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The Journal of Immunology, 2008, 180, 3889 -3899
Copyright © 2008 by The American Association of Immunologists, Inc.
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Lipopolysaccharide-Activated IL-10-Secreting Dendritic Cells Suppress Experimental Autoimmune Uveoretinitis by MHCII-Dependent Activation of CD62L-Expressing Regulatory T Cells1

Annie W. T. Lau*, Sabine Biester*, Richard J. Cornall{dagger} and John V. Forrester*,2

* Department of Ophthalmology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, United Kingdom; and {dagger} Nuffield Department of Clinical Medicine, Welcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC) are key regulators of immune responses. Mature DC are traditionally considered to be immunogenic, although there is accumulating evidence that they can also be tolerogenic and induce Ag-specific regulatory T cells (Tregs). However, the mechanism of this Treg induction and the site of Treg action in vivo are yet to be defined. In this study, using the experimental model of interphotoreceptor retinoid-binding protein peptide (1–20)-induced experimental autoimmune uveoretinitis, we show that s.c. inoculation of IRBP-peptide-pulsed IL-10-producing LPS-activated mature DC (IL-10-DC) at one site (the cervical region) suppresses autoimmunity induced at a separate site (the inguinal region). Our data show that s.c. IL-10-DC correlates with an increase in the number of CD4+CD25+Foxp3+ Tregs at the DC-draining lymph nodes (DC-dLN). However, although MHCII–/– IL-10-DC also induces Treg expansion at this DC-dLN, they failed to suppress experimental autoimmune uveoretinitis. Furthermore, unlike wild-type IL-10-DC, MHCII–/– IL-10-DC did not correlate with an increase in the percentage of Tregs expressing CD62L at the DC-dLN, nor did they associate with an increase in Treg number at a distal site. Similar effects were also observed after s.c. hen egg lysozyme-pulsed IL-10-DC, which produced a strong reduction in the number and activation of proliferating Ag-specific CD4+ 3A9 T effector cells. We therefore propose that IL-10-DC require MHCII-dependent Ag presentation, and hence TCR ligation, to promote CD62L-mediated trafficking of Tregs to the site of T effector cell priming, where they suppress autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulation of autoimmune responses is achieved through several routes, including thymic deletion, ignorance, anergy, and control by regulatory T cells (Tregs).3 Tregs come in various functional phenotypes, the most well studied being the CD4+CD25+Foxp3+ T cell (1, 2). Frequently considered a natural Treg because they are generated in the thymus and occur in the periphery in the naive organism (3), the CD4+CD25high Treg can also be expanded in vitro in the presence of appropriate APCs, IL-2, and specific Ag (4, 5). This approach has been adopted to generate large numbers of Tregs in vitro in the experimental model of diabetes in which not only can the disease be prevented by prior administration of Tregs, but they can also modulate active disease when adoptively transferred after disease induction/onset (6). More recently, it has been reported that Ag-specific Tregs can be induced from naive CD4+CD25– cells using dendritic cells (DC) and the immunomodulatory cytokine TGF-β1, and these induced Tregs can also suppress diabetes (7). This has significant potential for the customized management of autoimmune disease generally.

Central to this procedure is the use of APCs at the appropriate stage of differentiation. Previous studies have shown that mature DC are considerably more effective in inducing Tregs than immature DC (for review, see Ref. 5). The precise role of DC in this interaction is highly context dependent. It is known that polyclonal Tregs can inhibit polyclonal T cell responses (8), but for Tregs to function at peak efficiency, they require stimulation via their specific TCR to induce suppression of particular autoimmune pathologies (9). Thus, mature DC generate more effective Ag-specific Tregs, whereas the effect of Tregs is to suppress activation of T effector cells (Teffs) possibly through their effects on immature DC during Ag processing and presentation (see Ref. 5 for review). However, although DC can induce Ag-specific Tregs, DC have also been shown to block the suppressive effect of Tregs on CD25– effector T cell proliferation by TLR-induced IL-6 production (10).

These studies help to explain some of the apparent contradictions in reports on the effect of various DC preparations in models of autoimmunity. Mature DC have in certain strains and protocols either induced or exacerbated autoimmune inflammation (2, 10, 11, 12, 13, 14, 15, 16), whereas in other circumstances mature, immature, and semimature DC have inhibited disease (3, 4, 17, 18, 19, 20, 21, 22). In addition, the cytokine profile of the DC (either IL-10 or IL-12 producing) seemed to affect the tolerizing effect of DC in certain models (23). In several models, administration of DC appears to lead to expansion of Tregs, whereas under other circumstances, they appear to suppress autoimmunity by shifting the Th1 cell profile toward the Th2 phenotype (18, 24).

Recent studies have identified certain additional properties of Tregs that mediate their effects on disease suppression in vivo. These include the expression of homing molecules such as CCR7 and CD62L, which permit Treg entry into the T cell area of the lymph node (LN), where DC-T cell interactions occur (25). Expression of CD62L appears to be particularly important for Treg function (26), implicating the draining LN (dLN) as the critical site for action of Tregs.

