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The Journal of Immunology, 2008, 181, 4638 -4647
Copyright © 2008 by The American Association of Immunologists, Inc.
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Intrinsic and Induced Regulation of the Age-Associated Onset of Spontaneous Experimental Autoimmune Encephalomyelitis1

Hong Zhang*, Joseph R. Podojil*, Xunrong Luo{dagger} and Stephen D. Miller2,*

* Department of Microbiology-Immunology and the Interdepartmental Immunobiology Center and the {dagger} Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis is characterized by perivascular CNS infiltration of myelin-specific CD4+ T cells and activated mononuclear cells. TCR transgenic mice on the SJL background specific for proteolipid protein (PLP)139–151 develop a high incidence of spontaneous experimental autoimmune encephalomyelitis (sEAE). We examined the intrinsic mechanisms regulating onset and severity of sEAE. CD4+ T cells isolated from the cervical lymph nodes, but not spleens, of diseased 5B6 transgenic mice are hyperactivated when compared with age-matched healthy mice and produce both IFN-{gamma} and IL-17, indicating that the cervical lymph node is the initial peripheral activation site. The age-associated development of sEAE correlates with a decline in both the functional capacity of natural regulatory T cells (nTregs) and in PLP139–151-induced IL-10 production and a concomitant increase in IL-17 production. Anti-CD25-induced inactivation of nTregs increased the incidence and severity of sEAE. Conversely, induction of peripheral tolerance via the i.v. injection of PLP139–151-pulsed, ethylcarbodiimide-fixed APCs (PLP139–151-SP) inhibited the development of clinical disease concomitant with increased production of IL-10 and conversion of Foxp3+ Tregs from CD4+CD25– progenitors. These data indicate that heterogeneous populations of Tregs regulate onset of sEAE, and that induction of peripheral tolerance can be exploited to prevent/treat spontaneous autoimmune disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis (MS)3 is considered to be an immune-mediated disease of the CNS characterized by perivascular CD4+ T cell and mononuclear cell infiltration (1, 2, 3, 4). The critical role of CD4+ T cells in the autoimmune pathogenesis of MS is based on multiple lines of evidence. Activated myelin-reactive CD4+ T cells are found in CNS lesions (5). Several CD4+ T cell-targeted therapies inhibit the progression of MS (5). Genomic studies have confirmed the associations of the HLA class II with MS susceptibility (6, 7). Lastly, experimental autoimmune encephalomyelitis (EAE), an animal model of MS, is induced by priming with MHC class II-restricted myelin peptides in CFA and can be transferred solely by CD4+ T cells, providing direct definitive evidence that CD4+ T cells can initiate immune-mediated CNS demyelinating disease (8, 9, 10, 11). Collectively, these observations support an important pathogenic role for CD4+ T cells in mediating myelin damage in MS.

Transgenic (Tg) mice expressing TCRs specific for myelin basic protein (MBP) or proteolipid protein (PLP) have been used to study spontaneous EAE (sEAE) without the requirement for immunization with myelin peptide and adjuvant. However, sEAE does not develop in MBPAc1–11-specific TCR Tg mice housed in specific pathogen-free (SPF) conditions (12). In the presence of nontolerant MBP-reactive T cells, regulatory T cells (Tregs) expressing endogenous {alpha}-chain contribute to the prevention of sEAE in MBP TCR Tg mice (13). These data indicate that the mere presence of self-specific CD4+ T cells does not ensure development of autoimmune disease, and suggest, but do not prove, that the balance of self-reactive pathogenic T cells and Tregs determine whether the mice will develop spontaneous autoimmune disease.

A protective role for Tregs has been reported in multiple autoimmune diseases. CD4+CD25+ Tregs (natural Tregs; nTregs) are the best-documented Treg population. Depletion of this cell population in neonatal mice results in the spontaneous induction of spontaneous autoimmune diseases (14, 15, 16, 17, 18, 19, 20). For example, multiorgan autoimmune diseases (such as thyroiditis, gastritis, insulitis, et al.) are induced in BALB/c athymic nude mice receiving syngeneic CD4+ T cells depleted of CD25+ T cells. Reconstitution of CD4+CD25+ T cells within a limited period of time after the transfer of CD25– T cells inhibited disease development (18). Similarly, CD4+CD25+ T cells from NOD mice delayed/prevented development of spontaneous diabetes in CD28-deficient NOD mice (21). It is noteworthy, however, that regulation of self-reactive T cells likely involves multiple populations of immunoregulatory T cells, including nTregs, IL-10-producing Tregs (TR1), and TGF-β-producing Tregs (TH3) (22, 23, 24, 25). Using an adoptive transfer system using MBP TCR Tg cells, Cabbage and colleagues (26) show that endogenous Tregs can induce the differentiation of naive MBP Tg T cells into a unique tolerized state in the presence of nonactivated APCs. These MBP-specific regulatory Tg T cells produced IL-10 and TGF-β, which inhibited disease induced by activated APCs. Furthermore, it was shown that both the initial differentiation and subsequent tolerant state required the presence of endogenous Tregs. These data demonstrate that nTregs along with induced Tregs (iTregs), including TR1 and TH3, play an important role in maintaining self-tolerance.

