are results typical of three individual experiments.
710
CROSS-TOLERANCE BY LN-RESIDENT DC
FIGURE 3. Ag uptake in rLNs depends on the MR, but not on CCR7. A, MR⫺/⫺ or wild-type mice were injected i.v with 6 ␮g/g body weight aOVA.
After 5 min, CD11c⫹ cells in kidney and rLN were analyzed for aOVA MFI (n ⫽ 3 mice). Background fluorescence intensity detected in DCs from
noninjected mice was subtracted from the MFI values. B, CCR7⫺/⫺ or wild-type mice (two per group) were injected i.v with 6 ␮g/g body weight aOVA.
After 5 min, total rLN and cLN cells were analyzed for CD11c expression and Ag uptake. Results are representative for two individual experiments.
T cells activated by filterable Ag possess a curtailed lifespan
To provide more evidence for tolerance induction, we determined
the lifespan of T cells activated by filterable Ag. To this end, OT-I
cells activated in the rLN of OVA-injected mice had to be sepa-
rated from those activated in other locations and from Ag. This
was achieved by adoptive transfer into RAG⫺/⫺ secondary recipient mice to examine survival of OT-I cells 2 wk after transfer
(experimental plan in Fig. 7A). The very small cell numbers in the
tiny rLN precluded isolation from other cell types. Therefore, total
rLN cells pooled from several mice were transferred, and subsequently OT-I cell proportions within transferred CD8⫹ T cells
were examined. As a control for survival after immunogenic priming, draining cLN cells from OT-I cell-injected mice injected with s.c.
with OVA/CFA were transferred. A further control for survival of
naive OT-I cells was necessary, because rLNs contained not only
OT-I cells activated by filterable Ag, but also nonactivated OT-I cells
(Figs. 4 and 5) that presumably had not yet encountered OVA-presenting DCs after entering the node. Such naive OT-I cells may survive, resulting in a background level below which OT-I cell levels
cannot fall even if all activated cells were deleted. To define this
background, we also transferred rLN cells from mice injected with
OT-I cells but not with OVA.
FIGURE 4. Enrichment of filterable Ag in rLN DCs increases specific CD8⫹ T cell activation. A, A total of 2 ⫻ 106 CFSE-labeled OT I cells were
injected into C57BL/6 mice. Eighteen hours later, different doses of OVA were i.v. injected. Forty-eight hours after OVA injection, the proliferation of OT
I cells was analyzed in spleen, cutaneous, and rLN. Histograms show the proliferation profiles of CFSE⫹CD8⫹ cells. The numbers indicate the division
index ⫾ SD (n ⫽ 3 mice). B, Statistical analysis of A. The ratio between the division indices in the spleen or in rLNs divided by that of the cLN was given
as a quantitative parameter for the superiority of spleen and rLNs in uptake of soluble Ag at different doses. C, Division indices of proliferating OT-I cells
were determined in the left and right rLN after left NX, and in the left rLN of sham-operated animals (n ⫽ 6 mice/group, rLNs from two mice were pooled
for analysis). Shown are results typical of two individual experiments.
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5, D and E). Thus, rapidly transported Ag was sufficient for imprinting OT-I cells for defective cytokine production, demonstrating once more that hypothetical DC-mediated Ag transport at later
time points was not required.
Next, we determined whether OT-I cells developed OVA-specific cytotoxic effector function after priming by filterable Ag. Cytotoxicity was evident neither in rLNs, nor in any other location
(Fig. 6). In contrast, cytotoxicity was detected in the draining cLN
of mice s.c. injected with OVA/CFA, and to a lower extent in other
LNs and in the spleen (Fig. 6), presumably due to recirculation of
activated OT-I cells. In summary, OT-I cells primed by filterable
Ag expressed reduced activation markers, lacked cytokine production and cytotoxic activity, consistent with tolerance induction.