We have in this study designed experiments that address this question, i.e., whether Tregs require to home to the site of T cell activation to suppress disease. We have expanded Tregs preferentially in the nape-draining LN (dLN; superficial and deep cervical) by s.c. injection of DC in the nape, whereas Teffs were induced in a second dLN (the inguinal) by inoculating Ag s.c. in the back of hind legs. The model we used is that of experimental autoimmune uveoretinitis (EAU), a CD4-T cell-mediated inflammation used to study the human sight-threatening disease posterior uveitis, induced in C57BL/6 mice by s.c. injection of interphotoreceptor retinoid-binding protein (IRBP) peptide 1–20 in CFA. We have first shown that the s.c. inoculation of IRBP peptide-pulsed IL-10-producing mature DC (IL-10-DC) can significantly reduce the severity of EAU, through a mechanism that is associated with an increase in the population of Tregs and is dependent on the presentation of Ag via MHC class II. Furthermore, this process of TCR ligation correlates strongly with the up-regulation of CD62L expression on Tregs, suggesting that the effect is mediated through the trafficking of these suppressive cells to the site of Teff induction. We have validated these data in an Ag-specific system using the hen egg lysozyme (HEL) TCR-transgenic (tg) 3A9 mouse model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Inbred 8- to 12-wk-old C57BL/6, GFP, MHCII–/–, B10BR, and 3A9 TCR-tg mice were provided by the Medical Research Facility at Aberdeen University Medical School. The GFP mice (on C57BL/6) have the enhanced GFP construct integrated into chromosome 14D1, and express enhanced GFP in all of their cells and tissues, as described previously (27). The MHCII–/– (on C57BL/6) have a null insertion at the invariant chain locus that leads to defective heterodimer {alpha}/β-chain assembly and a haplotype-specific (H2b) failure to present Ag (28, 29, 30). The 3A9 TCR-tg line (B10BR background) expresses TCR that recognizes the HEL epitope 46–61 in the context of I-Ak (31). The procedures adopted conformed to the regulations of the Animal License Act (U.K.) and to the Association for Research in Vision and Ophthalmology statement for The Use of Animals in Ophthalmic and Vision Research.

Isolation and culture of bone marrow DC

DC were prepared and cultured using a method described previously (32). In brief, the tibias and femurs were removed from mice and flushed to obtain bone marrow cells. The red cells were first lysed with ammonium chloride/Tris solution, then depleted of T cells, B cells, and MHCII+ cells using rat anti-mouse mAbs (CD4 (clone GK 1.5), CD8 (clone 53-6.7), CD45R, and MHCII (clone P7/7), all from BD Pharmingen, except MHCII, which was from Serotec) and Dynabeads coated with sheep anti-rat IgG (Dynal Biotech). The remaining cells were then cultured at 7.5 x 105/ml (22.5 x 105/well) in 12-well plates with complete RPMI 1640 supplemented with rGM-CSF (R&D Systems) (0.5 µg/100 ml medium) for 6 days, at 37°C and 5% CO2. The culture was fed daily from day 2 by gently aspirating 75% medium and adding fresh medium described above. On day 6 of culture, the medium was withdrawn and the loosely adherent clusters were flushed, collected in PBS, and depleted of contaminating granulocytes using anti-mouse Gr1 mAb (clone RB6-8C5; BD Pharmingen) and Dynabeads. A single-cell suspension of remaining cells was then cultured immediately in 24-well plates at 106 cells/well. LPS (Sigma-Aldrich; Escherichia coli) was added either immediately (IL-10-DC) or 24 h later (IL-12-DC) at 1 µg/ml. IRBP1–20 peptide/HEL was also loaded at 30 µg/ml to induce an Ag-specific response. After addition of LPS and/or peptide, the cells were cultured overnight before being harvested and washed for phenotypic analysis (flow cytometry) or adoptive transfer.

Flow cytometry

Harvested DC were surface stained for Abs against CD11c, CD11b, CD40, CD86, MHCII, CD4, CD8, TLR4-MD2, and CD14. All Abs were used as color conjugates (BD Pharmingen), and four-color FACS analysis was performed (FACSCalibur; BD Biosciences).

For the calculation of absolute Treg number and characterization of their phenotype, LN were harvested and passed through a 100-µm sieve to obtain a single-cell suspension. The cells were first blocked with serum to prevent nonspecific binding, then surface stained for Abs against CD4, CD25, CD62L, CCR7, and CD103 (BD Pharmingen, except CCR7 from eBioscience), fixed and permeabilized twice with 0.1% saponin, and stained with Ab against Foxp3 (eBioscience), and six-color FACS analysis was performed (FACS LSR; BD Biosciences). A known number of nonfluorochrome-conjugated beads (BD Pharmingen) was added to each sample for the calculation of absolute number of cells. Nonparametric analysis of the results was performed by Mann-Whitney U test (Prism). Value of p < 0.05 was considered statistically significant.

Disease induction and evaluation

EAU was induced by immunizing s.c. with 500 µg of IRBP 1–20 (GPTHLFQPSLVLDMAKVLLD; purity >95%; Sigma Genosys) emulsified in CFA supplemented with 2.5 mg/ml Mycobacterium tuberculosis strain H37Ra (Difco) at the back of hind legs. Pertussis toxin (1 µg) was also administrated i.p. at the time of IRBP immunization.

Mice were sacrificed 28 days post-IRBP immunization by asphyxiation in CO2, and eyes harvested were immediately fixed in 2.5% phosphate-buffered glutaraldehyde and then embedded in glycol methacrylate resin for sectioning. Sections (3 µm) of each globe were taken at seven different levels. The sections were subsequently stained with H&E. Severity of disease was scored on a scale of 0 (no disease) to 4 (maximum disease) in half-point increments, according to a semiquantitative scoring system described previously (33). Nonparametric analysis of the EAU grades was performed by Mann-Whitney U test (Prism). Value of p < 0.05 was considered statistically significant.

Adoptive transfer experiments

A total of 106 DC was injected s.c. into the nape of the neck of each mouse. For studying the effects of DC on EAU, mice were immunized with IRBP 24 h after DC transfer. For the study of DC-induced Tregs, the LN (submandibular, superficial cervical, deep cervical, and inguinal) were harvested 3 days post-DC transfer.

