B cells limit epitope spreading and reduce severity of EAE induced with PLP peptide in BALB/c mice

Abstract

The role of B cells and antibody in Experimental Autoimmune Encephalomyelitis (EAE) appears to differ based on the identity and state (protein vs. encephalitogenic peptide) of the inducing antigen and the strain of mouse utilized. The involvement of B cells in the induction of EAE by peptides of proteolipid protein (PLP) in BALB/c mice was investigated. Wild-type and B cell-deficient (B cell−/−) mice on the BALB/c background were immunized with overlapping PLP peptides, and the disease course was followed. Although incidence and onset of PLP_180–199-induced EAE was similar in WT and B cell−/− mice, the clinical course was more severe in B cell−/− mice. During acute disease, proliferation and interferon-γ production by lymphoid cells from both strains were similar and were elicited predominantly in response to the immunizing antigen. However, during chronic disease lymphoid cells isolated from B cell−/− mice proliferated to a greater extent and produced more interferon-γ in response to the overlapping peptide PLP_185–206 and to the smaller internal peptide PLP_185–199 than did WT mice. These data suggest that B cells regulate PLP-induced EAE in BALB/c mice through control of epitope spreading.

1. Introduction

The role of B cells and antibody (Ab) in multiple sclerosis (MS) and its primary animal model, experimental autoimmune encephalomyelitis (EAE), remains controversial. Several factors suggest a role for B cells/Ab in MS pathogenesis. Plasma cells are numerous in active [1] and chronic [2] lesions. Oligoclonal immunoglobulin (Ig) in the cerebrospinal fluid is a hallmark of MS, the presence of which correlates with worse prognosis [3–5]. Furthermore, Ab specific for myelin proteins has been observed bound to demyelinating axons in active MS [6] and EAE [7] lesions.

Early studies of myelin protein-induced EAE with rats and mice depleted of B cells from birth supported a role for B cells/Ab in disease pathogenesis [8–11], while subsequent studies of peptide-induced EAE using genetically modified B cell deficient mice failed to support a role for B cells and their products in EAE [12–14]. More recent studies from this group have supported a role for B cells/Ab in protein-induced, but not peptide-induced EAE, in C57BL/6 mice [15, 16].

BALB/c (H-2^d) mice have traditionally been considered resistant to EAE [17, 18]. However, we recently demonstrated EAE induction in BALB/c mice upon immunization with proteolipid protein (PLP) and PLP peptides, aa180–199 and aa185–206 [19]. It is probable that the role of B cells in EAE pathogenesis varies depending on the genetic background of the animal strain and the myelin protein used for disease induction. Thus, we sought to characterize the role of B cells in the pathogenesis of PLP peptide-induced EAE in wild-type (WT) and B cell-deficient (B cell−/−) BALB/c mice.

2. Materials and Methods

Mice

Pathogen-free female WT and B cell−/− BALB/c mice were maintained in microisolator cages in accordance with University and NIH guidelines. WT mice were purchased from Taconic (Germantown, NY). B cell−/− mice on the BALB/c background were deficient in B cells due to a targeted disruption of the Jh locus of the Ig heavy chain JhD; [20], and were either purchased from Taconic or bred in-house from breeding pairs generously provided by M. Shlomchik (Yale University). There were no differences in EAE induction in B cell−/− mice purchased from Taconic and those bred in-house. All mice were used in experiments at 6–8 weeks of age. Sentinel mice showed no infection.

Antigens

PLP aa185–206 was synthesized (S-I-A-F-P-S-K-T-S-A-S-I-G-S-L-C-A-D-A-R-M-Y) by the Peptide Synthesis Facility, Washington University, St. Louis, MO. PLPaa180–199 (W-T-T-C-Q-S-I-A-F-P-S-K-T-S-A-S-I-G-S-L) and PLP185–199 (S-I-A-F-P-S-K-T-S-A-S-I-G-S-L) were synthesized by Sigma-Genosys (The Woodlands, TX). Purity of all peptides was confirmed by HPLC.

