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The Journal of Immunology, 2007, 179, 958 -966
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Inflamed Central Nervous System Drives the Activation and Rapid Proliferation of Foxp3+ Regulatory T Cells1

Richard A. O’Connor, Katy H. Malpass and Stephen M. Anderton2

University of Edinburgh, Institute of Immunology and Infection Research, School of Biological Sciences, Kings Buildings, West Mains Road, Edinburgh, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Resolution of experimental autoimmune encephalomyelitis requires a large cohort of Foxp3+ regulatory T cells (Tregs) within the CNS. In this study, we have used the passive transfer of murine experimental autoimmune encephalomyelitis using myelin-reactive T cells to study the development of this Treg response. Rapid proliferation of Tregs within the CNS (which is not seen in lymphoid organs) drives a switch in the balance of CNS proliferation from T effectors to Tregs, correlating with recovery. This proliferative burst drives a local over-representation of Vbeta8+ Tregs in the CNS, indicative of an oligoclonal expansion. There is also evidence for a small, but detectable, myelin oligodendrocyte glycoprotein-reactive Treg component expanded without prior immunization. Furthermore, CNS-derived Tregs, taken during recovery, suppressed the proliferation of CNS-derived effectors in response to myelin oligodendrocyte glycoprotein. Under these conditions, Tregs could also limit the level of IFN-{gamma} production, but not IL-17 production, by CNS-derived effectors. These data establish the CNS as an environment that permits extensive Treg proliferation and are the first to demonstrate Treg expansion specifically within the tissues during the natural resolution of autoimmune inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many studies have illustrated the importance of regulatory T cells (Tregs)3 in limiting the development of autoimmune disease, in setting thresholds for the expansion of autoreactive T cells, and in promoting recovery from clinical disease (reviewed in Ref. 1). There remain basic aspects of Treg biology that are unresolved, however. For example, whether Tregs are functionally suppressive at inflammatory sites or act solely to limit the priming of naive T cells in the lymph nodes (LNs) remains a controversial issue (2). Furthermore, where and when disease-relevant Tregs are selected and expanded during the natural resolution of inflammation is not well defined. These are key considerations for the development of Treg-based therapeutic strategies.

Experimental autoimmune encephalomyelitis (EAE) shares many pathological and histological features with multiple sclerosis (MS). Myelin-reactive T cells initiate inflammation within the CNS, resulting in an ascending paralysis. Increasing the numbers of Tregs has shown beneficial effects in EAE (3, 4, 5, 6), a strategy that may be of particular clinical relevance because Treg function is reported to be compromised in MS patients (7). The potential exists for Tregs to influence the course of EAE by affecting priming in the LN and/or the function of effector cells in the target organ itself. Adoptive transfer of polyclonal, peripherally derived CD25+ Tregs can protect mice against the development of clinical EAE (3, 4). The transferred Tregs were found to home to peripheral LNs and could not be recovered from the CNS, suggesting that, in these studies, their primary function in vivo was to limit the expansion of autoreactive T cells during priming (3).

In contrast, we recently reported an accumulation of IL-10-producing Foxp3+ Tregs in the CNS, which correlated with recovery from EAE. These CNS-derived Tregs displayed regulatory activity ex vivo and were able to transfer protection against EAE more efficiently than CD25+ Tregs derived from the periphery of naive mice (8). Several other groups have now confirmed the presence of Tregs in the CNS during recovery from EAE (9, 10). These were important observations because they highlighted, for the first time, that Tregs made an indispensable contribution to the natural resolution of autoimmune inflammation in the CNS. By extension, they highlight a need for sampling Tregs from the CNS as well as the blood in MS, although access to CNS is of course restrictive, making comparisons extremely difficult.

An important question arising from our previous studies is whether the increased numbers of Tregs present in the CNS during recovery from EAE results from a sequestration of polyclonal Tregs from the blood, from an influx of Ag-reactive Tregs primed initially in the lymphoid organs, or from the in situ activation and proliferation of Tregs specifically in the CNS? This question cannot be addressed satisfactorily with the actively induced EAE models used to date, because these involve a residual Ag depot at the site of immunization that might provoke the Treg response. We therefore followed the development of a Treg response to local tissue damage orchestrated by preformed effector T cells in a passive transfer model of EAE. In response to this, a highly elevated rate of proliferation was evident among Tregs specifically in the CNS, and this was not apparent in the LNs. At the peak of this proliferative response, there was a significant enrichment of Vbeta8+ Foxp3+ Tregs in the CNS, indicative of a degree of selective activation. Interestingly, however, it seems that only a minority of these CNS Tregs were responsive to the 35-55 peptide of myelin oligodendrocyte glycoprotein (pMOG), the autoantigen recognized by the pathogenic T cell population. Our data are the first to identify the inflamed CNS as an environment for the initial activation and proliferation of Tregs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, Ag, and tissue culture medium

C57BL/6 (CD45.2+, CD45.1+, or CD90.1+), OT-II, and 2D2 mice (all or the H-2b background) were bred under specific pathogen-free conditions at the University of Edinburgh (Edinburgh, U.K.). The 2D2 founder mice bearing a transgenic TCR recognizing myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (11) were a gift from Dr. V. K. Kuchroo (Harvard University, Boston, MA). Foxp3-GFP reporter mice were provided by Dr. A. Rudensky (University of Washington, Seattle, WA) (12). OT-II mice express a transgenic TCR recognizing the 323-339 peptide of OVA (pOVA) (13). Experiments received University of Edinburgh ethical approval and were performed under U.K. legislation.