Cytokine secretion and intracellular signaling analyses

Cytokine profiles in DC culture supernatants were evaluated using the cytometric bead multiplex array system (BD Biosciences) for IL-12 and IL-10, and cytokine ELISAs (R&D Systems) for IL-2 and TGF-β. Phosphorylation of intracellular ERK1 was measured using ERK1 ELISA (R&D Systems).

In vitro proliferation and suppression assays

LN were harvested from 3A9 TCR-tg mice, and single-cell suspensions were made by passing tissues through 70/100-µm-pore cell strainers. CD4+CD25+/CD25– cells were isolated using the MACS isolation kit (Miltenyi Biotec). IL-10-DC were prepared from non-tg B10BR mice, and then pulsed or not pulsed with 30 µg/ml HEL overnight before use. To test the ability of DC to expand Tregs/Teffs, 104 isolated CD4+CD25+/CD25– T cells were plated out per well on a 96-well flat-bottom plate. IL-10- DC+/–HEL were added at various quantities, as appropriate. A total of 100 U/ml IL-2 (PeproTech) and/or 30 µg/ml HEL was added per well for stimulation. [3H]Thymidine was added after 72-h culture and measured 12–16 h later. To test the suppressive ability of in vitro DC-expanded Tregs, 3A9 TCR-tg Tregs were expanded as above at 1:1 DC:Treg ratio for 5 days before harvest. Tregs were then isolated by depletion of the CD11c+ cells using magnetic microbeads (Miltenyi Biotec). A total of 104 freshly isolated 3A9 TCR-tg CD4+CD25– cells was plated per well on 96-well flat-bottom plate with appropriate number of DC-expanded Tregs, 5 x 104 CD4+/CD8+ depleted and mitomycin-treated splenocytes, and 30 µg/ml HEL. [3H]Thymidine was added at 72 h, and uptake by proliferating lymphocytes was measured 12–16 h later.

In vivo proliferation of CFSE-labeled lymphocytes

LN were collected from 3A9 TCR-tg mice, and single-cell suspensions were made by passing tissues through 70/100-µm cell strainers, as above. The cells were then labeled with 5 µM CFSE (Molecular Probes) and injected i.v. at 107 cells per animal (day 0). Animals were then transferred with 106 IL-10-DC+HEL the following day (day 1) and immunized with 500 µg of HEL in CFA supplemented with 2.5 mg/ml mycobacterium s.c. in the back of hind legs. In addition, 1 µg of pertussis toxin was also administered i.p. on day 2. Tissues were harvested on day 5 for FACS analyses of CFSE dilution. To assess the activation of CFSE-labeled Teffs, the harvested tissues were stained with various fluorochrome-conjugated Abs, and the Foxp3– CD4+ cells were evaluated for their CD25 expression as a marker of T cell activation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Differential cytokine secretion by bone marrow-derived DC (BMDC) in response to LPS correlates with TLR4/CD14 expression levels and ERK1 signaling

We have previously demonstrated that LPS-activated BMDC secrete a different set of cytokines by varying the timing of LPS activation of freshly purified immature DC from B10RIII (H2k) mice (17, 23). In this study, we confirm this result in C57BL/6 (H2b) mice: freshly purified BMDC are CD11C+lowCD11b+CD4–CD8a– expressing low levels of CD40, MHCII, and CD86 (Fig. 1, A and B, and data not shown). Freshly purified DC cultured immediately with LPS for 24 h (IL-10-DC) or cultured for 48 h with LPS added after a delay of 24 h (IL-12-DC) (see legend to Fig. 1 for definition of IL-10 and IL-12 DC) led to equal levels of up-regulation of CD40, MHCII, and CD86, but to differential secretion of IL-10 (immediate addition of LPS) and IL-12 (delayed addition of LPS), as shown previously (Fig. 1C). Interestingly, IL-12-DC secreted higher levels of IL-2 than IL-10-DC or immature DC, and also produced significant levels of TGF-β, unlike IL-10-DC or immature DC, both of which failed to secrete TGF-β (Fig. 1C).


Figure 1
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  		FIGURE 1. Differentially LPS-activated DC are phenotypically similar, but functionally different. A, FACS analyses showing the purity of DC (90% CD11c+) and all DC coexpress CD11b, indicating a myeloid cell type. B, FACS histograms showing the expression of cell surface markers on differentially activated DC. Non-LPS-stimulated immature DC after 24-h (dotted green line) or 48-h incubation (dotted red line) both express low levels of CD40, MHCII, CD83, and CD86, whereas IL-10 (solid green line) and IL-12 (solid red line) LPS-activated DC express equally up-regulated levels of all markers. C, Although immature DC failed to secrete any of the cytokine examined, DC activated with LPS immediately after preparation secrete high IL-10 and low IL-12 (IL-10DC), whereas DC activated 24 h after initial purification produce low IL-10 and high IL-12 (IL-12DC). IL-10 and IL-12 DC both secrete IL2, whereas only IL-12 DC secrete TGF-β.