The Journal of Immunology
711
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FIGURE 5. T cells activated by filterable Ag in rLN show a tolerized phenotype. A, A total of 2 ⫻ 106 CFSE-labeled OT I cells were injected into
C57BL/6 mice. After 18 h, either 10 ␮g/g body weight of OVA was injected i.v. or OVA-CFA s.c. Forty-eight hours later, expression of CD44, CD25,
and CD62L on OT-I cells proliferating in response to these two Ag forms or in unprimed control mice was determined. Quantitative analysis of activation
marker expression, expressed as MFI for each cell division, is shown on the right (n ⫽ 3 mice/group). B, Intracellular IL-2 and IFN-␥ staining of
CFSE-labeled OT-I cells proliferating in response to filterable OVA or to OVA/CFA as described in A. C, Quantitative analysis of B, showing the
proportions of cytokine-producing OT-I cells in different organs (three to four mice per group). D, Same experimental setup as in B, except that left NX
was performed 90 min after injection of OVA. Shown are the intracellular IL-2 and IFN-␥ content of OT-I cells proliferating in the left and right rLN and
in the left rLN of sham-operated animals. E, Quantitative analysis of D, showing the proportions of cytokine-producing OT-I cells (six mice per group, rLNs
from two mice were pooled for analysis). Shown are results typical of two individual experiments.
Before this secondary transfer, OT-I cells were more abundant
in mice primed with soluble OVA or with OVA/CFA than in
unprimed animals (Fig. 7B), as a consequence of proliferation in
response to Ag (Figs. 4 and 5). Two weeks after transfer into
secondary recipients, the frequency of OT-I cells primed by filterable Ag was reduced to the background level of naive OT-I cells
(Fig. 7B). In contrast, the frequency of OT-I cells activated by
OVA/CFA had not declined, but showed a tendency toward expansion (Fig. 7B), indicating that immunogenically primed OT-I
cells had been programmed to survive, as opposed to those primed
by filterable Ag. Their loss was Ag specific, because the frequency
of V␣2⫹V␤5⫺CD8⫹ cells, which were examined as examples for
712
CROSS-TOLERANCE BY LN-RESIDENT DC
FIGURE 6. T cells activated by filterable Ag in rLNs do not show cytotoxic activity. Mice were injected with 1 ⫻ 104 naive OT I cells and after
18 h, with either 10 ␮g/g body weight of soluble OVA i.v. or of OVA-CFA
s.c. Five days later, target cells were injected and 4 h later specific in vivo
cytotoxicity was determined in different organs (n ⫽ 3 mice). Shown are
results typical of two individual experiments.
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non-OVA-specific CD8⫹ T cells, was not significantly altered in
any experimental group (unprimed 6.6 ⫾ 2.8, filterable Ag 3.5 ⫾
1.4, OVA/CFA 3.3 ⫾ 0.3% of total CD8⫹ T cells, n ⫽ 3, p ⬎ 0.17
for all comparisons).
We also followed the fate of OT-I cells activated by filterable
Ag in RAG-competent secondary recipients, in which they had to
compete with endogenous T cells for entry into the pool of recirculating lymphocytes. As we expected this to be far more difficult,
we used as first and secondary recipients Thy1.1 congenic mice, in
which OT-I cells expressing Thy1.2 are easier to track. In addition,
we transferred pooled rLN cells from six primary recipients into a
single secondary recipient (precluding statistical analysis) and analyzed already on day 5 (experimental protocol in Fig. 7C). In this
experiment, we recovered from the secondary recipient only 2.2%
from the transferred OT-I cells primed by filterable Ag, as compared with the background of 6.1% recovery of transferred naive
OT-I cells from unprimed controls (Fig. 7D). In contrast, 16% of
the OT-I cells primed by OVA/CFA were retrieved (Fig. 7D),
consistent with better survival after immunogenic priming as compared with naive OT-I cells. OT-I cells from the spleen were almost completely lost (0.15% recovery, Fig. 7D), confirming its
dominant role in tolerance induction against soluble Ag. When we
examined IFN-␥ production as a functional parameter, 41% of the
OT-I cells primed by OVA/CFA produced this cytokine, as opposed to only 1.9% of the cells primed by filterable Ag in the rLN
(Fig. 7E). A total of 12.5% of OT-I cells primed in the spleen did
so, but this corresponded to a very low absolute number of cells,
due to their almost complete loss (Fig. 7E).
Next, we examined OT-I cells primed by filterable Ag for
direct evidence for apoptosis. To this end, we used the fluorescence reagent, FAM, which covalently binds to active caspase
3 and 7. Indeed, after priming with soluble OVA more apoptotic
OT-I cells were present in the rLN than in the spleen, or in the
cLN after immunogenic priming with OVA/CFA (Fig. 8). The
number of apoptotic cells detectable at one time point was
small, consistent with rapid clearance of apoptotic cells in vivo,
as opposed to the in vitro situation, in which apoptosis mechanisms are often studied. Taken together, the tolerized phenotype, lack of cytotoxicity, reduced lifespan and the higher proportion of apoptotic OT-I cells supported tolerance induction by
filterable Ag in the rLN.