The pMOG and pOVA were obtained from the Advanced Biotechnology Centre, Imperial College (London, U.K.). Tissue culture medium was RPMI 1640 medium, supplemented with 2 mM L-glutamine, 100U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10–5 M 2-ME (all from Invitrogen Life Technologies) and 5% FCS (Sigma-Aldrich).

Passive and active induction of EAE

Each donor mouse received 100 µg of pMOG emulsified in CFA containing 50 µg of heat-killed Mycobacterium tuberculosis H37Ra (Sigma- Aldrich) at a final volume of 100 µl injected s.c. into the hind legs. Seven to eleven days postimmunization, cells from the draining LNs (both inguinal and para-aortic) were processed and cultured at 4 x 106 cells/ml with 10 µg/ml pMOG, 10 U/ml IL-2 (R&D Systems), 25 ng/ml rIL-12 (R&D Systems), and 25 ng/ml rIL-18 (Marine Biological Laboratory). After 48 h, cultures were split as required, and the final concentration of IL-2 was increased to 100 U/ml. Cells were harvested after a 72-h culture and washed twice in serum-free PBS, and 4 x 106 blasts were transferred i.v. to each host animal. In some experiments, donor cells from GFP– littermates were transferred into male Foxp3-GFP reporter mice. On the day of transfer, each mouse also received 200 ng of pertussis toxin (Health Protection Agency, Dorset, U.K.) in 0.5 ml PBS i.p. In some experiments, EAE was induced by active immunization with pMOG as described previously (8). Clinical signs of EAE were assessed daily with the following scoring system: 0, no signs; 1, flaccid tail; 2, impaired righting reflex and/or gait; 3, partial hind limb paralysis; 4, total hind limb paralysis; 5, hind limb paralysis with partial front limb paralysis; and 6, moribund or dead.

Preparation of CNS-infiltrating mononuclear cells

Mice were sacrificed by CO2 asphyxiation and perfused with cold PBS. Spinal cords were removed by intrathecal hydrostatic pressure. Brain and spinal cords were cut into small pieces and digested with 2.5 mg/ml collagenase (Worthington Biochemical) and 1 mg/ml deoxyribonuclease (Sigma-Aldrich) for 25 min at 37°C followed by mechanical disaggregation. Single cell suspensions were washed once in tissue culture medium. Mononuclear cells were prepared from the interface of a 30:70% discontinuous Percoll gradient after centrifugation for 20 min at 850 x g.

Abs and FACS analysis

Cells were stained for FACS using the following Abs (all from eBioscience, except where stated): anti-CD4-allophycocyanin, anti-CD4 PE, anti-CD4 PerCP (BD Pharmingen), anti-CD8{alpha}-PE, anti-CD45.1-FITC/PE/PerCP/allophycocyanin, anti-CD25 FITC/PE (clone 7D4, Miltenyi Biotec), anti-CD25 PE/allophycocyanin (PC61; BD Pharmingen), CD62L FITC, CD44 PE, anti-IFN-{gamma}-FITC, anti-IL-17 PE, rat IgG1 FITC, and rat IgG1 PE. Anti-Foxp3-FITC/PE/allophycocyanin staining kits and appropriate isotype controls (rat IgG2a-FITC/PE/allophycocyanin) were purchased from eBioscience and used according to the manufacturer’s instructions. FACS data were collected on either a FACSCalibur or LSR flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).

For intracellular cytokine staining, cells were resuspended at 1 x 107 cells/ml in the presence or absence of 25 µg/ml pMOG. After overnight culture, 1 µg/ml of brefeldin A (1000 x stock; eBioscience) was added carefully before the last 4 h of culture. Cells were washed once in FACS buffer (PBS, 2% FCS, and 0.01% NaN3) and resuspended in 100 µl of permeabilization buffer (FACS buffer plus 0.1% saponin; Sigma-Aldrich) containing the anti-cytokine Abs or relevant isotype controls and incubated at room temperature in the dark for 40 min. Cells were washed in FACS buffer, surface stained on ice for 10 min, washed, and fixed in 1% paraformaldehyde before analysis.

Levels of apoptosis were measured using the Annexin V assay. After staining for cell surface markers, samples (1–2 x 105 cells/tube) were washed with 300 µl Annexin binding buffer (0.01M HEPES, 140 mM NaCl, and 2.5MM CaCl2). Supernatants were removed and 2 µl Annexin V-FITC per 105 cells (BD Pharmingen) was added to each pellet in 10 µl Annexin binding buffer. Cells were stained for 15 min in the dark at room temperature. Immediately before collection, 300 µl binding buffer and 10 µl 7-aminoactinomycin D (BD Pharmingen) were added to each sample.

BrdU staining

Mice were treated with 2 mg BrdU (Sigma-Aldrich) in PBS i.p. at various times as described. Following harvest, BrdU incorporation was detected using a FITC-BrdU Flow Kit (BD Pharmingen) largely as described in the manufacturer’s instructions. To costain optimally for BrdU and Foxp3, it was essential to incorporate the use of the eBioscience fix/perm buffer (from the Foxp3 staining kit). The adjustment was made as follows. Samples were surface stained as normal (e.g., CD4/CD25), washed in FACS buffer, and resuspended in 1 ml of eBiosciences fix/perm buffer and incubated for at least 30 min at 4°C. From this point onward, the anti-BrdU kit instructions were followed from the point after initial incubation in BD cytofix-cytoperm buffer.