The current study focused on PLP139–151-specific 5B6 TCR Tg (5B6 Tg) mice on the highly EAE-susceptible SJL background. Unlike MBP peptide-specific TCR Tg mice that developed sEAE only when raised in a conventional animal facility or when crossed to RAG 1-deficient (RAG–/–) background to remove Tregs, 5B6 Tg mice on a RAG+/+ background develop sEAE at a high incidence under barrier SPF conditions (27). The 5B6 Tg SJL mice thus provide a valuable model to determine endogenous mechanisms regulating spontaneous development of autoimmune disease in susceptible mice with an intact immune system. We show that initial development of clinical disease correlates with the appearance of hyperactivated CD4+ T cells in the cervical lymph nodes (cLN), but not spleens, of clinically affected 5B6 Tg mice compared with age-matched healthy controls and with infiltration of CD4+ T cells into the lumbar spinal cord. Interestingly, nTregs from 80-day-old 5B6 Tg mice exhibit decreased suppressive capacity compared with 40-day-old mice, and depletion/inactivation of Tregs using anti-CD25 mAb treatment between 30 and 40 days of age led to a significant increase in disease incidence and severity. Furthermore, PLP139–151-activated splenocytes from 48-day-old and 116-day-old 5B6 Tg mice produce significantly less IL-10 than 27-day-old Tg, suggesting an age-associated decrease in iTreg/TR1 activity. Lastly, induction of peripheral immune tolerance using peptide-pulsed, ethylcarbodiimide (ECDI)-fixed APCs (Ag-splenocytes; Ag-SP) in 30–40-day-old 5B6 mice prevented or delayed onset of disease concomitant with increased production of IL-10 and induction of Foxp3+ Tregs from CD4+CD25– 5B6 progenitors. Collectively, these findings suggest that multiple regulatory mechanisms contribute to the regulation of the age-associated sEAE in 5B6 Tg mice.


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

PLP139–151-specific 5B6 TCR Tg mice were obtained from Dr. Vijay Kuchroo (Harvard Medical School, Boston, MA) and crossed to SJL CD90.1 congenic mice. All mice used in this study were female 5B6 Tgs. The mice were housed under specific pathogen-free conditions in the Northwestern University Center for Comparative Medicine Barrier Facility. Paralyzed animals were provided easier access to food and water. All protocols were approved by Northwestern University Animal Care and Use Committee.

Peptides

PLP139–151 (HSLGKWLGHPDKF) and OVA323–339 (ISQAVHAAHAEINEAGR) were purchased from Genemed Synthesis.

Clinical evaluation of EAE

Mice were observed for clinical symptoms of sEAE three times a week. Mice were scored on a scale of 0–5 as follows: 0 = no abnormality; 1 = limp tail or hind limb weakness; 2 = limp tail and hind limb weakness; 3 = partial hind limb paralysis; 4 = complete hind limb paralysis; 5 = moribund. The data are plotted as the mean daily clinical score for all animals in a particular experimental group.

Immunohistochemistry

CNS immunohistological evaluation was performed as previously described (28). In brief, brain and spinal cord were removed by dissection from anesthetized, perfused mice. Blocks of brain halves and 2 to 3 mm sections of spinal cord were frozen in OCT (Miles Laboratories) in liquid nitrogen and stored at –80°C. The 6 µM thick cross-sections from spinal cord and 10 µM cross or sagittal sections from brain were stained using tyramide signal amplification direct kit (NEN) according to manufacturer’s instructions. Slides were examined using a Leica DM500B fluorescent microscope and images captured using the SPOT RT camera (Diagnostic Instruments) and SPOT imaging software. Three serial sections from each fragment per examined mouse were analyzed at x2 and x40 magnification.

Ag-specific delayed-type hypersensitivity (DTH) responses

DTH responses were measured using a 24-h ear-swelling system as previously described (28). The increase in ear thickness was determined 24 h after ear challenge by injecting 10 µg of respective peptide (in 10 µl of saline) into the dorsal surface of the ear. Results are expressed in units of 10–4 inches ± SEM.

Flow cytometry

Single-cell suspensions were blocked for 10 to 15 min with anti-CD16/32 before staining with a fluorescently tagged Ab-mixture directed against surface markers CD4 (RM4–5) and CD25 (PC61) (BD Pharmingen or eBioscience). Intracellular Foxp3 was stained using eBioscience Foxp3 staining buffer set and Abs according to the manufacturer’s instruction. Data were acquired on an LSR II cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Intracellular cytokine staining

Mice were perfused and CNS mononuclear cells were isolated as described (50). Cells were activated for 5 h with 5 ng/ml phorbol 12-myristate 13-acetate and 500 ng/ml ionomycin in the presence of GolgiStop (BD Biosciences), followed by staining with LIVE/DEAD fixable dye (Molecular Probes; Invitrogen) and the fluorescently tagged Ab against CD4 (RM4–5). Intracellular IL-17 and IFN-{gamma} were stained according to the eBioscience Intracellular Cytokine Staining Protocol.

In vitro CD4+CD25+ CFSE dilution T cell suppression assay

Sorted CD4+ responders were labeled with CFSE. A fixed number of responder T cells was cultured with titrated numbers of CD4+CD25+ T cells in the presence of PLP139–151 and irradiated splenocytes as APC. Cells were cultured for 72 h at 37°C in HL-1 medium (BioWhittaker) supplemented with 50 µM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Proliferation was determined by the dilution of CFSE. Discrimination of responders and Tregs was based on staining with CD90.1 and CD90.2.

Measurement of cytokine secretion

Spleen cell suspensions were cultured as described above. Triplicate wells were stimulated with the indicated doses of Ag and incubated for 72 h. Supernatants were collected and cytokine production was tested using the Beadlyte mouse multicytokine detection system (Millipore) and analyzed using Luminex 100 IS software.