 
Because LPS activates innate immune cells via TLR4 and CD14, we next investigated the effects on TLR4 and CD14 expression as well as the subsequent intracellular signaling events that are involved in the LPS-induced activation of IL-10-/IL-12-DC, and whether these correlate with their differential cytokine production. Contrary to previous reports in which LPS induces the up-regulation of TLR4 on DC, leading to their maturation and activation (34), our flow cytometric data showed that the expression of TLR4-MD2 complex was significantly down-regulated on both IL-10-DC and IL-12-DC upon LPS stimulation (Fig. 2A). Interestingly, although both IL-10- and IL-12-DC showed an increase in their surface expression of CD14 upon LPS stimulation, the increase was significantly greater in the IL-12-DC compared with the IL-10-DC. In addition, the level of phosphorylated ERK1 (MAPK downstream of TLR4 signaling (35)) was greatly enhanced in IL-12-DC, but not in IL-10-DC. It is possible, therefore, for the full activation of DC and IL-12 production, both the expression of surface CD14 and the up-regulation of ERK1 phosphorylation are required.


Figure 2
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  		FIGURE 2. IL-10/IL-12 secretion by differentially LPS-activated DC correlates with the expression of TLR4 and CD14 and their intracellular level of phosphorylated ERK1. A, The level of TLR4 and CD14 expression on different types of DC. The expression of TLR4 is greatly reduced after LPS activation (II (IL-10-DC) (solid green line) and IV (IL-12-DC) (solid red line) compared with their immature counterparts (I (broken green line) and III (broken red line)), whereas the expression of CD14 is greatly increased on IL-12-DC (IV) compared with IL-10-DC (II). B, Only LPS-activated IL-12-DC produce a significant level of phosphorylated ERK1.

 
The s.c. transfer of LPS-activated IL-10-secreting DC inhibits the induction of EAU in a MHCII-dependent manner

To investigate the role of Ag presentation by IL-10-DC, we prepared BMDC from MHCII–/– mice, in which a null mutation has been inserted at the invariant chain locus, resulting in defective heterodimer {alpha}/β-chain assembly and a haplotype-specific (H2b) failure to present Ag (28, 29, 30).

We found that LPS-early activated DC from MHCII–/– mice (MHCII–/– IL-10-DC) also secreted a large amount of IL-10 and minimal IL-12 (Fig. 3A). However, s.c. transfer of MHCII–/– IL-10-DC failed to suppress EAU, whereas IRBP peptide-pulsed wild-type (WT) IL-10-DC suppress the disease significantly (Fig. 3, B–D). This suggests that IL-10 secretion by DC alone is not sufficient for the induction of immune tolerance. More importantly, the data show that Ag-specific engagement of the TCR is required to inhibit disease.


Figure 3
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  		FIGURE 3. WT IL-10-DC suppress the induction of EAU in a MHCII-dependent manner. Although both early LPS-activated WT and MHCII–/– DC (WT and MHCII–/– IL-10-DC) secrete high IL-10, MHCII–/– IL-10-DC fail to suppress EAU, whereas WT IL-10-DC can significantly suppress the disease. A, MHCII–/– IL-10-DC produce similar levels of IL-10 and IL-12 compared with WT IL-10-DC. B, 106 WT or MHCII–/– IL-10-DC were s.c. inoculated in the nape before IRBP immunization in the hindlegs to induce EAU. Each full circle represents one mouse. Data shown are representative of two different experiments showing similar results. C and D, Histopathology of harvested eyes. C, MHCII–/– IL-10-DC, extensive damage of the entire retina, including photoreceptor destruction, vasculitis, vitritis, and granuloma formation. D, WT IL-10-DC, most of the retina is intact, with minor signs of inflammation such as retinal folds.

 
Both WT and MHCII–/– IL-10-DC induce the expansion of CD4+CD25+Foxp3+ Tregs at the cervical dLN

We have previously suggested that the suppressive function of IL-10-producing DC may be due to their ability to expand Tregs (23), because the s.c. injections of IRBP peptide-pulsed IL-10-producing DC into the neck led to an increase in the percentage of CD4+CD25+GITR+ cells in the cervical dLN compared with PBS control, an effect that appeared to peak 48–72 h after DC transfer. In this study, we have investigated this further by assessing the absolute number of CD4+CD25+Foxp3+ Tregs at the dLN after s.c. inoculation of DC.

We have found that inoculation of WT IRBP peptide-pulsed IL-10 DC correlated with an increase in the absolute number of CD4+CD25+Foxp3+ Tregs selectively at the site-specific skin-draining cervical LN (Fig. 4, A and B), and not at other sites, e.g., at the juxtaposed submandibular eye dLN (Fig. 4C) (36). This supports the notion that the suppression of EAU induced by IL-10-DC is associated with expansion of Tregs in the dLN. However, MHCII–/– IL-10-DC also correlated with an increase in the population of CD4+CD25+Foxp3+ Tregs at the dLN (Fig. 4, A and B), despite the fact that they failed to suppress EAU (Fig. 3). This indicates that Treg expansion alone at this site was insufficient to inhibit EAU, and suggested that there may be a defect in the trafficking of Tregs in MHCII–/– IL-10-DC-treated mice to the distal site of Teff priming.


Figure 4
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  		FIGURE 4. WT or MHCII–/– IL-10-DC are both associated with an increase in the absolute numbers of CD4+CD25+Foxp3+ Tregs at the DC-dLN. The absolute numbers of CD4+CD25+Foxp3+ Tregs are increased by both WT or MHCII–/– IL-10-DC at the DC-draining superficial cervical (A) (**, p = 0.0043, 0.0076) and deep cervical (B) (**, p = 0.0043, 0.0011) LN, but not at the non-dLN, i.e., the submandibular (C) (p = 0.409, 0.3496) (see Fig. 5). WT or MHCII–/– IL-10-DC were adoptively transferred s.c. in the neck 3 days before the harvest of tissues. The absolute numbers of CD4+CD25+Foxp3+ Tregs induced by WT IL-10-DC, MHCII–/– IL-10-DC, and PBS control were detected using flow cytometry. Data shown are representative of two different experiments showing similar results.