In vitro BrdU assays

CD4+CD25– cells (4 x 104) and/or the same number of CD4+CD25+ cells and 2 x 105 irradiated (30 Gy) splenic APC were cultured in the presence or absence of 10 µg/ml pMOG with or without addition of 0.1 ng/ml rIL-2 (R&D Systems). BrdU was added to a final concentration of 10 µM for the final 12 h as indicated. Cells were recovered from culture surface stained and processed for BrdU and Foxp3 staining as described above.

CD4+CD25+ purification

For purification of CD25–/+ populations from the periphery, CD4+ cells from the spleen and pooled LNs were purified by magnetic selection using MACS anti-CD4 microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. The CD4+ cells obtained were then surface stained for CD4 and CD25 and sorted on a FACSAria (BD Biosciences). CNS-infiltrating mononuclear cells were surface stained with CD4 and CD25 before FACS-sorting. Purity of CD4+CD25+ cells was routinely >93%.

In vitro suppression assays

CD4+CD25– cells (2 x 104 cells/well) were cultured for 96 h with 1 x 105 irradiated (30 Gy) splenic APC, 2 µg/ml anti-CD3 (clone 145.2C11; eBioscience), and with or without an equal number of CD4+CD25+ cells. [3H]TdR (0.5 µCi; Amersham Biosciences) was added for the final 18 h of culture and incorporation was measured using a liquid scintillation beta-counter (Wallac). In some assays 2 x 104 2D2 or OT-II CD4+ T cells were used as responders and were stimulated with 10 µg/ml pMOG or pOVA, respectively. Mean cpm of triplicate cultures are shown.

Statistical analysis

Statistical analysis of results was performed using a two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Acute, monophasic disease after transfer of pMOG-reactive T cell populations

To assess the influence of Tregs on the development and resolution of passively induced EAE, we used a model that reliably induced a single acute episode of paralytic disease followed by full recovery (Fig. 1A). Encephalitogenic donor populations were generated from the draining LNs of pMOG-immunized C57BL/6 mice by in vitro restimulation with pMOG in the presence of IL-2, IL-12, and IL-18 in a protocol adapted from that of Ito et al. (14). This induction protocol gave a highly reproducible disease (90% incidence) with a mean maximum clinical score of 3.17 ± 0.82 (n = 188). The peak of disease consistently fell between days 9 and 12, with the onset of recovery from day 13 onwards, and clinical resolution within 21 days. Wherever possible, donor cells bearing isotypic markers (CD45.1 or CD90.1) were used to allow discrimination of the host and donor populations (Fig. 1B).


Figure 1
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  		FIGURE 1. Passive EAE disease course and donor cell contribution to the CNS infiltrate. EAE was passively induced as described in Materials and Methods. A, Mean clinical score ± SD (n = 7). B and C, Cells were recovered at 12 days postinduction and assessed by FACS analysis for donor cell (CD45.1+) distribution. D, IFN-{gamma} production by CNS-infiltrating T cells cultured with or without pMOG (10 µg/ml). One experiment representative of three is shown. Bar charts, mean ± SD of three individual mice.

 
The local enrichment of donor CD4+ cells in the CNS was apparent when compared with their relative paucity in the LNs (Fig. 1C). Interestingly, even at the peak of disease, the pMOG-reactive IFN-{gamma}-producing cells were contained almost entirely within the CNS-infiltrating donor population (Fig. 1D). Indeed, we found no evidence of significant pMOG-induced IFN-{gamma} production by host-derived cells. Given the current interest in the role of IL-17 in driving EAE (15), it was notable that the frequency of T cells producing IL-17 in response to pMOG (either at the time of transfer, or upon reisolation from the CNS) was never greater than a few percent.

EAE resolution is associated with concentration of host-derived Tregs within the CNS

We determined the relative representation of Foxp3+ Tregs in the CNS and lymphoid tissues during the course of disease. The cervical LNs were monitored to see whether any changes in Treg numbers in the CNS were preceded, accompanied, or followed by similar changes in the regional LNs. The distal, inguinal LNs were included as a negative control. Comparable proportions of Foxp3+ cells were recovered from the cervical and inguinal LNs at all time points, and these levels did not change over the period observed (Fig. 2A). In contrast, there were dynamic fluctuations in the proportion of Foxp3+ cells present in the CNS (Fig. 2A). When disease was in the ascendancy (days 10 and 12), Foxp3+ cells were significantly underrepresented in the CNS compared with the periphery. On entry to the recovery phase, however, numbers of Foxp3+ cells in the CNS increased, reaching levels significantly higher than those seen in the LNs. Finally, at the resolution of clinical disease, the percentage of Foxp3+ cells in the CNS was equivalent to peripheral levels.


Figure 2
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  		FIGURE 2. CNS-infiltrating Tregs are host derived. EAE was induced as described in Materials and Methods. A, Percentage of Foxp3+ cells within the CD4+ population in the CNS ({blacksquare}), cervical LN ({triangleup}), and inguinal LN ({blacktriangleup}). Each data point illustrates the mean ± SD of three individual mice. * and ** = significantly different to LN values. B, FACS plots illustrating the contribution of donor (CD45.1+) and host (CD45.1–) CD4+ T cells to the CNS infiltrate and the level of CD25 expression on host vs donor cells, gated as shown. C, Overlay plot illustrating the comparative expression of Foxp3 in CNS-infiltrating CD4+ T cells of host (dotted line) and donor (solid line) origin. D, Foxp3 expression among host (•) and donor ({circ}) CD4+ T cells in the CNS. One experiment representative of three is shown.