Ag-coupled cell tolerance

Single cell suspensions of RBC-free splenocytes were coupled with PLP139–151 or OVA323–339 peptides using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (ECDI; Calbiochem-Behring) as previously described (29). A total of 5 x 107 Ag-coupled splenocytes were injected i.v. into the lateral tail veins of recipient 5B6 Tg mice at 30–40 days of age.

Statistical analysis

Comparisons of cytokine production and DTH responses between the various groups were analyzed by unpaired Student’s t test. Disease incidence was compared via {chi}2 analysis using Fisher’s exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Development of sEAE in 5B6 Tg mice

A total of 73 female 5B6 Tg mice housed in SPF conditions were monitored for the development of clinical signs of EAE for 160 days (Fig. 1A). Over 80% of these mice developed sEAE. No signs of EAE were observed in any animals under 42 days of age, and very few mice over the age of 100 days developed clinical disease. We refer to mice under 42 days of age as young mice and mice older than 42 days of age as old mice. The disease first presented with the loss of tail tone and extended to weakness of hind limbs. In a few cases, hind limb paralysis developed, but remarkably the disease symptoms were generally mild and none of the mice reached a moribund state (score 5). Unlike PLP139–151/CFA-induced EAE in wild-type SJL mice, no sustained remissions or overt relapses were observed (Fig. 1B). In a few cases some improvement was observed; however, it never went beyond improvement of more than one clinical grade.


Figure 1
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  		FIGURE 1. Incidence and onset pattern of clinical signs of spontaneous EAE. A total of 73 female 5B6 PLP139–151-specific TCR Tg mice housed in the Northwestern University Barrier Facility were monitored at least three times/week for clinical symptoms of sEAE for 160 days after birth. A, The cumulative percentage of mice with disease symptoms (solid line) and number of the mice with disease onset within the indicated age brackets (bars) are plotted. B, A total of 56 mice with disease symptoms were monitored for severity of clinical symptoms and the data plotted as the mean daily clinical score vs age.

 
Correlation of clinical symptoms with the CD4+ T cell infiltration in lumbar spinal cord

To begin to identify potential mechanisms of pathogenesis of sEAE in 5B6 Tg mice, histopathological analysis was performed on cerebellum, brain stem, and spinal cord tissues from five to six representative Tg mice with or without clinical disease symptoms, respectively. Spinal cords were divided into upper cervical, lower cervical, upper thoracic, middle thoracic, lower thoracic, upper lumbar, and lower lumbar sections. Three serial sections were analyzed from each CNS region. CD4+ T cells (red) were observed throughout the whole CNS from cerebellum to lumbar spinal cord in mice with clinical symptoms (Fig. 2A). The T cell infiltration mainly affected white matter. Interestingly, CD4+ T cells were also found in some of the Tg mice without clinical signs (Fig. 2A) as early as 24 days of age (data not shown). The infiltration in healthy mice mainly affected the upper spinal cord, cerebellum, and brain stem, but very few or no CD4+ cells were found in lumbar spinal cord, whereas the lumbar spinal cord was heavily infiltrated in mice displaying clinical disease (Fig. 2, A and B). Overall, the numbers of CNS-infiltrating CD4+ T cells were significantly greater in mice with clinical symptoms than healthy mice (Fig. 2C). To determine the cytokine profiles of CD4+ T cells in the CNS during disease pathogenesis, we isolated CNS cells from the pooled brain and spinal cord of clinically affected mice for analysis of IL-17 and IFN-{gamma} production. Upon in vitro PMA/ionomycin stimulation, CNS-infiltrating CD4+ T cells isolated from diseased animals exhibited a relatively equal distribution of Th17 and Th1 cells (Fig. 2D) with a Th17/Th1 ratio of 0.95 in this particular experiment. Our preliminary data show similar 1:1 Th17/Th1 ratio in infiltrating CD4+ T cells isolated separately from the brain and spinal cord of clinically affected mice. Taken together, these data indicate that clinical symptoms of sEAE correlate with the CD4+ T cell infiltration in lumbar spinal cord, and it is likely that both Th17 and Th1 cells are involved in the pathogenesis.


Figure 2
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  		FIGURE 2. Correlation of disease severity with CNS infiltration of CD4+ T cells. A, Five to six female 5B6 PLP139–151-specific TCR Tg mice with or without symptoms of sEAE were examined for the infiltration of CD4+ T cells (red) in the cerebellum, brain stem, and cervical, thoracic, and lumbar spinal cord. Tissues were also stained for myelin using anti-PLP (green) and counterstained with DAPI (blue). B, The CD4+ T infiltration in brain and lower lumbar spinal cord of five individual sick and healthy mice was scored on a scale of 0–3 as follows: 0 = no infiltration; 1 = meninges affected; 2 = meninges and white matter affected; 3 = meninges, white matter and gray matter are affected. The data are graphed as percentage of the mice with each score. C, CNS cells were isolated from four to five 5B6 TCR Tg mice without (healthy) or with (sick) symptoms of sEAE. The percentage of CD4+ T cells were determined by flow cytometry. *, The number of CD4+ T cells in sick mice was significantly greater than those in healthy mice, p < 0.02. D, CNS cells isolated from a mouse with clinical symptoms of sEAE were stimulated for 4h with PMA and ionomycin in the presence of Golgistop for analysis of intracellular IL-17 and IFN-{gamma}. Cells were gated on live CD4+ T cells. Data are representative of three separate experiments.

