Intrinsic and induced regulation of the age-associated onset of spontaneous experimental autoimmune encephalomyelitis

Abstract

Multiple sclerosis (MS) is characterized by perivascular CNS infiltration of myelin-specific CD4⁺ T cells and activated mononuclear cells. T cell receptor (TCR) transgenic mice on the SJL background specific for proteolipid protein (PLP)_139-151 (5B6 Tg mice) 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 (cLN), but not spleens, of disease 5B6 Tg mice are hyper-activated when compared to age-matched healthy mice and produce both IFN-γ and IL-17 indicating that the cLN 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 PLP_139-151-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 PLP_139-151-pulsed, ECDI-fixed APCs (PLP_139-151-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

Multiple sclerosis (MS)² is considered to be an immune-mediated disease of the central nervous system (CNS) characterized by perivascular CD4⁺ T cell and mononuclear cell infiltration (1-4). 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 human leukocyte antigen (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 complete Freud’s adjuvant and can be transferred solely by CD4⁺ T cells providing direct definitive evidence that CD4⁺ T cells can initiate immune-mediated CNS demyelinating disease (8-11). Collectively, these observations support an important pathogenic role for CD4⁺ T cells in mediating myelin damage in MS.

Transgenic 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 MBP_Ac1-11-specific TCR Tg mice housed in specific pathogen free (SPF) conditions (12). In the presence of non-tolerant MBP-reactive T cells, regulatory T cells (Tregs) expressing endogenous α 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 (nTregs) are the best-documented Treg population. Depletion of this cell population in neonatal mice results in the spontaneous induction of spontaneous autoimmune diseases (14-20). For example, multi-organ 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 (T_R1) and TGF-β-producing Tregs (T_H3) (22-25). Employing an adoptive transfer system using MBP TCR Tg cells, Cabbage and colleagues (26) show that endogenous Tregs can induce the differentiation of naïve MBP Tg T cells into a unique tolerized state in the presence of non-activated 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 T_R1 and T_H3, play an important role in maintaining self-tolerance.

The current study focused on PLP_139-151-specific 5B6 TCR Tg mice on the highly EAE susceptible SJL background (5B6 Tg mice). Unlike MBP peptide-specific TCR Tg mice which developed sEAE only when raised in a conventional animal facility or when crossed to recombination-activation-gene 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). 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 hyper-activated CD4⁺ T cells in the cervical lymph nodes (cLN), but not spleens, of clinically affected 5B6 Tg mice compared to 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 to 40 day-old mice and depletion/inactivation of Tregs using anti-CD25 mAb treatment between 30-40 days of age led to a significant increase in disease incidence and severity. Furthermore, PLP_139-151-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/T_R1 activity. Lastly, induction of peripheral immune tolerance using peptide-pulsed, ECDI-fixed APCs (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

Mice

PLP_139-151-specific 5B6 TCR Tg mice were obtained from Dr. Vijay Kuchroo (Harvard Medical School) and crossed to SJL CD90.1 congenic mice. All mice employed 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 (ACUC).

Peptides

PLP_139-151 (HSLGKWLGHPDKF) and OVA_323-339 (ISQAVHAAHAEINEAGR) were purchased from Genemed Synthesis, San Francisco, CA.

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). Briefly, 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; Elkhart, IN) in liquid nitrogen and stored at −80°C. 6 μM thick cross-sections from spinal cord and 10 μM cross or sagital sections from brain were stained using Tyramide Signal Amplification (TSA) Direct kit (NEN, Boston, MA) according to manufacturer’s instructions. Slides were examined using a Leica DM500B fluorescent microscope and images captured using the SPOT RT camera (Diagnostic instruments, Sterling Heights, MI) and SPOT imaging software. Three serial sections from each fragment per examined mouse were analyzed at ×2 and × 40 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 15min with anti-CD16/32 before staining with a fluorescently tagged antibody-cocktail 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 antibodies according to manufacturer’s instruction. Data were acquired on an LSR II cytometer (BD) and analyzed with FlowJo software (Tree Star, Ashland, OR).

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 of phorbol 12-myristate 13-acetate and 500 ng/ml of ionomycin in the presence of Golgistop (BD) followed by staining with LIVE/DEAD fixable dye (Molecular Probes; Invitrogen) and the fluorescently tagged antibody against CD4 (RM4-5). Intracellular IL-17 and IFN-γ 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 Carboxy Fluoroscein Succinimidyl Ester (CFSE). A fixed number of responder T cells was cultured with titrated numbers of CD4⁺CD25⁺ T cells in the presence of PLP_139-151 and irradiated splenocytes as APC. Cells were cultured for 72 h at 37° C in HL-1 medium (BioWhittaker, Walkersville, MD) 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 antigen and incubated for 72 h. Supernatants were collected and cytokine production was tested using the Beadlyte mouse multi-cytokine Detection System (Upstate, Waltham. MA) and analyzed using Luminex 100 IS software (Luminex, Austin, TX).