 
Transfer of CD45-disparate cells allowed the assessment of the relative contribution of host and donor cells to the Treg pool. The majority of CD4+CD25+ T cells within the CNS were clearly host derived (Fig. 2B). Furthermore, donor cells were almost entirely Foxp3–, while during recovery, up to ~40% of the host CD4+ compartment was Foxp3+ (Fig. 2, C and D). Over time, the changes in the numbers of Foxp3+ cells in the CNS resulted entirely from changes within the host cell infiltrate. Notably, there was no fluctuation in the proportion of Foxp3+ cells within the donor population. Thus, we find no evidence to suggest the conversion of precommitted encephalitogenic cells to a regulatory phenotype.

Treg expansion within the CNS includes a small MOG (35-55)-responsive component

We next used in vitro assays to test whether CNS-derived effector cells or Tregs could respond to defined myelin Ags. To assess the importance of immunization, we tested cells from mice that were undergoing active vs passive for disease. CNS T cells were sorted into CD25– and CD25+ fractions and cultured in the presence or absence of pMOG (Fig. 3A), and with or without exogenous IL-2. Proliferation was measured by BrdU uptake and was detected using FACS to allow for the determination of both Foxp3 expression and proliferation. CNS-derived Foxp3– T cells displayed a strikingly high level of background (medium only) proliferation in vitro, with around 50% of cells incorporating BrdU during the last 12 h of a 72-h culture (Fig. 3B). This was the case whether the CD25– cells were sourced from mice with active or passive disease. Analysis of CD25+ cells from mice with active EAE revealed a low background level of BrdU incorporation among Foxp3+ cells that was greatly elevated by the addition of pMOG to the cultures. In contrast, Foxp3+ cells from mice with passive disease only showed marginal increases in BrdU incorporation in the presence of pMOG (Fig. 3, A and B). Nevertheless, this small increase was consistently observed over several experiments. For both cell populations, neither the level of background proliferation, nor the magnitude of the Ag-induced response, were influenced by the addition of exogenous IL-2 (data not shown). As yet, we have not been able to detect pMOG-driven cytokine production by CNS-derived Tregs (data not shown).


Figure 3
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  		FIGURE 3. Specificity and in vitro suppressive potential of CNS-derived Tregs. Sorted CNS-derived effectors (CD25–) or Tregs (CD25+; day 14) were cultured in the presence or absence of pMOG (10 µg/ml). A, FACS plots show BrdU incorporation by Foxp3+ cells derived from mice with passively induced (top) or actively induced (bottom) EAE during the last 12 h of a 72-h culture. B, BrdU incorporation by Foxp3– and Foxp3+ T cells from mice with passively induced EAE. C, CNS-derived Tregs were cocultured with CD4+CD25– cells from naive B6, 2D2, or OT-II mice; cultures were stimulated with 2 µg/ml anti-CD3, 10 µg/ml pMOG, or 10 µg/ml pOVA, respectively. The degree of suppression is illustrated as a percentage reduction in the maximal (effectors only) response (32300, 55641, and 24530 mean cpm, respectively). D and E, Sorted CD25– and CD25+ CNS-infiltrating CD4+ T cells were cultured separately or at the ratios indicated in the presence or absence of 10 µg/ml pMOG. D, Proliferation as assessed by incorporation of [3H]TdR during the last 16 h of a 96-h culture. E, BrdU incorporation during the last 12 h of culture was assessed by FACS analysis (gated on Foxp3– cells). All cultures contained irradiated B6 splenic APC. One experiment representative of three is shown.

 
From these data, we conclude that Foxp3+ cells derived from the CNS of mice with active disease contain a significant fraction of pMOG-reactive cells, presumably generated in response to immunization with pMOG. However, the Foxp3+ cells that accumulate in the CNS of mice that were not previously immunized (passive EAE) show a much smaller cohort of pMOG-reactive cells.

Thus, pMOG-responsive T cells made up, at best, a few percent of the total CNS Foxp3+ complement in passive EAE. Nevertheless, we sought to test their capacity to suppress the pMOG responses of naive and effector T cells in vitro by coculture of sorted CNS CD4+CD25+ cells with CD25– cells (Fig. 3, C–E). CNS-derived CD25+ cells were able to suppress the anti-CD3-driven proliferative response of CD4+CD25– T cells taken from naive mice (Fig. 3C). Analysis of their effects on naive TCR transgenic T cells revealed that CNS Tregs suppressed the peptide-driven proliferation of pMOG-reactive 2D2 T cells, but not of OT-II T cells (bearing a TCR that recognizes the 323-339 pOVA; Fig. 3C). This further indicated that at least some of the host-derived CNS Tregs were responsive to pMOG.

Coculture of CNS-derived CD25+ with CNS-derived CD25– T cells reduced the proliferative response of the latter to pMOG (Fig. 3D). Use of BrdU staining confirmed that proliferation of the Foxp3– fraction was suppressed under these conditions (Fig. 3E). However, it is important to note that this suppression was only evident in a 1:1 coculture (Fig. 3D). This was in contrast to the profound suppression of naive responses achieved with CNS Tregs at much lower frequencies (Fig. 3C). Thus, CNS Tregs do show suppressive capacity upon CNS effectors, but these in vitro assays appear to be at the limit of their function.