 
Onset of clinical symptoms sEAE correlates with enhanced activation of CD4+ T cells in the cervical LNs of 5B6 Tg mice

Peptide-specific in vivo DTH and in vitro recall responses were used to determine whether the onset of clinical symptoms of sEAE in clinically affected mice correlated with increased functional activation of PLP139–151-specific T cells and the anatomic site of the primary activation of the Tg T cells. Compared with age-matched healthy Tg mice, animals with clinical disease displayed enhanced DTH responses to ear challenge with PLP139–151 (Fig. 3A), CD4+ T cell CNS infiltration (Fig. 2C), and enhanced production of both IFN-{gamma} and IL-17 in both the spleen and cLNs in response to in vitro stimulation with PLP139–151 (Fig. 3B) indicating enhanced development of both effector Th1 and Th17 cells. To identify where the Tg T cells were initially activated, we measured the recall proliferation by CFSE dilution of CD4+ T cells from spleens and cLNs of nonsymptomatic controls in comparison to mice with recent onset disease (Fig. 3C). As anticipated, robust proliferation of CD4+ T cells was observed in both groups of mice with no detectable difference of proliferation of spleen CD4+ T cells. However, CD4+ T cells from the cLNs of mice with recent onset sEAE proliferated more robustly than mice without disease symptoms, although no significant difference of the cLN cell numbers was observed (Fig. 3D). In summary, these data suggest that mice with recent onset clinical sEAE have more Ag-experienced CD4+ T cells than mice without disease symptoms, and these activated CD4+ T cells appear to be enriched in cLNs, a site previously associated with CNS Ag drainage (30, 31, 32, 33).


Figure 3
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  		FIGURE 3. CD4+ T cells from the cLN of 5B6 Tg mice with new-onset sEAE are hyperresponsive to Ag stimulation. A, DTH responses were determined in four female 5B6 PLP139–151-specific TCR Tg mice either with or without symptoms of sEAE as well as in two naive littermate (LM) controls using a 24 h ear swelling assay. Mice were challenged with 10 µg of PLP139–151 in the dorsal surface of the left and right ears and the increase in ear thickness measured 24 h later. The data are expressed as mean change in ear swelling in units of 10–4 inches ± SEM. DTH levels in sick Tg mice were significantly greater than those in healthy Tg mice, *, p < 0.05. B, Sorted CD4+ T cells from cLNs or spleens of 5B6 TCR Tg mice with (sick) or without (healthy) symptoms of sEAE were stimulated with PLP139–151-pulsed, irradiated wild-type SJL/J splenocytes for 3 days. Supernatants were assayed for the production of IFN-{gamma} and IL-17. Data are representative of two independent experiments. C, Sorted CD4+ T cells from cLNs or spleens were labeled with CFSE and stimulated with PLP139–151-pulsed, irradiated wild-type SJL/J splenocytes for 3 days. Proliferation was determined by dilution of CFSE. The numbers indicate the percentage of undivided cells. Data are representative of three separate experiments. D, Numbers of T cells from the spleen and cLN of healthy 5B6 TCR Tg mice or 5B6 mice with new-onset symptoms were determined. Data are presented as mean cell number of three mice from each group and pooled from three separate experiments.

 
Age-related decline in the suppressive capacity of nTregs from 5B6 Tg mice

Lafaille et al. (13) reported that MBP TCR Tg mice on RAG+/+ background did not develop spontaneous EAE unless Tregs were removed by crossing these mice onto a RAG–/– background. In contrast, 5B6 TCR Tg mice on a RAG+/+ background develop sEAE in an age-dependent manner (Ref. 27 ; Fig. 1), but when crossed to the RAG–/– background they develop such severe disease that the line cannot be maintained (27). We thus wished to determine whether development of spontaneous clinical disease in 5B6 TCR+/+ was associated with an age-related deficiency in the numbers and/or suppressive function of Treg cells. Although we found that 5B6 Tg mice generally have a lower percentage of Foxp3+CD4+ Tregs as compared with wild-type SJL controls, the numbers of Tregs in the spleens and cLNs of 5B6 Tg mice did not significantly vary with age, nor were the numbers different when comparing clinically affected mice vs nonaffected controls of a similar age (Fig. 4A). Although sorted CD4+CD25+ T cells from 5B6 Tg mice at various ages were found to suppress the proliferation of effector CD4+CD25– 5B6 T cells at high Treg-Teff ratios (where Teff is effector T cell) (data not shown), the suppressive function of sort purified splenic CD4+CD25+ T cells was decreased in older mice as illustrated by the significantly reduced ability of Tregs from 80-day-old as compared with 40-day-old mice to suppress CFSE dilution at a 1:1 Treg-Teff ratio (Fig. 4B). Purification of the nTregs was equivalent in the two age groups (Fig. 4C).