 
MHCII Ag presentation by DC is required to significantly increase the trafficking of Tregs to the distal inguinal LN

As indicated above, because Treg expansion appeared unaffected by the lack of functional MHCII on the inducing DC, yet Ag presentation through MHCII was necessary for EAU suppression, we speculated that specific Ag may be required to induce homing signals in the expanded Treg population. Our experimental design of IL-10-DC s.c. inoculated at one site (cervical dLN) and Ag immunization at another site (inguinal dLN) allowed us to explore this hypothesis.

We first wished to verify that the s.c. inoculation of IL-10-DC in the nape of the neck was associated with selective trafficking of the inoculated DC to the site-specific (superficial cervical, deep cervical) dLN. IL-10-DC were cultured from congenic GFP tg mice (see Materials and Methods) and inoculated s.c. in the nape, and various lymphoid tissues were harvested on days 1, 3, and 6 after adoptive transfer. We found that there was a progressive accumulation of IL-10-DC in the draining superficial and deep cervical LN over 6 days (Fig. 5, A and B), but no migration of DC to other secondary lymphoid tissues such as the eye dLN (submandibular, Fig. 5C) or the distal inguinal LN (Fig. 5D).


Figure 5
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  		FIGURE 5. IL-10 DC selectively track to their dLN. Subcutaneously administered GFP IL-10-DC (106 cells at the nape) selectively traffic to the superficial cervical (A) and deep cervical (B) LN. No GFP DC could be detected at the eye dLN (submandibular (C)) or the distal inguinal LN (D). Tissues were harvested at days 1, 3, and 6 after DC transfer, and GFP cells were detected using flow cytometry.

 
We next examined whether s.c. inoculation of IL-10-DC expands CD4+CD25+Foxp3+ Tregs at distal non-DC dLN as well as the DC-draining cervical LN (Fig. 5). If this were so, it would indicate that the IL-10-DC-induced population of Tregs at the site-specific dLN had the capacity to traffic to distal LN. Our data showed that s.c. inoculation of WT IL-10-DC is associated with a significant increase in the population of CD4+CD25+Foxp3+ Tregs at distal sites, whereas MHCII–/– IL-10-DC failed to do so (Fig. 6A). This suggests that presentation of specific Ag is required to permit trafficking of Tregs to distant sites and particularly to the site of T cell activation (the inguinal dLN of the immunization site).


Figure 6
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  		FIGURE 6. WT IL-10-DC, but not MHCII–/– IL-10-DC, correlates with an increase in the population of Tregs at the distal inguinal LN and the expression of CD62L on Tregs at the DC-dLN. A, 106 WT or MHCII–/– IL-10-DC were inoculated s.c. in the neck 3 days before the harvest of cells from the distal inguinal LN. The absolute numbers of CD4+CD25+Foxp3+ Tregs after s.c. transfer of WT IL-10-DC, MHCII–/– IL-10-DC, and PBS control were detected using flow cytometry. B, Only WT IL-10-DC is associated with a significant increase in the expression of CD62L on CD4+CD25+Foxp3+ Tregs. The cervical dLN were harvested 3 days after the s.c. injection of 106 WT or MHCII–/– IL-10-DC in the neck.

 
This was further supported by data showing that the percentage of CD4+CD25+Foxp3+ Tregs expressing CD62L was significantly increased when IRBP peptide-loaded WT IL-10-DC were s.c. inoculated, compared with PBS controls (p < 0.05), whereas MHCII–/– IL-10-DC failed to associate with a significant increase in CD62L expression on Tregs (Fig. 6B). Because CD62L is a lymphoid-homing molecule previously shown to be critical for the in vivo suppressive function of Tregs (25, 37, 38, 39, 40, 41), our data would imply that, in addition to the increase in the population of Ag-specific Tregs, MHCII Ag presentation by tolerogenic IL-10-DC appears to mediate the appropriate trafficking of Tregs to distal non-DC dLN via the induction of their CD62L expression.

Presentation of specific Ag is required for the optimal expansion of Ag-specific Tregs, as well as for their Ag-specific suppressive function and CD62L expression

To demonstrate Ag specificity, we turned to the HEL tg mouse model because there is no equivalent TCR-tg model for IRBP peptide. Using the CD4+ 3A9 TCR-tg mouse (in which the majority of CD4+ T cells express the 3A9 TCR that specifically recognize the HEL epitope 46–61 in the context of I-Ak (31)), we show that the proliferation and activation of Ag-specific Teffs can be regulated by LPS-activated IL-10-DC.