Antigen-coupled cell tolerance

Single cell suspensions of RBC-free splenocytes were coupled with PLP_139-151 or OVA_323-339 peptides using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (ECDI, Calbiochem-Behring Corp., La Jolla, CA) as previously described (29). 5×10⁷ Ag-coupled splenocytes were injected intravenously into the lateral tail veins of recipient 5B6 Tg mice at 30-40 d 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 X² analysis using Fisher’s exact test.

Results

Development of sEAE in 5B6 Tg mice

Seventy-three female 5B6 Tg mice housed in SPF conditions were monitored for the development of clinical signs of EAE for 160 days (Figure 1A). Over 80% of these mice developed sEAE. No signs of EAE were observed in any animals under 42 d of age and very few mice over the age of 100 d developed clinical disease. We refer to mice under 42 d of age as young mice and mice older than 42 d 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 PLP_139-151/CFA-induced EAE in wildtype SJL mice, no sustained remissions or overt relapses were observed (Figure 1B). In a few cases some improvement was observed, however it never went beyond improvement of more than one clinical grade.

Figure 1. Incidence and onset pattern of clinical sings of spontaneous EAE.

Seventy-three female 5B6 PLP_139-151-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) Fifty-six 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 5-6 representative transgenic 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. 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 (Figure 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 (Figure 2A) as early as 24 d 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 (Figure 2A,B). Overall, the numbers of CNS-infiltrating CD4⁺ T cells were significantly greater in mice with clinical symptoms than healthy mice (Figure 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-γ 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 (Figure 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, this data indicates 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. Correlation of disease severity with CNS infiltration of CD4⁺ T cells.

(A) Five to Six female 5B6 PLP_139-151-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 4-5 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-γ. 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 employed to determine whether the onset of clinical symptoms of sEAE in clinically affected mice correlated with increased functional activation of PLP_139-151-specific T cells and the anatomic site of the primary activation of the Tg T cells. Compared to age-matched healthy Tg mice, animals with clinical disease displayed enhanced DTH responses to ear challenge with PLP_139-151 (Figure 3A), CD4⁺ T cell CNS infiltration (Figure 2C), as well as enhanced production of both IFN-γ and IL-17 in both the spleen and cervical LNs (cLNs) in response to in vitro stimulation with PLP_139-151 (Figure 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 non-symptomatic controls in comparison to mice with recent onset-disease (Figure 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 (Figure 3D). In summary, these data suggest that mice with recent onset clinical sEAE have more antigen-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 antigen drainage (30-33).

Figure 3. CD4⁺ T cells from the cLN of 5B6 Tg mice with new-onset sEAE are hyper-responsive to antigen stimulation.

(A) DTH responses were determined in four female 5B6 PLP_139-151-specific TCR Tg mice either with or without symptoms of sEAE as well as in two naïve litter mate controls (LM) using a 24 h ear swelling assay. Mice were challenged with 10 μg of PLP_139-151 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 PLP_139-151-pulsed, irradiated wild-type SJL/J splenocytes for 3d. Supernatants were assayed for the production of IFN-γ and IL-17. Data is representative of two independent experiments. (C) Sorted CD4⁺ T cells from cLNs or spleens were labeled with CFSE and stimulated with PLP_139-151-pulsed, irradiated wild-type SJL/J splenocytes for 3d. 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 3 mice from each group and pooled from 3 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 (MBP T/R⁺) did not develop spontaneous EAE unless Tregs were removed by crossing these mice onto a RAG^−/− background (MBP T/R⁻). In contrast, 5B6 TCR Tg mice on a RAG^+/+ background develop sEAE in an age-dependent manner (27) (Figure 1), but when crossed to the RAG^−/− background 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 to wildtype 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. non-affected controls of a similar age (Figure 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 (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 to 40 day-old mice to suppress CFSE dilution at a 1:1 Treg:Teff ratio (Figure 4B). Purification of the nTregs was equivalent in the two age groups (Figure 4C).

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 to those in littermate controls (LM). 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 (grey shade) 5B6 Tg mice without disease symptoms were co-cultured with CFSE-labeled responder CD4⁺CD25⁻ T cells from a 60-day-old 5B6 Tg mice at the indicated ratio of nTreg to responders in the presence of PLP_139-151-pulsed, irradiated naïve splenocytes for 3d. 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 d old (young) and 80 d old (old) Tg mice were similar as determined by the percentage of Foxp3⁺ cells. (D) 30-40 day old 5B6 TCR Tg mice were treated with rat IgM control antibody (n=9) or 7D4 anti-CD25 antibody (n=10) for 7 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 if in vivo depletion of CD25⁺ T cells in young 5B6 Tg mice would affect the incidence or severity of sEAE. 30-40 day old mice were treated with monoclonal anti-CD25 antibody (7D4) for 7 treatments at two days interval and monitored for development of clinical EAE for an additional 30 days. Figure 4D shows mice treated with anti-CD25 antibody have higher incidence (6/10 = 60%) vs. 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 if 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-α, IFN-γ, (Figure 5) and TGF-β (data not shown) in response to in vitro stimulation with PLP_139-151. 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-α and IFN-γ production between the different groups (Figure 5 E-F) was observed, while 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 (Figure 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 exhibit 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 non-clinically affected younger mice (Figure 5G). This indicates that a pathologic population of PLP_139-151-specific T cells arises as regulation is lost.