Figure 4
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  		FIGURE 4. Age-associated decline of suppressive capacity of nTregs in 5B6 Tg mice. A, Spleen and cLN T cells were isolated from 5B6 TCR Tg mice of the indicated ages and the percentages of CD4+Foxp3+ T cells were determined by flow cytometry and compared with those in littermate (LM) controls. Data are presented as mean of two mice per group and are representative of two separate experiments. B, Sorted CD4+CD25+ T cells from the spleen of 40-day-old (black line) or 80-day-old (gray shade) 5B6 Tg mice without disease symptoms were cocultured with CFSE-labeled responder CD4+CD25– T cells from 60-day-old 5B6 Tg mice at the indicated ratio of nTreg to responders in the presence of PLP139–151-pulsed, irradiated naive splenocytes for 3 days. In vitro suppressive capacity was determined by the efficiency of suppressing proliferation (CFSE dilution) of the responder CD4+ T cells. Data are representative of three separate experiments. C, The purity of the sorted cells from the 40-day-old (young) and 80-day-old (old) Tg mice were similar as determined by the percentage of Foxp3+ cells. D, The 30–40-day-old 5B6 TCR Tg mice were treated with rat IgM control Ab (n = 9) or 7D4 anti-CD25 Ab (n = 10) for seven treatments administered at three-day intervals and monitored for clinical signs of disease for an additional 30 days. The maximal disease score of each individual mouse is plotted as well as the mean clinical score of all mice in each group (horizontal line). Disease incidence in anti-CD25-treated mice is significantly greater than control Ig-treated mice, *, p < 0.02. The data are pooled from three separate experiments.

 
To further define the role of nTregs in the regulation of sEAE, we determined whether in vivo depletion of CD25+ T cells in young 5B6 Tg mice would affect the incidence or severity of sEAE. A total of 30–40-day-old mice were treated with monoclonal anti-CD25 Ab (7D4) for 7 treatments at two days interval and monitored for development of clinical EAE for an additional 30 days. Fig. 4D shows mice treated with anti-CD25 Ab have higher incidence (6/10 = 60%) than the mice treated with control IgM (1/9 = 11%, p < 0.05). Taken together, these findings indicate that nTregs from 5B6 Tg mice play a functional role in suppressing disease in young mice, but that their suppressive capacity declines with age.

Production of IL-10 by 5B6 splenic CD4+ T cells declines with age

We were also interested in determining whether the age-associated development of sEAE in the 5B6 Tg mice was associated with a change in the pattern of peptide-induced cytokine production. Spleen cells isolated from the 5B6 Tg mice at 27, 48, and 116 days of age were analyzed for the production of IL-10, IL-4, IL-5, IL-6, TNF-{alpha}, IFN-{gamma}, (Fig. 5), and TGF-β (data not shown) in response to in vitro stimulation with PLP139–151. Lymphocytes from individual mice were analyzed to avoid pooling cells from mice having preclinical sEAE with cells from healthy mice. No conclusion can be drawn regarding TGF-β production due to the inconsistency from experiment to experiment (data not shown). No significant differences in TNF-{alpha} and IFN-{gamma} production between the different groups (Fig. 5, E and F) was observed, whereas IL-10, IL-4, IL-5, and IL-6 production was generally higher in young 27-day-old mice vs mice at 48 and 116 days of age (Fig. 5, A–D). Interestingly, IL-10 production was consistently significantly higher in the younger mice. Thus regulatory cytokine production, especially IL-10, is decreased as mice age and exhibits an enhanced incidence of clinical disease. In a separate experiment, splenocytes from 5B6 Tg mice at 29, 55, and 97 days of age were similarly analyzed for the production of IL-17, and 97-day-old mice were found to produce significantly more peptide-induced IL-17 than nonclinically affected younger mice (Fig. 5G). This indicates that a pathologic population of PLP139–151-specific T cells arises as regulation is lost.


Figure 5
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  		FIGURE 5. Cytokine profiles of peptide-stimulated splenic T cells of 5B6 Tg mice of varying ages. Splenocytes from 5B6 Tg mice of the indicated ages were stimulated with PLP139–151 for 72 h. Supernatants were assayed for the production of IL-10 (A), IL-4 (B), IL-5 (C), IL-6 (D), TNF-{alpha} (E), and IFN-{gamma} via LiquiChip analysis (F). Data from one of three representative experiments is shown. *, Levels of peptide-induced IL-10 were significantly higher in 27-day-old 5B6 mice than in 48- or 116-day-old mice, p < 0.01. G, In a separate analysis, splenocytes from two to three individual 5B6 Tg mice at 29, 55, and 97 days of age were stimulated with PLP139–151 for 72 h and supernatants assayed for the production of IL-17 by ELISA. **, Levels of peptide-induced IL-17 were significantly higher in 97-day-old 5B6 mice than in 29- or 55-day-old mice, p < 0.05.

 
Ag coupled-cell tolerance inhibits the development of sEAE by enhancing IL-10 production and conversion of Foxp3+ iTregs

The above findings indicate that the functional decline of nTreg activity as well as peptide-induced IL-10 production in 5B6 Tg mice correlates with the age-related development of clinical sEAE. We next tested whether development of spontaneous disease could be prevented or delayed by the induction of peptide-specific tolerance induced by the i.v. injection of PLP139–151-pulsed, ECDI-fixed syngeneic splenocytes (Ag-SP), which we have shown can successfully prevent and treat both actively induced and adoptive EAE in conventional mice (34, 35, 36, 37, 38, 39, 40, 41, 42, 43). The 5B6 mice were tolerized with PLP139–151-SP or OVA323–339-SP between 30 and 40 days of age and observed for development of clinical disease for an additional 30–40 days. Tolerization significantly reduced peptide-specific DTH responses (Fig. 6A) and delayed onset of clinical sEAE (Fig. 6B). Furthermore, splenocytes from PLP139–151-SP tolerized 5B6 mice produced significantly more IL-10 than OVA323–339-SP tolerized mice upon peptide recall in vitro (Fig. 6C). Again, the TGF-β results were inconsistent from experiment to experiment and no conclusions could be drawn.