First, we investigated the ability of IL-10-DC to expand Ag-specific T cells in vitro. To study the role of specific Ag, we have used HEL-pulsed IL-10-DC, in the presence or absence of the cognate Ag HEL and/or IL-2. We have isolated the separate populations of Tregs/Teffs from the LN of TCR-tg mice according to their CD25 expression using MACS magnetic columns, and confirmed by flow cytometry that this method gives a good purity of Tregs (~70% Foxp3+) and Teffs (>90% Foxp3–) (Fig. 7A). By culturing these separate populations of Tregs/Teffs with different quantities of IL-10-DC (pulsed or not pulsed with HEL), we have found that HEL-pulsed IL-10-DC can induce the proliferation of both Tregs and Teffs (Fig. 7, B and C). Furthermore, the level of proliferation directly correlates with the numbers of DC in culture, and both CD4+CD25+ (Treg) and CD4+CD25– (Teff) populations require the presence of HEL for maximum proliferation. Of particular interest is the strong proliferation of Tregs induced by IL-10-DC and HEL (Fig. 7B) because these cells have previously been shown to be anergic and failed to proliferate unless exogenous IL-2 is added in vitro. However, we have shown above that IL-10-DC secrete significant amounts of IL-2 (Fig. 1C), which probably provides the stimulus for overcoming Treg anergy, allowing them to proliferate in response to specific Ag, i.e., HEL. Interestingly, the combination of both HEL and IL-2 appears to have an additive effect on the amount of Treg proliferation. In contrast, non-HEL-pulsed IL-10-DC incubated with IL-2 failed to induce any significant proliferation of Teffs, although HEL-pulsed IL-10-DC with IL-2 together seems to induce proliferation that is higher than that induced by HEL-pulsed IL-10-DC alone. Note that there appears to be a decline in proliferation at 1:1 DC:Treg ratio compared with 1:2 ratio, which may be explained by excessive TCR stimulation.


Figure 7
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  		FIGURE 7. IL-10-DC require cognate Ag for the optimal expansion of 3A9 T cells. CD4+CD25high Tregs and CD4+CD25– populations were isolated from 3A9 lymphocytes using MACS isolation. A, The expression of Foxp3 in the two cell populations confirm that CD25high Tregs are mostly Tregs, whereas most CD25– cells are Foxp3–. B and C, When cultured with HEL-pulsed or non-Hel-pulsed LPS-activated IL-10-DC at various DC:T cell ratios, the data show that although IL-2 can induce some proliferation of Tregs, as previously documented (B), the presentation of HEL Ag greatly improves the optimal expansion of both populations of Tregs (B) and Teffs (C). Data shown are representation of three experiments showing similar results.

 
In addition to testing the ability of IL-10-DC to induce the proliferation of Tregs described above, we have also harvested the cocultured Tregs and used flow cytometry to phenotype these cells. Although we found all sets of Tregs maintain similar level of Foxp3 after culture irrespective of the treatment (data not shown), interestingly, only those Tregs that have been expanded in the presence of HEL retained their expression of CD62L, compared with freshly isolated TCR-tg Tregs, or DC or DC plus IL-2 cocultured Tregs (Fig. 8A). This is in good agreement with the data above, which suggested that the presentation of specific Ag via MHCII by DC is required to up-regulate the expression of CD62L (Fig. 6). We propose this to be a key function of tolerizing DC, because the up-regulation of CD62L expression on the expanded Treg population allows them entry to other lymphoid tissues, where they can exert their suppressive function.


Figure 8
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  		FIGURE 8. Specific Ag (HEL) is required for in vitro expansion of CD4+CD25+ Tregs that retain their expression of CD62L and their ability to mediate Ag-specific suppression. A, Flow cytometry analyses showing the requirement of specific Ag HEL to retain the expression of CD62L on DC-expanded 3A9 Tregs. B, Suppression assays demonstrating that DC-induced Tregs must be expanded in the presence of HEL to retain its potent Ag-specific suppressive function.

 
In addition to the ability of DC-expanded Tregs to maintain their expression of Foxp3 and CD62L, it is also important that they retain their ability for Ag-specific suppression. We have therefore isolated the in vitro DC-expanded Tregs by depleting CD11C+ cells, then using these Tregs to suppress the proliferation of HEL-induced 3A9 CD25– Teffs. By adding different quantities of Tregs to Teffs, we investigated the suppressive function of HEL-specific Tregs. Our data show that although the presence of DC plus IL-2 alone can expand Tregs (Fig. 7B), this set of expanded Tregs only showed minimal suppressive ability, i.e., slightly more than that of freshly isolated unstimulated Tregs (Fig. 8B). In contrast, both DC plus HEL- and DC plus HEL plus IL-2-expanded Tregs are equally potent at inhibiting the proliferation of 3A9 Teffs upon presentation of the cognate Ag; even at a ratio of 1:64 Treg:Teff, both these expanded Tregs suppress proliferation by 50% (Fig. 8B). These data demonstrate that, although IL-2 plus HEL appears to have an additive effect on the level of Treg proliferation (Fig. 7B), it is the activation by specific Ag, i.e., HEL, that is essential to determine the ability of the expanded Tregs to retain their Ag-specific suppressive function.

IL-10-DC+HEL administered to one LN drainage site (the cervical nodes) inhibit priming of HEL-specific Teffs induced at a distal LN (the inguinal node)

Finally, we wished to demonstrate this suppressive activity of IL-10-DC in vivo in an Ag-specific model. To achieve this, we first transferred CFSE-labeled 3A9 lymphocytes to non-tg B10BR mice before the administration of DC at the one site of drainage (cervical nodes; see Fig. 5 above), followed by the activation of 3A9 Teffs by HEL/CFA immunization at the draining site of the inguinal LN.

Our data clearly show that the s.c. administration of IL-10-DC at the neck is associated with a strong reduction in both the number and activation of proliferating 3A9 Teffs in vivo (Fig. 9). It is evident that although this effect can be observed in all the LN studied, the effect is far more pronounced in the inguinal LN, i.e., the LN draining the site of HEL/CFA immunization and therefore the site of T cell priming. We believe the data provide compelling evidence that the administration of IL-10-DC+Ag at one site, i.e., the neck, which we have demonstrated traffic only to the site-specific dLN (cervical) (Fig. 5), can potently reduce the activation of Teffs at a separate site, i.e., Ag-draining inguinal LN. These data also correlate with the up-regulation of CD62L expression on Tregs, and we suggest that CD62L expression, which has previously been demonstrated as a requirement for Treg trafficking (25, 37, 38, 39, 40, 41), is induced in an Ag-specific manner by tolerizing IL-10-DC; thus, these two events are probably causally linked.