CNS Tregs can suppress pMOG-driven production of IFN-{gamma}, but not IL-17

As shown above, the dominant effector cytokine produced by the transferred T cell population in response to pMOG was IFN-{gamma}, with very few IL-17 producers evident. We used coculture experiments to test whether CNS Tregs could affect the production of these two cytokines by CNS-derived effectors (Fig. 4). In cultures containing only CD25– cells, the proportion of Foxp3– cells producing IFN-{gamma} in the absence of pMOG increased, suggesting that Tregs limit "spontaneous" IFN-{gamma} production (Fig. 4A, top left and middle). This was confirmed by the reintroduction of CD25+ cells, which returned the spontaneous IFN-{gamma} production to levels seen with cultures containing total (unseparated) CD4+ cell populations. When pMOG was included, IFN-{gamma} production was again greater in the CD25– cells cultured alone compared with the total CD4+ population, particularly in the IFN-{gamma}high population (Fig. 4A, lower left and middle). Again, addition of CD25+ cells to the cultures caused a reduction in this pMOG-induced IFN-{gamma} production by Foxp3– cells (Fig. 4A, lower right). In contrast, we did not find any increased levels of spontaneous IL-17 production by Foxp3– cells cultured in the absence of CD25+ cells. However, there was a small, but clearly detectable, IL-17+ population upon culture with pMOG (Fig. 4B). Strikingly, this pMOG-induced IL-17 production was not suppressed by the inclusion of the CNS CD25+ cells (Fig. 4, B and C).


Figure 4
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  		FIGURE 4. CNS Tregs can suppress the pMOG-driven production of IFN-{gamma}, but not IL-17. CD4+ T cells were FACS sorted from the CNS of mice recovering from EAE (day 14). Total CD4+ (containing both CD25– and CD25+ cells), CD25– alone, or CD25– and CD25+ cells (recombined at a 1:1 ratio) were cultured overnight in the presence or absence of 25 µg/ml pMOG (all cultures contained irradiated B6 splenic APC). Production of IFN-{gamma} (A) or IL-17 (B) by CD4+Foxp3– T cells was assessed by FACS. C, Bar graph, pooled IL-17 data from two individual experiments. For IFN-{gamma}, one experiment representative of three similar experiments is shown.

 
Local enrichment of Vbeta8-expressing Tregs within the CNS

EAE has commonly been associated with oligoclonal expansion of myelin-reactive T cells using Vbeta8 genes. This is also true in the C57BL/6 model (16), with pMOG-reactive "public" TCRs using Vbeta8 (17). Therefore, if Treg accumulation in the CNS results from an expansion of myelin-reactive clones, this should lead to local skewing of the TCR repertoire toward Vbeta8. To assess this, we compared Vbeta expression in the CNS and the periphery using a panel of anti-Vbeta Abs.

At the peak of disease, Foxp3– and Foxp3+ T cells in the cervical LN displayed similar patterns of Vbeta usage (Fig. 5A, top). Vbeta usage in the CNS differed from that seen in the periphery. Most notably, there was an over-representation of Vbeta8.1/2+ cells among the Foxp3– population (Fig. 5A, bottom). At this time point, the representation of Vbeta8.1/2+ cells among the Foxp3+ population in the CNS was equal to that seen in the LN.


Figure 5
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  		FIGURE 5. Vbeta usage is skewed in the CNS in both T effectors and Tregs. EAE was induced as described in Materials and Methods. A, At the peak of disease, cells from the cervical LNs and CNS-infiltrating mononuclear cells were extracted for analysis of Vbeta expression among Foxp3– ({square}) and Foxp3+ cells ({blacksquare}). B, EAE was induced in male Foxp3-GFP reporter mice by transfer of encephalitogenic cells derived from nontransgenic (GFP–) littermates. Expression of Vbeta8.1/2 on Foxp3– ({circ}, {blacksquare}, and {triangledown}) and GFP+Foxp3+ (host-derived Tregs, {blacktriangleup}, {blacktriangledown}, and •) cells in the LN ({blacksquare} and {blacktriangleup}), spleen ({triangledown} and {blacktriangledown}), and the CNS ({circ} and •) at 9 and 14 days postinduction of EAE (peak of disease and during recovery); *, p < 0.003 compared with the proportion of cells expressing V-beta8.1/2 in the periphery. Data are representative of three similar experiments. In naive mice, the frequency of Foxp3+ cells within the peripheral lymphoid organs that are Vbeta8.1,2+ consistently lies between 15 and 19%.

 
EAE was induced in Foxp3-GFP reporter mice by transfer of encephalitogenic cells derived from nontransgenic littermates to allow the identification of host-derived Tregs on the basis of GFP positivity. During recovery (day 14), Vbeta8.1/2+ cells were significantly over-represented in the GFP+ fraction within the CNS. Thus, between peak and recovery, the proportion of Vbeta8.1/2+ GFP+ in the CNS T cells had increased to an elevated level equivalent to that seen among their Foxp3– counterparts (Fig. 5B). Over this same time period, Vbeta usage among Foxp3+ and Foxp3– cells in LN and spleen remained stable. Furthermore, there was no increase in the expression of any of the other Vbeta tested in the CNS or the periphery (data not shown).