EAE Induction and Clinical Scoring

Mice were immunized (s.c.) with PLP_180–199 (200µg/mouse) or PLP_185–206 (400µg/mouse) or PLP_185–199 (200µg or 500µg/mouse) emulsified (1:1) in IFA containing 0.5mg/mouse Mycobacterium tuberculosis, strain H37RA, 0.1ml emulsion/mouse. Emulsions were prepared using an Omni-Mixer (Omni International, Warrenton, VA). Mice also received 300ng pertussis toxin (List Biological Laboratories, Campbell, CA) intravenously at the time of immunization and 72h later. The development of EAE was followed and graded on a scale of 0–5 by a blinded observer as previously described [21].

Histology

Mice were perfused with buffered 4% paraformaldehyde, the CNS dissected, and 1mm CNS slices were prepared. The CNS tissue was then embedded in paraffin, and four micron thick CNS tissue sections were placed on glass slides. Slides were stained with H&E stain to assess inflammation and axonal loss, and Luxol Fast Blue to assess degree of demyelination. Scoring was done by a blinded observer (AHC) on a scale of 0–5 as published [21].

Proliferation Assays

Spleen and lymph node cells were isolated from immunized mice. Cells from 3–5 mice were pooled from each respective organ and cultured in quadruplicate at 2.5×10⁶ cells/ml with antigen [PLP_180–199 or PLP_185–206 or PLP_185–199] or mitogen (ConA, 1µg/ml) and 5% FBS in complete RPMI-1640. ³H-thymidine (0.5 µCi/well) was added during the final 18h, and its incorporation counted (Betaplate 1205; Wallac, Gaithersburg, MD).

Cytokine ELISA

Spleen and lymph node cells were isolated from immunized mice and cultured at 2.5×10⁶ cells/ml with the indicated peptide antigen at 10µg/ml and 5% FBS in complete RPMI-1640. Cell culture supernatants were collected at the indicated times and frozen at −80°C until assayed. Supernatant fluids were analyzed in duplicate for IFNγ (detection limit: 2pg/ml), TNFα (detection limit: 5.1pg/ml), IL-17 (detection limit: 5pg/ml) and IL-10 (detection limit: 4pg/ml) by QuantikineM ELISA (R&D Systems, Minneapolis, MN) according to manufacturer’s instructions.

Serum antbody ELISA

Microtiter plates (Nunc, Rochester, NY) were coated with 10µg/ml PLP_180–199 or PLP_185–206 in bicarbonate coating buffer (pH 9.0) overnight at 4°C. Plate were washed in PBS-0.05% Tween-20 (PBST) and blocked with 3%BSA at room temperature (RT) for 2h. Serum samples isolated from WT or B cell−/− BALB/c mice were diluted 1:100 and 1:400 in dilution buffer (PBS/0.5% BSA/0.05% Tween 20) and added to wells (100µl) and incubated 1h at RT. Plates were washed with PBST and horseradish peroxidase-coupled secondary antibody diluted in dilution buffer (100µl; anti-IgM [1:500]; anti-IgG [1:40 000]; both from Jackson ImmunoResearch, West Grove, PA) was added to the appropriate wells for 1h at RT. Plates were washed and 100µl of substrate (OptEIA; BD Biosciences) was added to each well. Reactions were stopped by the addition of 100µl of 2.5N sulfuric acid (LabChem, Pittsburgh, PA). The absorbance at 450nm was read on a ChemWell microplate analyzer (Awareness Technology, Palm City, FL). Results are reported as the Abs450 of diluted serum samples incubated with peptide minus the Abs450 of diluted serum samples incubated in the absence of peptide. Background values were typically Abs450 <0.07. Background-subtracted results of Abs450 >0.1 were considered positive.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 3.0 (San Diego, CA). Fisher’s Exact Test, unpaired t-test, Mann-Whitney test and 2-way ANOVAs were applied, as indicated. P<0.05 was considered significant.

3. Results

B cell-deficient BALB/c mice are more susceptible to EAE induced by PLP peptides

To investigate the role of B cells in PLP-induced EAE in BALB/c mice, WT and B cell−/− mice were immunized with PLP_180–199 emulsified in CFA. Both groups of mice presented with clinical signs at similar times post-immunization (Table 1). However, EAE was worse in B cell−/− mice, reflected in a more severe and prolonged disease course (Figure 1A; P<0.0001 by two way ANOVA) and a higher median maximal score (Table 1; P=0.021). There was a trend for a higher disease incidence in B cell−/− mice compared to WT mice (P=0.06). Similar results were obtained when animals were immunized with PLP_185–206, but disease incidence was lower in both WT and B cell−/− mice, as observed previously (Figure 1B, Table 1; [19]).