Figure 9
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  		FIGURE 9. The s.c. transfer of IL-10-DC+HEL at the neck is associated with a strong reduction in both the number and activation of proliferating 3A9 Teffs induced at the distal inguinal LN. CFSE-labeled 3A9-TCR lymphocytes were transferred to non-tg B10BR mice, followed by the s.c. transfer of HEL-pulsed IL-10-DC at the neck and the active immunization of HEL/CFA at the inguinal. Lymphoid tissues were harvested, and CFSE cells were detected by flow cytometry 3 days later; data shown were gated on CD4+ T cells. Our data show that CD4+ T cell proliferation (CFSE dilution (A)) and activation (CD25 expression (B)) are most pronounced, as expected, at the Ag-draining inguinal LN, and the s.c. transfer of HEL-pulsed IL-10-DC can greatly reduce this effect. C, Mean values of i, the total number of CFSE cells and ii, the percentage of CFSE-proliferating cells expressing CD25 (upper left quadrants of B) at various LN studied from the HEL/CFA-immunized or IL-10-DC+HEL-treated and HEL/CFA-immunized animals. Data shown are representative of two separate experiments with two to four animals per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Naturally occurring Tregs are generated in the thymus, and under steady-state conditions populate the T cell areas of secondary lymphoid organs, where they constitute ~5–10% of CD4+ T cells. Ag-specific Tregs can be expanded from this initial pool by induction with peptide-loaded DC in vitro and in vivo, and have been shown to be highly effective in preventing and inhibiting autoimmune conditions such as diabetes (4, 5, 6). A central question that arises is where is the site of Treg action? Evidence in the NOD mouse indicates that there is preferential localization to the lymphoid tissues draining the site of inflammation (the pancreatic LN) (42), but because these experiments were performed after adoptive transfer of Ag-specific Tregs i.v. in which there would be general distribution of the Tregs, it can be concluded either that there was preferential recruitment of recirculating Tregs to the pancreatic LN or there was further expansion of Tregs locally in the LN in response to the autoimmune inflammation.

We have formally addressed the requirement for homing in our model of EAU, in which we have induced Tregs at one site (the nape cervical LN) using s.c. inoculation of DC, while inducing Teff at a second site using s.c. IRBP peptide immunization (the inguinal LN). We have shown that MHCII–/– IL-10-DC have the potential to increase the population of Tregs at the LN draining the site of skin DC inoculation, but fail to inhibit EAU induced by IRBP peptide immunization at a distal site. Moreover, this effect correlates with the failure of MHCII–/– DC to increase the expression of CD62L on Tregs at the dLN or the absolute number of Tregs at the distal LN, compared with WT IL-10-DC (Fig. 6). The requirement for the presentation of specific Ag for the induction of CD62L expression on Tregs was further confirmed with our in vitro studies using CD4+CD25+ Tregs from the CD4+ 3A9-TCR-tg model (Fig. 8). Together these data strongly suggest that the known requirement for CD62L expression and homing by Tregs (25, 37, 38, 39, 40, 41) for their suppressive activity is dependent on Ag-specific TCR ligation.

Our experimental design raises a number of questions. First, how robust is the concept that s.c. inoculation of Ag-loaded DC leads to selective homing of DC to the site dLN? There is considerable literature on this, indicating that tissue-derived myeloid DC migrate only to the dLN and do not recirculate, despite recent evidence that there may be a minor recirculating population (43, 44). In addition, we have formally tested this by inoculating GFP-labeled DC into the skin and shown a highly selective drainage of DC to the specific LN by flow cytometry (Fig. 5) (36).

A further criticism may be raised by contending that soluble Ag, coadministered as part of the emulsified IRBP peptide in the CFA, has the potential to be distributed widely through the lymph to all secondary lymphoid tissues, where it may be taken up by resident DC and induce either Teffs or Tregs at several secondary lymphoid sites. Previous work by Catron et al. (43) and others has indeed shown that soluble Ag can reach dLN very rapidly after injection, but that cell (DC)-associated Ag takes several hours to reach such sites. However, soluble Ag alone has no capacity to generate Tregs, and we have confirmed this by direct testing (data not shown).

Our data therefore demonstrate the disease-inhibiting potential of DC-induced Ag-specific Tregs and indicate that a significant part of this effect is dependent on preferential homing of the Tregs to the site of Teff induction. This was further supported by our in vivo study using the systemic transfer of CFSE-labeled Ag-specific 3A9 CD4+ T cells, followed by HEL/CFA immunization at the inguinal, in which the s.c. adminstration of IL-10-DC+HEL in the neck is associated with a strong reduction in both the absolute number and activation of proliferating CD4+ 3A9 TCR cells at the distal Ag dLN (Fig. 9). The mechanism of this potent reduction in Teff number and activation is currently unclear, although recently it has been reported that Tregs can induce the apoptosis of Teffs (45), a hypothesis that may be applicable in this work and is presently under further study. Previous studies in a colitis model in which Tregs were directly administered i.v. have shown that Tregs migrate to the mesenteric LN and to the site of inflammation in the colon (46, 47). Other studies have also shown that Tregs can recirculate from the LN to inflammatory sites (26). However, most of these studies have supplied Tregs exogenously or have used chimeric/congenic mice. In the present study, we have expanded endogenous Tregs in vivo in immunocompetent mice at one site using s.c. Ag-loaded DC and have asked what is required of the expanded Tregs to inhibit EAU induced by Teffs at another site.