Treg accumulation reflects their selective proliferation within the CNS

Thus far, we had observed a sharp increase in the frequency of host-derived Foxp3+ cells within the CNS during the resolution of EAE, and that this correlated with an increase in the frequency of Tregs expressing Vbeta8, suggestive of a response to myelin autoantigen(s). The next question was what was driving this effect? Were the Tregs proliferating and, if so, where? Would increased proliferation in the regional LN precede the appearance of recently divided Tregs in the CNS, or would the trigger for Treg proliferation be provided by the CNS itself? To address this, we combined in vivo BrdU-labeling and ex vivo Foxp3 staining using a cumulative dosing approach. We confined our observations to the period of interest identified in previous experiments (see Fig. 2, A and B) and compared levels of BrdU incorporation in the CNS, the cervical LNs, and the distal inguinal LNs. It was immediately apparent that elevated levels of proliferation among both Foxp3– and Foxp3+ T cells were entirely restricted to the CNS (Fig. 6, A and C). From 10 days posttransfer onwards, the proportion of CD4+ T cells having proliferated, as indicated by incorporation of BrdU, was significantly greater in the CNS than in the LNs (Fig. 6A). As would be expected with a cumulative dosing strategy, the proportion of BrdU+ cells increased over time. In the CNS, the proportion of BrdU+Foxp3+ T cells increased to a higher level than was seen among Foxp3– cells. On day 14, the majority (63.3 ± 1.12%) of CNS Tregs had proliferated within the preceding 6 days (Fig. 6A). The absolute number of T effectors, which had recently proliferated, peaked at day 12 before declining (Fig. 6B). Thus, peak effector proliferation preceded peak Treg proliferation which, in turn, coincided with physical recovery. Notably, the burst of Treg proliferation in the CNS was synchronous with the rapid increase in Treg numbers identified in our earlier experiments (Fig. 2), indicating that in situ proliferation underlies this phenomenon.


Figure 6
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  		FIGURE 6. Treg proliferation occurs specifically within the inflamed CNS. EAE was induced as described in Materials and Methods. A and B, Mice were given 2 mg BrdU i.p. on days 8, 10, 12, and 14. A, The percentage of Foxp3– ({square}, {triangleup}, and {circ}) and Foxp3+ cells ({blacksquare}, {blacktriangleup}, and •), which had incorporated BrdU in the CNS ({square} and {blacksquare}), the cervical LN ({triangleup} and {blacktriangleup}), and the inguinal LN ({circ} and •). B, The absolute number of BrdU+Foxp3– ({square}) and BrdU+Foxp3+ ({blacksquare}) in the CNS over time. C and D, Mice were given BrdU i.p. 24 h before sacrifice. C, BrdU incorporation among Foxp3– and Foxp3+ T cells in the cervical LN and the CNS measured on day 14 post EAE-induction. D, BrdU incorporation among Foxp3– ({square}) and Foxp3+ cells ({blacksquare}) in the CNS. E, Passive EAE in Foxp3-GFP mice following transfer of donor cell from GFP– littermates. The proportion of CNS-infiltrating GFP– (Foxp3–; {circ}) and GFP+ (Foxp3+; •) staining with Annexin V and 7-aminoactinomycin D is shown. Each data point represents the mean ± SD of three individual animals.

 
The level of BrdU incorporation in the cervical and inguinal LNs was equivalent throughout the period observed, giving no indication of increased proliferation outside the CNS itself (Fig. 6A). Foxp3+ cells consistently displayed a higher turnover rate than their Foxp3– counterparts (Fig. 6, A, C, and D). This was true even in the steady state in naive mice (data not shown), confirming observations previously made using CD25 as a Treg marker (18).

For a more detailed account of the daily balance of proliferation, we used a 24-h pulse approach, giving BrdU the day before sacrifice, to give a snapshot of proliferation within the previous 24 h. As noted above, proliferation in the CNS was significantly higher than in the LN and higher among Foxp3+ cells than their Foxp3– counterparts (Fig. 6D). Kinetic analysis revealed a shift in the balance of proliferation in the CNS in favor of Tregs from day 12 onward. The initially high level of proliferation among Foxp3– cells in the CNS dropped, while Foxp3+ cells continued to proliferate (Fig. 6D).

Using encephalitogenic cells derived from nontransgenic littermates to induce EAE in Foxp3-GFP reporter mice allowed us to assess the rate of apoptosis among host-derived Tregs in the CNS vs the periphery. There was a generally higher level of apoptosis in the CNS than the periphery and, in the CNS, Foxp3+ cells had a higher rate of death than their Foxp3– counterparts at all time points through disease onset, peak, and into recovery (Fig. 6E). These data argue against the shift in balance toward Tregs in recovery being due to an excessive death of effectors in the CNS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have previously described the critical role of Foxp3+ Tregs within the CNS during recovery from EAE (8). In this study, we have investigated how this Treg population arises. Our data provide a picture of Tregs entering the CNS in response to inflammation and initiating a dramatic proliferative burst. This involves a small, but detectable, pMOG-reactive component, suggesting that at least some Tregs can naturally mirror the Ag reactivity of the effector cells they suppress. A recent report using a combination of Foxp3-GFP reporter mice and Ab-pMOG multimers has quantified the pMOG-reactive Treg compartment in mice immunized for active EAE (19). This fraction correlates well with the proportion that we see in the CNS during active disease using the BrdU-labeling technique. As yet, multimers have not been used to assess pMOG-reactive CNS Tregs in passive disease as we have attempted in this study. We believe that the BrdU technique that we have developed in this study may well be of use to other investigators who wish to test panels of candidate Ags for stimulatory activity on Foxp3+ cells extracted from inflammatory sites.

Studies using active induction protocols (involving immunization with autoantigen in CFA) have highlighted the influence of Tregs on the development of EAE, illustrating an influence of Tregs on the priming and polarization of T cell effectors (3, 4, 8, 20, 21). However, autoimmune diseases such as MS are not recognized in time to allow therapeutic interventions aimed at suppressing the early stages in the differentiation of autoreactive T cells. In this sense, the capacity of Tregs to influence ongoing inflammation, orchestrated by precommitted effector T cells, is of particular relevance. We and others have observed increased numbers of Foxp3+CD25+ T cells in the CNS during the resolution of EAE, suggesting Treg function in the target organ may be more relevant than in the LN (8, 10). In these actively induced models of EAE, however, it is difficult to determine where the Tregs are exerting their influence. To overcome this complication, we used the passive transfer of preformed effector cells to induce EAE, removing the possibility of Treg priming by a peripheral Ag-adjuvant depot.