Table 1.

PLP peptide-induced EAE in WT vs. B cell−/− BALB/c mice

Antigen	Incidence	Day of Onseta (dpi) Mean ± S.D.	Max. Scoreb Median (range)
PLP180–199
WT	6/14d	16 ± 9.1e	0 (0–4.0)f
B cell−/−	12/15d	11.2 ± 3.1e	4.0 (0–5.0)f
PLP185–206
WT	2/10c	17.5 ± 2.1e	0 (0–4.0)g
B cell−/−	7/10c	15.0 ± 2.3e	3.5 (0–4.0)g
PLP185–199
200µg/mouse
WT	1/4	47	0.6
B cell−/−	1/4	40	0.7
500µg/mouse
WT	4/4	20.2 ± 9.8e	2.5 (2.0–4.0)e
B cell−/−	3/4	15 ± 1.2e	3.75 (0–4,0)e

Results expressed as median (range).

^aof mice presenting with clinical signs

^bof all mice

^cP=0.07 (Fisher’s Exact Test)

^dP=0.06 (Fisher’s Exact Test)

^enot significant (unpaired t-test [day of onset] or Mann-Whitney test [median max score])

^fP=0.021 (Mann-Whitney test)

^gP=0.043 (Mann-Whitney test); dpi: day post immunization

Figure 1. PLP peptide-induced EAE in WT and B cell−/− BALB/c mice.

WT (■ ) and B cell−/− (□) BALB/c mice were immunized with (A) PLP_180–199 [WT N=14 through d44; N=5 d45–80; B cell−/− N=15 through d44; N=5 d45–80], (B) PLP_185–206 [WT N=10; B cell−/− N=10], or (C) PLP_185–199 [WT N=4; B cell−/− N=4] in CFA and administered pertussis toxin. All mice were graded daily on a scale of 0–5 for signs of EAE. Error bars represent S.E.M. Results were analyzed by two-way ANOVA: p<0.0001 WT vs. B cell−/− (A&B); p=not significant (C).

The above data indicate that B cell−/− mice on the BALB/c background develop worse clinical signs with PLP peptide-induced EAE than their WT counterparts. Previous data from this laboratory demonstrated that the core PLP_185–199, common to both PLP_180–199 and PLP_185–206, was not encephalitogenic in WT mice when mice were followed for 35d post immunization [19]. Thus, WT and B cell−/− BALB/c mice were immunized with the core PLP_185–199 peptide and followed for a longer period of time to determine if the B cell−/− would be susceptible to EAE induction with this peptide. This peptide was found to be weakly encephalitogenic in both strains with onset in one of four B cell−/− mice at 40dpi, and in one of four WT mice at 47dpi (Table 1). Disease severity was similar in both strains. After development of EAE, the WT mouse remained at a grade 2.0 for the duration of the experiment (62d). The B cell−/− mouse recovered fully from the first episode by 47dpi, relapsed at 50dpi and subsequently succumbed to EAE at 58dpi. To determine if a higher dose of PLP_185–199 would be more effective at disease induction, a second group of mice was immunized with 500µg peptide emulsified in CFA (Figure 1C). This higher peptide dose resulted in greater disease incidence with similar severity in WT and B cell−/− mice (Table 1).

Histologic characterization of CNS pathology

Antibody is generally considered important to the process of demyelination. To determine whether B cell−/− mice would display less demyelination or other differences in histopathology compared to WT mice, CNS sections were prepared from immunized mice and observed by light microscopy. The amount of inflammation and demyelination were consistent with clinical severity in both WT and B cell-deficient mice (Table 2).

Table 2.

Histologic characterization of CNS tissue from PLP_180–199 immunized mice during acute EAE

Strain	Clinical Score	Clinical Stage	Inflammation	Demyelination	Remyelination	Axonal loss
WT	1	Onset	2	1	1	0
WT	4	Peak Acute	3	3	1	1
B cell−/−	1.5	Onset	0.5	0	0	0
B cell−/−	4	Peak Acute	3	2.5	0	2.5

Detection of serum antibody specific for PLP peptides

To demonstrate the activation of a PLP peptide-specific B cell response in WT mice with EAE, ELISAs were performed on serum samples isolated from WT BALB/c mice 14d post immunization with PLP_180–199 or PLP_185–206 (Figure 2). In each case, low titers of IgM and higher titers of IgG were detected to the immunizing antigen, but not to the overlapping peptide. As would be expected, no serum antibody was detected in B cell−/− mice (not shown).