Finally, we studied whether DCs in the rLNs that had captured
filterable Ag showed any particularities that may be related to tolerance induction. B7-H1 (PD-L1) expression was unchanged in
any site (data not shown), whereas aOVA⫹ DCs in the rLN and the
FIGURE 7. T cells activated by filterable Ag in rLNs possess a curtailed
lifespan. A, Experimental plan for B: 2 ⫻ 106 CFSE-labeled OT I cells were
injected into C57BL/6 mice. After 18 h, mice were injected with either 10 ␮g/g
OVA i.v. or OVA-CFA s.c. After 48 h, single-cell suspensions from the rLNs
or the cLNs were transferred into RAG⫺/⫺ secondary recipient mice. rLN cells
were pooled from six to eight first recipient mice. Twelve days later, spleen
and LN cells from RAG⫺/⫺ mice were analyzed for the frequency of OT-I
cells. B, Proportions of V␣2⫹V␤5⫹ OT-I cells in all CD8⫹ cells before and
after transfer into RAG⫺/⫺ mice (two experiments using two to three mice per
group). C, Experimental plan for D and E: 2 ⫻ 106 CFSE-labeled OT I cells
(Thy.1.2) were injected into C57BL/6 Thy 1.1 congenic mice. After 18 h, mice
were injected with either 10 ␮g/g OVA i.v. or OVA-CFA s.c. After 48 h,
single-cell suspensions from the rLNs, cLNs, or spleen were transferred into
RAG-competent secondary recipient mice (C57BL/6.Thy1.1). To this end,
rLN cells from six first recipient mice were pooled. After 5 days, the spleens
and LNs from secondary recipient mice were taken and analyzed for surviving
Thy1.2⫹ OT-I cells. D, The proportions of OT-I cells recovered from secondary recipients per transferred OT-I cells. E, The proportions of IFN-␥-producing cells among recovered OT-I cells.
The Journal of Immunology
713
cause only very few DCs had captured aOVA there (Fig. 2, gray
areas).
Discussion
spleen expressed more B7-DC (PD-L2), a negative regulator of T
cell activation (44, 45), than other DCs (Fig. 9). Analysis of
B7-DC on Ag-bearing DCs in other LNs was not possible be-
FIGURE 9. Expression of B7-DC by T cells activated by filterable Ag.
C57BL/6 mice were injected i.v with 6 ␮g/g body weight aOVA. After 45
min, CD11c⫹ DC in the rLN, cLN, mesenteric LN (mLN), spleen, and
kidney were analyzed for Ag uptake and B7-DC expression. The black line
indicates unstained cells, the black shaded line total CD11c⫹ DCs, and the
gray line with gray area the aOVA⫹ and CD11c⫹ cells.
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FIGURE 8. Apoptosis of T cells activated by filterable Ag in rLNs. A,
A total of 2 ⫻ 106 Far-red-labeled OT I cells were injected into C57BL/6
mice. After 18 h, mice were injected with either 10 ␮g/g OVA i.v. or
OVA-CFA s.c. After 48 h, single-cell suspensions from the rLNs, the
cLNs, and the spleen were prepared and caspase 3/7 activity was determined on OT-I cells identified by gating for CD8⫹ Far-red⫹ events.
FAM⫹7AAD⫺ represented early apoptotic and FAM⫹7AAD⫹ late apoptotic/dead OT-I cells. Statistical analyses of apoptotic cells are shown (n ⫽
3 mice/group). B, A representative dot plot is shown.
Systemic injection of soluble Ag devoid of inflammatory stimuli
has been widely used to study mechanisms of T and B cell tolerance (23–25). This system is thought to mimic presentation of
innocuous self-serum proteins and possibly also orally administered Ag, against which immunity is not required or may even be
harmful. It is generally assumed that such tolerance is induced in
the spleen, which presents soluble Ags to T cells particularly well
(26 –28). However, splenectomy usually does not result in autoimmunity against otherwise ignored soluble Ags, suggesting that
further sites may exist where tolerance can be induced. Here, we
propose the kidney-rLN system as one such additional site.