Of interest, data from a recent study indicate that circulating Treg numbers in aged human populations are maintained by a very rapid turnover of CD4+CD45RO+CD25highFoxp3+ cells (22). In this study, we found a remarkable rate of proliferation in Foxp3+ T cells specifically within the inflamed CNS. The expected turnover of the transferred effector T cells in the CNS appeared to provoke this Treg proliferation, which was sustained as the proliferation of the pathogenic cohort waned. Although Tregs present an anergic phenotype in vitro, they can proliferate in vivo while maintaining their suppressive function (23, 24, 25). In contrast to these previous studies describing Treg proliferation in the lymphoid system, in this study, we found no elevation in Treg proliferation in the cervical LNs during the course of passively induced EAE. Rather, we show a robust proliferative response in the CNS itself. As such, our study is the first to demonstrate that the proliferation of CD25+Foxp3+ regulatory T cells appears to be restricted to the tissue, rather than in the draining LNs, during the natural resolution of inflammation.

The over-representation of Vbeta8+ Foxp3+ T cells in the CNS relative to the LNs suggests some degree of restriction on which Tregs proliferate in the CNS (although 25% of CNS Tregs bearing Vbeta8+ clearly cannot totally account for a turnover rate of 60%). Another interesting point is that, although pMOG-reactive T cells favor the use of Vbeta8, the small percentage of pMOG- reactive Tregs that appear in the CNS during passive disease cannot account for the larger increase in Vbeta8+ Tregs. As yet, we cannot account for this discrepancy, although T cells recognizing other myelin autoantigens have also been reported to favor Vbeta8 usage. This possibility requires further investigation.

Recent studies have demonstrated the presence of CNS-derived Ags in APC in the cervical LNs and the trafficking of dendritic cells (DCs) from the CNS to these nodes (26, 27). These findings raised the possibility that propagation of the autoaggressive lesion follows peripheral presentation of CNS-derived Ags. Miller and colleagues (28) addressed this issue and found that proliferation in response to a "spread" epitope was occurring specifically within the CNS and not in the cervical LNs or other peripheral lymphoid organs. Our results extend the notion of the CNS as an environment that triggers initial T cell activation and clonal expansion to include the triggering of Tregs. There is clear evidence that, during the course of EAE, CNS-resident DCs efficiently process and present CNS-derived Ags (28, 29) (30). It is also known that mature DCs can efficiently stimulate the proliferation of Tregs (25, 31), and so it seems likely that DCs infiltrating the inflamed CNS promote the expansion of Tregs in situ. It will be interesting to determine whether Tregs in the CNS are located in particular areas at particular times. In common with other studies (9, 19), preliminary immunohistological data do not indicate any discrete localization of the Treg infiltrate, but rather a widespread distribution within cerebellar lesions (data not shown). However, a complete picture will require exhaustive analysis of the entire CNS throughout the course of the disease, which is beyond the scope of this report.

TGF-beta is an important cytokine in Treg biology, maintaining the suppressor function and peripheral survival of natural Tregs in vivo (32, 33), as well as inducing de novo expression of Foxp3 and functional differentiation into Tregs in vitro (34). TGF-beta is expressed within the inflamed CNS, and its production correlates with recovery from clinical EAE (35). A recent report suggested that an interaction between neurons and T cells promotes the conversion of encephalitogenic T cells to Foxp3+ Tregs (10). These results are in conflict with our own observations. We found no evidence for the conversion of the encephalitogenic donor cells to a Treg phenotype (they were consistently <1% Foxp3+ at the time of transfer and upon reisolation). Although Liu et al. (10) showed up to 27% Foxp3-expression among encephalitogenic donor cells recovered from the CNS, we would argue that this most likely reflects an expansion of the ~7% of Foxp3+ cells already present in their donor population at transfer. The recent report by Korn et al. (19) supports our view.

The conditional expression of transgenic TGF-beta in the islets has been shown to inhibit the spontaneous development of autoimmune diabetes (36). Early expression of TGF-beta correlated with an increased frequency of CD4+CD25+ cells (that showed Foxp3 mRNA expression) in the islets, to levels similar to those we see in the host compartment in the CNS during recovery from EAE. BrdU analysis revealed that around 20% of these CD4+CD25+ cells were proliferating, indicating that a proliferative burst of Tregs can also impact on the pathology in the pancreas (although delayed TGF-beta expression also inhibited disease without an increase in CD4+CD25+ numbers) (36). Thus, a Treg proliferative burst may be required if they are to prevent development of disease (36), as well as play a role in recovery from autoimmune disease, shown in this study.

The relative proliferative rates of Foxp3+ and Foxp3– cells in situ in the CNS (Foxp3+ > Foxp3–) were reversed upon isolation and in vitro culture (see Fig. 3). This suggests an in vivo interaction in which a mediator in the inflamed CNS (derived either from the T effectors or from innate cells) drives the proliferation of Tregs which, in turn, inhibit T effector proliferation and IFN-{gamma} production. Upon isolation in vitro, T effectors are released from this and proliferate rapidly in the absence of additional Ag. In contrast, Tregs become comparatively unresponsive even in the presence of pMOG. The nature of such an in vivo driver of Treg proliferation is as yet unknown. IL-2 is an obvious candidate, but the lack of Treg proliferation upon addition of IL-2 to in vitro cultures suggests otherwise. Of course, rather than being a soluble mediator, the driver of Treg proliferation might be a particular cell type present in the CNS that is not contained within our CD4+ populations.