Figure 6
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  		FIGURE 6. Inhibition of spontaneous EAE by Ag-coupled cell tolerance (Ag-SP). The 30–40-day-old 5B6 TCR Tg mice were left untreated or i.v. tolerized with 5 x 107 OVA323–339-SP or 5 x 107 PLP139–151-SP. A, Seven days posttolerization, mice from each group were ear challenged with 10 µg PLP139–151, and swelling was measured 24 h later. Data from one of three representative experiments are shown. DTH levels in PLP139–151-SP tolerized mice were significantly less than those in OVA323–339-SP injected mice, *, p < 0.0001. B, The incidence of clinical disease was monitored for an additional 35 days after tolerization. Data are presented as cumulative incidence of 46 PLP139–151-SP treated Tg mice and 45 OVA323–339-SP treated Tg mice pooled from three separate experiments. *, Disease incidence significantly less than that in OVA323–339-SP tolerized mice, p < 0.05. C, On day 6 posttolerization, splenocytes from five separate PLP139–151-SP and OVA323–339-SP tolerized 5B6 TCR Tg mice or littermate (LM) controls were stimulated with PLP139–151 for 72 h. Supernatants were assayed for the production of cytokines. Production of IL-10 was significantly higher in PLP139–151-SP treated mice, *, p < 0.05. Data are representative of two separate experiments.

 
To determine whether the protective effects of PLP139–151-SP treatment on clinical disease development were associated with activation and/or generation of peptide-specific Tregs, the effects of i.v. administration of PLP139–151-SP vs OVA323–339-SP on both bulk CD90.1+CD4+ and FACS-sorted CD90.1+CD25– 5B6 TCR Tg T cells were determined following adoptive transfer into wild-type CD90.2 SJL mice. Interestingly, peptide-specific tolerance resulted in an increase of 7-fold and 2-fold, respectively, in the percentage of CD90.1+CD25lowFoxp3+ and CD90.1+CD25highFoxp3+ CD4+ T cells in wild-type SJL recipients of bulk donor 5B6 CD90.1+CD4+Tg T cells 3 days after treatment (Fig. 7A). PLP139–151-SP tolerization also induced expression of Foxp3 in recipients of purified naive donor CD4+CD25– Tg T cells (Fig. 7, B and E) and CD25 expression on these donor CD90.1+Foxp3+ T cells was lower than on recipient CD90.2+ Foxp3+ T cells (Fig. 7, C and D), indicating conversion/activation of an adaptive Foxp3+ iTreg by Ag-SP tolerance. Collectively, the data indicate that Ag-SP tolerance inhibits induction of sEAE by inducing various Treg subsets including adaptive and IL-10-producing Foxp3+ Tregs.


Figure 7
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  		FIGURE 7. Ag-SP tolerance induces generation of Ag-specific Foxp3+ T cells. A total of 5 x 106 bulk CD90.1+CD4+ (A) or FACS-sorted CD90.1+CD4+CD25– (B–E) T cells from 5B6 TCR Tg mice were transferred to naive CD90.2+ SJL mice on day –1, followed by injection of 5 x 107 PLP139–151 -SP or OVA323–339 -SP on day 0. A, A total of 14, 38, 62 h after tolerance induction, expression of Foxp3 and CD25 on the donor CD90.1+CD4+ T cells was determined by flow cytometry and the data plotted as the mean ratio of CD90.1+ Foxp3+CD25high and Foxp3+CD25low cells in PLP139–151-SP tolerized vs OVA323–339 tolerized mice, n = 5 per group. The data is representative of three separate experiments. Ratio of Foxp3+CD25+ cells is significantly greater than in OVA323–339-SP injected control values, **, p < 0.01, ***, p < 0.001. On day 5, splenocytes from tolerized recipients of CD90.1+CD4+CD25– Tg T cells were tested for the expression of Foxp3 and CD25. Foxp3 staining on gated donor CD90.1+CD4+ T cells (B) and the relative fluorescence of CD25 on both donor and recipient CD4+Foxp3+ T cells from PLP139–151-SP tolerized mice (C) is shown. Isotype control (filled histogram); donor CD90.1+ CD4+ Foxp3+T cells (black line); and recipient CD90.2+ CD4+ Foxp3+ T cells (gray line). D, Summary of geometric mean, mean and median fluorescence intensity of CD25 on both donor and recipient CD4+Foxp3+ T cells from PLP139–151-SP (n = 3) and OVA323–339-SP (n = 3) tolerized mice. Values on donor C90.1+ T cells significantly less than on recipient CD90.2+ T cells in PLP139–151-SP treated mice, *, p < 0.05, **, p < 0.01. E, The number of donor CD4+CD90.1+Foxp3+ T cells from three individual PLP139–151-SP and OVA323–339-SP tolerized mice were determined. The data are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been previously reported that 5B6 TCR Tg mice specific for PLP139–151 on a conventional RAG+/+ background developed a high incidence of sEAE when raised in SPF conditions (27). In the current study, we extended the previous study by describing the immunological changes correlating with disease onset and determining that spontaneous disease development was associated with the age-related decline in several intrinsic regulatory mechanisms involved in maintenance of self-tolerance.