Figure 5. Cytokine profiles of peptide-stimulated splenic T cells of 5B6 Tg mice of varying ages.

(A-F) Splenocytes from 5B6 Tg mice of the indicated ages were stimulated with PLP_139-151 for 72 h. Supernatants were assayed for the production of (A) IL-10, (B) IL-4, (C) IL-5, (D) IL-6, (E) TNF-α, and (F) IFN-γ via LiquiChip analysis. 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 2-3 individual 5B6 Tg mice at 29, 55 and 97 days of age were stimulated with PLP_139-151 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.

Antigen 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 PLP_139-151-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 (34-43). 5B6 mice were tolerized with PLP_139-151-SP or OVA_323-339-SP between 30-40 days of age and observed for development of clinical disease for an additional 30-40 days. Tolerization significantly reduced peptide-specific DTH responses (Figure 6A) and delayed onset of clinical sEAE (Figure 6B). Furthermore, splenocytes from PLP_139-151-SP tolerized 5B6 mice produced significantly more IL-10 than OVA_323-339-SP tolerized mice upon peptide recall in vitro (Figure 6C). Again the TGF-β results were inconsistent from experiment to experiment and no conclusions could be drawn.

Figure 6. Inhibition of spontaneous EAE by antigen-coupled cell tolerance (Ag-SP).

30-40 day old 5B6 TCR Tg mice were left untreated, or i.v. tolerized with 5×10⁷ OVA_323-339-SP or 5×10⁷ PLP_139-151-SP. (A) Seven days post-tolerization, mice from each group were ear challenged with 10 μg PLP_139-151, and swelling was measured 24 h later. Data from one of three representative experiments are shown. DTH levels in PLP_139-151-SP tolerized mice were significantly less than those in OVA_323-339-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 PLP_139-151-SP treated Tg mice and 45 OVA_323-339-SP treated Tg mice pooled from three separate experiments. *Disease incidence significantly less than that in OVA_323-339-SP tolerized mice, p < 0.05. (C) On day 6 post-tolerization, splenocytes from five separate PLP_139-151-SP and OVA_323-339-SP tolerized 5B6 TCR Tg mice or littermate controls (LM) were stimulated with PLP_139-151 for 72 h. Supernatants were assayed for the production of cytokines. Production of IL-10 was significantly higher in PLP_139-151-SP treated mice, *p<0.05. Data is representative of two separate experiments.

To determine if the protective effects of PLP_139-151-SP treatment on clinical disease development were associated with activation and/or generation of peptide-specific Tregs, the effects of i.v. administration of PLP_139-151-SP vs. OVA_323-339-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⁺CD25^lowFoxp3⁺ and CD90.1⁺CD25^highFoxp3⁺ CD4⁺ T cells in wild type SJL recipients of bulk donor 5B6 CD90.1⁺CD4⁺Tg T cells 3 days after treatment (Figure 7A). PLP_139-151-SP tolerization also induced expression of Foxp3 in recipients of purified naïve donor CD4⁺CD25⁻ Tg T cells (Figure 7B, E) and CD25 expression on these donor CD90.1⁺Foxp3⁺ T cells was lower than on recipient CD90.2⁺ Foxp3⁺ T cells (Figure 7 C,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. Ag-SP tolerance induces generation of antigen-specific Foxp3⁺ T cells.

5×10⁶ bulk CD90.1⁺CD4⁺ (A) or FACS-sorted CD90.1⁺CD4⁺CD25⁻ (B-E) T cells from 5B6 TCR Tg mice were transferred to naïve CD90.2⁺ SJL mice on day −1, followed by injection of 5×10⁷ PLP_139-151 -SP or OVA_323-339 -SP on day 0. (A) 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⁺CD25^hi and Foxp3⁺CD25^low cells in PLP_139-151-SP tolerized vs. OVA_323-339 tolerized mice, n = 5 per group. The data is representative of three separate experiments. Ratio of Foxp3⁺CD25⁺ cells is significantly greater than in OVA_323-339-SP injected control values, **p<0.01, ***p<0.001. (B-E) 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 PLP_139-151-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 (grey line). (D) Summary of geometric mean, mean and median fluorescence intensity of CD25 on both donor and recipient CD4⁺Foxp3⁺ T cells from PLP_139-151-SP (n=3) and OVA_323-339-SP (n=3) tolerized mice. Values on donor C90.1⁺ T cells significantly less than on recipient CD90.2⁺ T cells in PLP_139-151-SP treated mice, *p<0.05, **p<0.01. (E) The number of donor CD4⁺CD90.1⁺Foxp3⁺ T cells from three individual PLP_139-151-SP and OVA_323-339-SP tolerized mice were determined. The data is representative of three separate experiments.