Whether or not Tregs are functional in sites of inflammation remains a controversial issue (37). TLR ligation and exposure to inflammatory cytokines might interfere with the suppressive capacity of Tregs limiting their activity in inflammatory lesions (38, 39). Other studies have illustrated the enhanced suppressive capacity of "effector/memory-like" CD103+ Tregs and attributed this to their ability to migrate to sites of inflammation (40). Functional Tregs have certainly been recovered from inflammatory sites in various experimental models (8, 36, 41) as well as from the synovial fluid of patients with rheumatoid arthritis (42, 43). Indeed, the results of a recent study suggest that the protective effects of Tregs in the target organ are of greater consequence than their actions in the LN (44). In this study, we find that the quality of Treg suppressive function in vitro is dependent on the source of the responders and on the response that is being measured. We focused on Ag-driven responses and found CNS Tregs suppress pMOG-induced proliferation of naive 2D2 T cells (whereas naive splenic Tregs do not, data not shown), but not pOVA-induced proliferation of naive OT-II cells, indicating a requirement for some degree of TCR engagement for Treg function in these assays. When pMOG-driven responses of CNS-derived effectors were analyzed, Tregs could limit both proliferation and IFN-{gamma} production, but IL-17 producers appeared resistant to the effects of Tregs. This is particularly striking, given the relative numbers of IFN-{gamma}- vs IL-17-producing cells involved in this model. Thus, when Tregs are included in these cultures, they can reduce the level of IFN-{gamma} that is produced by 20–30% of the Foxp3– cells, but fail to control IL-17 production by a comparatively much smaller population. These data are consistent with the recent report that Tregs are less effective at suppressing the differentiation of Th17 vs Th1 cells in a model of graft-vs-host disease (45) and extend this differential capacity to the regulation of preformed T effectors from an inflammatory site. Korn et al. (19) used proliferation as a readout for suppressive capacity of CNS Tregs upon CNS effectors and reported a lack of suppression at the peak of active disease, although this was observed in recovery. Similarly, in our hands, suppression of naive responses is highly effective, whereas CNS Tregs are only active on proliferation of CNS effectors when cultured at a 1:1 ratio. Our data using IFN-{gamma} as a readout show that, when Tregs are present at their natural frequency (i.e., in unfractionated CNS CD4+ cells), they limit the IFN-{gamma} production by Foxp3– cells both in the presence and absence of additional pMOG (Fig. 4a).

Finally, there is the issue of whether the Treg population needs to recognize tissue-specific Ag(s) to exert their protective effects on the CNS. CNS-derived Tregs were potent in vitro inhibitors of naive 2D2 T cells and (to a lesser extent) of CD25– CNS effectors in response to pMOG. This suggests that, although the pMOG-reactive Treg contingent is probably <5% of all CNS Foxp3+ cells, it can have a significant impact on suppressive function. In support of this notion, Foxp3-GFP+ cells sorted from spleens of pMOG-immunized mice effectively suppressed naive 2D2 T cell responses in vitro, even though as few as 2% of that Treg population bound to Ab-pMOG multimers (19). Alternatively, this might reflect a global suppressive effect of Tregs that clearly have recently been activated in vivo. The fact that CNS Tregs do not suppress naive OT-II T cell responses to pOVA argues against this. Although these are positive results, in that we have been able to identify a self-Ag to which some CNS-derived Tregs can respond without prior immunization, they, of course, beg the question what do the majority of these cells recognize: A different myelin Ag, or perhaps a non-CNS restricted self-Ag that is up-regulated during inflammation? Although it will be a challenge to identify the Ag(s) recognized, importantly, it could provide a route to Ag-based regimens to maintain/expand the numbers and/or functions of these cells.


   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 work was supported by grants from the U.K. Medical Research Council. S.M.A. is a Senior Research Fellow of the Medical Research Council. Back

2 Address correspondence and reprint requests to Dr. Stephen M. Anderton, University of Edinburgh, Ashworth Laboratories, Kings Buildings, West Mains Road, Edinburgh, United Kingdom. E-mail address: steve.anderton{at}ed.ac.uk Back

3 Abbreviations used in this paper: Treg, regulatory T cell; LN, lymph node; EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; MOG, myelin oligodendrocyte glycoprotein; pMOG, MOG (35-55) peptide; pOVA, OVA (323-339) peptide; DC, dendritic cell. Back

Received for publication January 25, 2007. Accepted for publication May 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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Cutting Edge: Th1 Cells Facilitate the Entry of Th17 Cells to the Central Nervous System during Experimental Autoimmune Encephalomyelitis
J. Immunol., September 15, 2008; 181(6): 3750 - 3754.
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P. P. Sarangi, S. Sehrawat, S. Suvas, and B. T. Rouse
IL-10 and Natural Regulatory T Cells: Two Independent Anti-Inflammatory Mechanisms in Herpes Simplex Virus-Induced Ocular Immunopathology
J. Immunol., May 1, 2008; 180(9): 6297 - 6306.
[Abstract] [Full Text] [PDF]

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R. K. Selvaraj and T. L. Geiger
Mitigation of Experimental Allergic Encephalomyelitis by TGF-{beta} Induced Foxp3+ Regulatory T Lymphocytes through the Induction of Anergy and Infectious Tolerance
J. Immunol., March 1, 2008; 180(5): 2830 - 2838.
[Abstract] [Full Text] [PDF]

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