Two major observations pertaining to the maintenance of tolerance are made in this study. First, CD4+CD25+ Treg cells from 80-day-old 5B6 Tg mice were found to be functionally less suppressive than Treg cells from isolated 40-day-old mice (Fig. 4B) correlating with the age-dependent increase in onset of clinical sEAE. In confirmation of a protective role of nTregs in young 5B6 Tg mice, we found that their inactivation by treatment of young animals with anti-CD25 led to a significant increase in disease incidence and severity (Fig. 4C), whereas their induction following tolerization with PLP139–151-SP led to a significant delay in disease onset (Fig. 6A). This is consistent with earlier reports showing an age-related decline in suppressive function of CD4+CD25+ Tregs in 16 wk-old as compared with 8 wk-old NOD mice corresponding with development of spontaneous type 1 diabetes (24, 44). The age-associated functional deficiency in suppressive function was demonstrated despite the observation that there was no observable difference in the frequency of CD25+Foxp3+ cells between young vs old 5B6 mice or between clinically affected vs unaffected mice of the same age (Fig. 4A). Second, splenic T cells from 48- and 116-day-old 5B6 mice produced significantly less IL-10 upon peptide recall (Fig. 5A) than 27-day-old Tg mice, a time point before onset of sEAE. IL-10 has been reported to play an important role in maintaining the nonencephalitogenic phenotype of the autoreactive T cells (45). As the Tr1 regulatory cell population is a major source of IL-10 (25), this observation is consistent with an important potential role for Tr1 cells in maintaining self-tolerance in young 5B6 mice. In support for a protective role of IL-10 in preventing disease onset, we found that tolerization of young 5B6 mice with PLP139–151-SP that led to a significant delay in disease onset (Fig. 6B) was associated with enhanced production of IL-10 (Fig. 6C). In addition, we previously reported that tolerization of mice with pre-existing PLP139–151-induced relapsing-EAE with PLP139–151-SP at peak of acute disease significantly increased the levels of production of the anti-inflammatory cytokines TGF-β and/or IL-10 in CD4+ T cells recovered from both the periphery and the CNS upon peptide restimulation in vitro (46). Together, these data support the hypothesis that the regulation of the age-related induction of sEAE in 5B6 Tg SJL mice is highly complex and likely involves a heterogeneous population of immunoregulatory T cells whose activity can be enhanced by tolerogenic administration of cognate peptide in the form of PLP139–151-pulsed, ECDI-fixed syngeneic splenocytes (PLP139–151-SP).

Tolerization with PLP139–151-SP resulted in a 7-fold and 2-fold expansion vs OVA323–339-SP injected controls, respectively, in the percentage of Foxp3+CD25low and Foxp3+CD25high T cells in recipients of bulk 5B6 CD4+ T cells (Fig. 7A) within 62 h following treatment. This rapid increase in Treg percentage indicates the generation of adaptive Tregs. In addition, PLP139–151-SP vs OVA323–339-SP tolerization of wild-type recipients of purified CD90.1+CD4+CD25– 5B6 TCR Tg cells led to the generation of a significant population of Foxp3+ T cells, the majority of which expressed a CD25low phenotype distinguishing them from CD25high nTregs (Fig. 7, B–E). This CD4+Foxp3+CD25low phenotype is a characteristic of adaptive Tregs that were recently reported to be induced from peripheral CD4+Foxp3+ precursors in CD28-deficient NOD mice by nonmitogenic anti-CD3 immunotherapy (47). Thus, tolerization with PLP139–151-SP apparently leads to the induction of multiple regulatory mechanisms including some expansion of CD4+CD25highFoxp3+ nTregs and the conversion of naive CD4+CD25– 5B6 T cells to CD4+CD25lowFoxp3+ adaptive Tregs, as well as inducing enhanced IL-10 production (Fig. 6) with the net outcome of significantly inhibiting/delaying onset of sEAE in 5B6 TCR Tg mice.

Another key observation made in this study is that cLN T cells, but not spleen T cells, from 5B6 Tg SJL mice with recent-onset sEAE are hyperresponsive to peptide stimulation compared with cLN T cells from mice without disease symptoms (Fig. 3C). The initial presence of hyperresponsive CD4+ T cells in cLNs supports the hypothesis that this lymphoid organ is the primary site for the peripheral activation of T cells specific to an immunodominant myelin proteinduring the development of sEAE. This finding is consistent with prior studies indicating that the cLNs are lymphatic sites draining some areas of the CNS (30, 31, 32, 33), and may suggest that initial activation of myelin-specific Tg T cells occurs as the result of leakage of myelin proteins from the CNS. Similar results have been found in the MBPAc1–11 TCR Tg mouse system wherein activated T cells are first noted in the cLNs preceding onset of clinical disease in 7–8-wk-old mice and surgical removal of the cLNs can delay the age of disease onset (54). The failure to identify peptide-specific hyperresponsive T cells in the spleen speaks against the possibility that the activated cells in the cLNs were initially activated in the CNS and simply "leaked" into the peripheral circulation through the compromised blood-brain barrier. In situ tolerance has been reported to occur in T cells entering the CNS during noninflammatory conditions. Indeed, CNS T cells from MBP TCR Tg mice without disease symptoms fail to respond to Ag, whereas T cells in the cLN and other non-CNS draining LNs proliferated robustly (48). In addition, CNS cells from asymptomatic MBP TCR Tg mice are able to suppress the proliferation of peripheral MBP-specific T cells in vitro. Other experiments have demonstrated that the interaction between neurons and T cells could convert encephalitogenic T cells to regulatory T cells, which presumably suppress activation of encephalitogenic T cells under noninflammatory conditions (49). These data suggest that the noninflamed CNS may normally be a "tolerogenic" milieu in the steady state. In contrast, in the inflamed CNS during progressive relapsing EAE in wild-type SJL mice, the CNS serves as the primary site where naive T cells specific for endogenously released spread epitopes become activated (50, 51).