Liquid reabsorption from the glomerular ultrafiltrate causes enrichment of filtrated molecules below albumin size in the kidney
tubular system (31). Unlike metabolic waste products, such proteins are not excreted with the urine, but instead are reabsorbed by
tubular epithelial cells. It has recently been reported that filterable
dextran molecules also reached DCs in the kidney (33). Our approach of simultaneous injection of small and large fluorescent
molecules allowed demonstration that uptake from the tubular filtrate was far more efficient than that from the circulation. Furthermore, it revealed selective enrichment of filterable protein Ag in
kDC and rLN DCs. Although the functional relevance of Ag accumulation in kDC remains to be addressed, the enrichment in
rLNs could be shown here to result in efficient activation of naive
CD8⫹ T cells at concentrations too low for priming in any other
LN. The cells that acquired such Ag were most likely DCs, because in our experiments, they could activate naive OT-I cells.
These DCs may be related to a recently described MR⫹ DC subset
located in the paracortex of peripheral LNs (46). Also the spleen
enriched soluble protein Ag, and even more efficiently so than
rLNs. This observation was consistent with the previous identification of a splenic conduit system that efficiently targeted soluble
small molecules to DCs in the splenic paracortex (28). Also, LNs
(47, 48) and the thymus (49) contain conduit systems. Such a system may have conveyed filterable Ag arriving with the lymph flow
toward resident MR⫹ DCs within rLNs. The concentration mechanism in the kidney may complement these anatomical structures
at improving presentation of soluble Ag to T cells in the LN.
Presentation of filterable Ag induced T cell tolerance, as evidenced by low activation markers, lack of significant cytokine production, absence of cytotoxic activity, higher apoptosis-indicating
caspase 3/7 activity, and a curtailed lifespan of OT-I cells, all of
which are characteristic for tolerized T cells (9, 18, 19, 30, 41– 43).
The few OT-I cells proliferating in nondraining cLNs in response
to nonenriched circulating Ag did not possess a reduced lifespan,
but showed a tolerized phenotype, suggesting that also other LNs
may contribute to tolerization, albeit to at a lesser extent because
they were not supplied with concentrated Ag by the kidney. Determining the lifespan of OT-I cells primed in individual LNs was
technically demanding, because not only activated, but also naive
OT-I cells that had not yet encountered filterable Ag were present
in these nodes. The extremely low cell numbers in the rLN precluded separation of naive and activated OT-I cells for determining
their individual lifespans. Nevertheless, transfer of unseparated
OT-I cells allowed demonstration of their decline to levels below
those observed after transfer of naive OT-I cells both in RAGdeficient and -competent recipient mice. This demonstrated that
OT-I cells activated by filterable Ag had been deleted, while the
naive ones in the rLNs survived. In contrast, the high proportions
of immunogenically activated OT-I cells had increased to some
714
triggered migration. Our approach of i.v. injection of tracers bona
fide allowed physiologic labeling of DCs and reproducibly did not
result in any detectable maturation. Under these conditions, tolerogenic Ag was relocated without the need for DC migration. These
findings do not exclude DC-mediated Ag transport to rLNs at later
time points, but our NX experiments demonstrated that such hypothetical steady-state migration was not required for tolerance in
our system.
Others have previously shown that LN-resident DCs could obtain viral Ag from skin- or lung-derived migratory DCs (10 –12).
Apart from the organ studied, these systems differ from ours by the
presence of an infectious agent that could mature DCs and induce
their migration. In contrast, innocuous Ag injected into the skin
reached the LN through lymph vessels before skin-derived DCs
had immigrated (56, 57). Also the kidney is connected with its
draining LN by lymph vessels, as demonstrated by cannulating
experiments in sheep (58). As cannulation of the tiny renal lymph
vessels of mice has not been achieved yet, we were unable to
directly prove that rapid cell-independent relocation of filterable
Ag occurred through lymph vessels. Nevertheless, our results demonstrate continual DC-independent relocation of innocuous circulating Ag from the kidney to the rLN and local uptake by tolerogenic resident DCs. This mechanism may contribute to preventing
unwanted immunity against innocuous foreign or self proteins in
the serum.
Acknowledgments
We thank M. C. Nussenzweig for MR⫺/⫺ mice and M. Lipp for CCR7⫺/⫺
mice. We acknowledge support by the Flow Cytometry Core Facility of the
Institute of Molecular Medicine and Experimental Immunology and by the
House for Experimental Therapy.
Disclosures
The authors have no financial conflict of interest.
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