An alternative possibility is that the homing-competent Tregs preferentially traffic to the site of inflammation, in this case the eye. The homing molecule CD62L binds preferentially to receptors on the high-endothelial venules in the T cell area of the LN (48), an interaction facilitating transendothelial migration into this area and T-APC congress. High-endothelial venule-like structures are also found in inflamed tissue, including the retina in EAU (49), and it is possible that Tregs could act locally particularly during ongoing inflammation. Indeed, Tregs have been detected at many sites of autoimmune inflammation (46), and this mechanism may explain how Tregs can inhibit ongoing disease. Whether or not Tregs home to the eye at the onset of disease and prevent local Ag presentation via tissue-derived APCs remains to be determined, but in initial experiments, we have been unable to detect any CD4+CD25+ T cells in the inflammatory exudates in the eye by flow cytometry (data not shown). The possibility that there may be subsets of Tregs homing respectively to the LN where T cells are initially activated or to sites of ongoing inflammation adds a further complexity to the potential role for expression of homing molecules in the regulation of disease (26). Some studies have suggested that CD62Llow {alpha}E integrinhigh E-/P-selectinhigh Tregs are more effective at controlling disease at the site of inflammation rather than in the dLN (50, 51). However, recent experiments suggest that after Ag challenge, there is marked proliferation of both Tregs and Teffs in the dLN with a several fold greater effect in the Treg population (52). Furthermore, the suppression of Teffs by Tregs appears to be a concentrated activity in the T cell area of LN under control of the chemokine CCR7, whereas Tregs at the site of inflammation appear to have much less effect (53). Our current data suggesting that DC-induced Ag-specific Tregs have their primary site of action in the dLN would support this latter view.

Although our data have addressed the site of Treg action and the requirement for Treg homing in the induction of immune tolerance by adoptively transferred IL-10-DC, the mechanism used by the induced Tregs to suppress remains to be defined. Although earlier studies have shown the ability of Tregs to directly inhibit Teffs (19, 54), more recently it has been suggested that Tregs can also suppress via inhibiting the activation of DC (55, 56). In our model, it is clear that IL-10-DC-induced Ag-specific Tregs can directly inhibit the proliferation of Ag-specific Teffs, as shown by our data with the in vitro expanded 3A9 Tregs (Fig. 8). However, our in vivo CFSE study (Fig. 9) also demonstrates that the s.c. transfer of IL-10-DC can suppress the priming (i.e., both proliferation and activation) of naive 3A9 cells, presumably by inhibiting the ability of DC to prime, as shown by a recent study by DiPaolo et al (57). Clearly, further studies are required to define the exact mode of DC-induced Treg-mediated suppression, but it is likely that there are multiple mechanisms involved in their function.

The data in this work also confirm that IL-10-DC, despite being LPS activated, retain the potential to induce Tregs. In a previous study, it has been shown that ligation of TLRs can permit DC to override their normal tolerizing function (10), but this may be context and strain dependent. In several recent reports, mature DC, activated by exposure to various TLRs or cytokines, have been shown to induce Ag-specific Tregs (4, 5, 19), and this might be expected because ligation of the TCR is an important step in this process. Interestingly, under the condition of the current experiments, LPS activation of DC led to a down-regulation of TLR4, which may have had an impact on the subsequent induction of Tregs after s.c. inoculation. It is possible that TLR4 up-regulation on mature DC while Ag is presented is one of a restricted set of innate immune conditions required to induce Ag-specific adaptive immunity as opposed to tolerance, and, from a therapeutic standpoint, for the management of human diseases, this is a highly important issue. The corollary may also be true, i.e., that TLR down-regulation (Fig. 2) promotes tolerance, thus emphasizing the importance of the conditions in which maturation of DC occurs.

The further questions that remain to be answered therefore relate to the fine control of DC activation for either Treg expansion or the generation of an immune response. Previously, the expression of costimulatory molecules, or the production of immunosuppressive vs immunostimulatory cytokines, were considered critical regulators of autoimmune inflammation. Although these elements are important, it is now apparent that there are many other components that fine-tune this response, and one of these is TCR ligation-dependent up-regulation of homing molecules. How this is achieved is not known at present, and, in addition, it is highly likely that there are other required signaling and cell surface molecules. For instance, CCR4 and CCR8 are considered important for Treg migration into inflammatory sites (58), whereas CCR7 is expressed on LN-homing Tregs and, as indicated above, appears to be essential for Treg function (25). Similarly, CCR2 has been shown to be important in Treg trafficking in a model of collagen arthritis (59). Clearly, there are many further subtleties in this process of immune regulation yet to be revealed.


   Acknowledgments
 
We thank Department of Histology, Rosie Dawson, and Julie Taylor (University of Aberdeen) for the histological sectioning and staining of murine eyes, and Liz Muckersie and Marie Robertson (Department of Ophthalmology, University of Aberdeen) for technical support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 project was supported by the Development Trust (University of Aberdeen) and the National Health Service Grampian Endowment Trust (07/49). Back

2 Address correspondence and reprint requests to John V. Forrester, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, U.K. E-mail address: j.forrester{at}abdn.ac.uk Back

3 Abbreviations used in this paper: Treg, regulatory T cell; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; dLN, draining lymph node; EAU, experimental autoimmune uveoretinitis; HEL, hen egg lysozyme; IRBP, interphotoreceptor retinoid-binding protein; LN, lymph node; Teff, T effector cell; tg, transgenic; WT, wild type. Back

Received for publication April 13, 2007. Accepted for publication January 10, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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