The specific sites of the CNS infiltration of encephalitogenic T cells are major determinants of disease symptoms and severity. Our data clearly shows expression of clinical symptoms of spontaneous EAE correlates with the infiltration of CD4+ T cells into the lumbar spinal cord region. Interestingly, significant infiltration of CD4+ T cells can be detected in brain and upper spinal cord in both mice with and without disease symptoms. However, only a very few mice without disease symptoms have detectable infiltration in lumbar spinal cord, which is observed in all the mice with symptomatic sEAE (Fig. 2). We were unable to compare the activity of CNS-infiltrating CD4+ T cells from mice with sEAE to mice without disease symptoms due to the difficulty of isolating sufficient numbers of CD4+ T cells from CNS of nonsymptomatic mice for functional analysis. However, T cells isolated from the CNS of 5B6 mice with recent-onset disease express a highly activated phenotype as indicated by the up-regulation of CD25, CD69, CD44, and CD11a and the down-regulation of CD45RB (data not shown). It is likely that both Th1 and Th17 cells may contribute to the disease pathogenesis based on the observation that both Th17 and Th1 were found at roughly a 1:1 ratio in CNS-infiltrating CD4+ T cells in diseased mice (Fig. 2D), and the observation that spleen and cLN (Fig. 3B) T cells from mice with disease symptoms produced significantly greater amounts of both IFN-{gamma} and IL-17 than mice without symptoms. Muller et al. (52) reported that the distribution of inflammatory cells correlated with disease symptoms using an atypical model of EAE induced in C3H/HeJ mice. These mice developed two distinct types of EAE, one characterized by ascending paralysis and the other by axial rotatory symptoms. Mice that exhibited only ascending paralysis showed inflammation preferentially in the spinal cord, whereas inflammation presented only in cerebellum and brainstem in mice that exhibited axial rotatory EAE. More recently, it was reported that T cells specific for different epitopes on myelin oligodendrocyte glycoprotein (MOG) generated different ratios of Th17/Th1 cells in two C3H congenic strains (53). T cell infiltration of the brain leading to atypical EAE characterized by proprioception defects, ataxia, spasticity, and hyperreflexivity was evident when Th17 cells were in excess of Th1 cells, whereas more classic EAE, characterized by ascending flaccid hindlimb paralysis and parenchymal inflammatory infiltration in the spinal cord and brain, was seen in mice exhibiting a ~1:1 Th17/Th1 ratio. These data suggest that T cell specificity may influence sites of inflammation by inducing different T cell effector cells. Our data indicate that infiltration of Th1 and Th17 cells in a 1:1 ratio to the lumbar spinal cord primarily contributes to the pathogenesis of a classic form of sEAE in 5B6 Tg mice, whereas infiltration in cerebellum and brain stem alone is not associated with disease symptoms (Fig. 2). Preferential finding of inflammatory infiltrates in the lumbar spinal cord may be attributed to the specificity and the naturally determined effector function of the Tg T cells (Th17 vs Th1) in our model.

In summary, we propose that the decline of multiple regulatory mechanisms with age contributes to the development of sEAE in 5B6 Tg mice. In the young mice, a balance between pathogenic T cells and a regulatory network including nTregs and induced Tr1 cells protects the animals from onset of clinical CNS disease. However, as suppressive capacity of nTregs and IL-10-producing Tr1 cells declines, the threshold of activation is decreased and proinflammatory PLP139–151-specific CD4+ Th1 and Th17 cells are activated in the cLNs and initiate clinical disease. Our results also indicate that peripheral induction of peptide-specific tolerance can reactivate these two T cell-mediated regulatory networks leading to prolonged protection from development of the spontaneous onset of overt clinical disease.


   Acknowledgments
 
We thank Dr. Vijay K. Kuchroo for the gift of the 5B6 TCR Tg mice, Samantha Bailey for helpful suggestions and discussions throughout the course of this work, Matt DeGutes and Gwen Goings for technical assistance with immunohistochemistry, and Terra Frederick for critical reading of the manuscript.


   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 National Institutes of Health R01 Grants NS-026543 and NS-048411, National Multiple Sclerosis Society Grants RG 3793-A-7 and RG 3965-A-8, and a grant from the Myelin Repair Foundation. Back

2 Address correspondence and reprint requests to Dr. Stephen D. Miller, Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611. Email address: s-d-miller{at}northwestern.edu Back

3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; Tg, transgenic; MBP, myelin basic protein; PLP, proteolipid protein; sEAE, spontaneous experimental autoimmune encephalomyelitis; SPF, specific pathogen-free; Treg, regulatory T cell; nTreg, natural Treg; iTreg, induced Treg; 5B6 Tg, 5B6 PLP139–151 TCR Tg; cLN, cervical lymph node; ECDI, ethylcarbodiimide; Ag-SP, Ag-splenocytes; DTH, delayed-type hypersensitivity. Back

Received for publication May 5, 2008. Accepted for publication July 18, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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G. C. Furtado, M. C. G. Marcondes, J.-A. Latkowski, J. Tsai, A. Wensky, and J. J. Lafaille
Swift Entry of Myelin-Specific T Lymphocytes into the Central Nervous System in Spontaneous Autoimmune Encephalomyelitis
J. Immunol., October 1, 2008; 181(7): 4648 - 4655.
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