Discussion

It has been previously reported that 5B6 TCR Tg mice specific for PLP_139-151 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 (Figure 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 (Figure 4C) while their induction following tolerization with PLP_139-151-SP led to a significant delay in disease onset (Figure 6A). This is consistent with earlier reports showing an age-related decline in suppressive function of CD4⁺CD25⁺ Tregs in 16 week-old as compared to 8 week-old NOD mice corresponding with development of spontaneous type 1 diabetes (24,44). The age-associated functional deficiency in suppressive function was demonstrated in spite of 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 (Figure 4A). Second, splenic T cells from 48 and 116 day-old 5B6 mice produced significantly less IL-10 upon peptide recall (Figure 5A) than 27 day-old Tg mice, a time point prior to onset of sEAE. IL-10 has been reported to play an important role in maintaining the non-encephalitogenic 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 PLP_139-151-SP which led to a significant delay in disease onset (Figure 6B) was associated with enhanced production of IL-10 (Figure 6C). In addition, we previously reported that tolerization of mice with pre-existing PLP_139-151-induced R-EAE with PLP_139-151-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 populations of immunoregulatory T cells whose activity can be enhanced by tolerogenic administration of cognate peptide in the form of PLP_139-151-pulsed, ECDI-fixed syngeneic splenocytes (PLP_139-151-SP).

Tolerization with PLP_139-151-SP resulted in a 7-fold and 2-fold expansion vs. OVA_323-339-SP injected controls, respectively, in the percentage of Foxp3⁺CD25^low and Foxp3⁺CD25^high T cells in recipients of bulk 5B6 CD4⁺ T cells (Figure 7A) within 62 h following treatment. This rapid increase in Treg percentage indicates the generation of adaptive Tregs. In addition, PLP_139-151 SP vs. OVA_323-339-SP tolerization of wildtype 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 CD25^low phenotype distinguishing them from CD25^high nTregs (Figure 7 B-E). This CD4⁺Foxp3⁺CD25^low 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 non-mitogenic anti-CD3 immunotherapy (47). Thus, tolerization with PLP_139-151-SP apparently leads to the induction of multiple regulatory mechanisms including some expansion of CD4⁺CD25^highFoxp3⁺ nTregs and the conversion of naïve CD4⁺CD25⁻ 5B6 T cells to CD4⁺CD25^lowFoxp3⁺ adaptive Tregs, as well as inducing enhanced IL-10 production (Figure 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 hyper-responsive to peptide stimulation compared to cLN T cells from mice without disease symptom (Figure 3C). The initial presence of hyper-responsive 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 protein during the development of sEAE. This finding is consistent with prior studies indicating that the cLNs are a lymphatic site draining some areas of the CNS (30-33) 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 MBP_Ac1-11 TCR Tg mouse system wherein activated T cells are first noted in the cLNs preceding onset of clinical disease in 7-8 week old mice and surgical removal of the cLNs can delay the age of disease onset (J. Lafaille, personal communication). The failure to identify peptide-specific hyper-responsive 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 non-inflammatory conditions. Indeed CNS T cells from MBP TCR Tg mice without disease symptoms fail to respond to antigen, while 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 non-inflammatory conditions (49). These data suggest that the non-inflamed CNS may normally be a ‘tolerogenic’ milieu in the steady-state. In contrast, in the inflamed CNS during progressive relapsing EAE in wildtype SJL mice, the CNS serves as the primary site where naïve 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 (Figure 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 non-symptomatic 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 upregulation of CD25, CD69, CD44, 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 (Figure 2D) and the observation that spleen and cLN (Figure 3B) T cells from mice with disease symptoms produced significantly greater amounts of both IFNγ 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, while 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 hyper-reflexivity was evident when Th17 cells were in excess of Th1 cells, while more classic EAE characterized by ascending flaccid hindlimb paralysis and parenchymal inflammatory infiltration in the spinal cord and brain was seen in mice exhibiting approximately a 1:1 Th17:Th1 ratio. This data suggests 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 ration to the lumbar spinal cord primarily contributes to the pathogenesis of a classic form of sEAE in 5B6 Tg mice, while infiltration in cerebellum and brain stem alone is not associated with disease symptoms (Figure 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 pro-inflammatory PLP_139-151-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.