Figure 2. Serum antibody specific for PLP peptides in EAE mice.

WT and B cell−/− mice were immunized (imm.) with PLP_180–199 or PLP_185–206 and 14d later serum was collected. Serum IgM and IgG specific for (A) PLP_180–199 and (B) PLP_185–206 was determined by ELISA using 1:100 and 1:400 dilutions of serum.

Characterization of the immune response to overlapping PLP peptides in WT and B cell ^−/− mice

We sought to characterize differences in the T cell response that may explain the increased disease severity in B cell−/− mice. WT and B cell−/− BALB/c mice were immunized with PLP_180–199, and lymph nodes and spleens were removed 10–14 days later, prior to disease onset. Single cell suspensions were prepared, and proliferation in response to PLP_180–199, PLP_185–206, and PLP_185–199 was assessed by ³H-thymidine incorporation. Data represents cells isolated from a total 6 six mice in two separate experiments. Lymph node and spleen cell populations from WT and B cell−/− strains responded similarly to the immunizing PLP_180–199 and was the predominant response detected in each case (Figure 3A and data not show). Lesser responses to the overlapping peptides were also detected, with a significantly greater response to the core PLP_185–199 peptide detected in B cell^−/− mice.

Figure 3. Proliferation of spleen cells isolated from PLP_180–199 immunized mice.

WT and B cell−/− BALB/c mice were immunized with PLP_180–199. Spleen cells were isolated (A) 10–14d post immunization (dpi) and (B) 60–70dpi. Proliferation in response to PLP_180–199, PLP_185–206, and PLP_185–199 was assessed by ³H-thymidine incorporation. Results expressed as Stimulation Index (S.I.; [cpm in presence of Ag]/[cpm in absence of Ag]) and are representative of 6 mice in each group analyzed in 2 separate experiments. Peak proliferation by each group in response to each peptide each group was compared by unpaired t-test. Only statistically significant results are noted.

Cytokines produced in response to the overlapping PLP peptides were characterized by ELISA of cell culture supernatants. Lymph nodes and spleens were isolated from WT and B cell−/− mice immunized 10–14d prior with PLP_180–199. Cells were cultured with PLP_180–199, PLP_185–206, or PLP_185–199, and cell culture supernatants harvested. Similar levels of IFNγ in response the immunizing PLP_180–199 peptide by spleen cells (Figure 4A) and lymph node cells (not shown). Lower, similar, amounts of IFNγ in response to the overlapping peptides was also noted (Figure 4A). TNFα was detected in response to all three peptides in supernatants from spleen (Figure 4B) and lymph node cultures (not shown), with significantly higher levels detected in WT mice compared to B cell^−/− mice. No significant differences in IL-17 expression were noted between WT and B cell−/− mice (not shown).7

Figure 4. Cytokine production by spleen cells isolated from PLP_180–199 immunized WT and B cell−/− mice during acute EAE.

WT and B cell−/− BALB/c mice were immunized with PLP_180–199. Production of (A) IFNγ and (B) TNFα by spleen cells in response to the indicated PLP peptides was characterized by ELISA at 10–14dpi. Results expressed as cytokine produced in the presence of peptide minus cytokine produced in presence of media alone from 6 mice in each group analyzed in 2 separate experiments. Peak cytokine production by each group in response to each peptide was compared by unpaired t-test, and significant results are noted.

Analysis of the immune response early in the disease process did not provide an explanation for more severe EAE in B cell−/− mice compared to WT BALB/c mice. Thus, cell proliferation and cytokine production were analyzed during the chronic stage of disease of PLP_180–199-immunized mice. Spleen cells from WT and B cell−/− mice responded similarly to the immunizing PLP_180–199 at this late time-point with proliferation (Figure 3B) and cytokine production (Figure 5). The difference in proliferation to PLP_185–199 by B cell−/− vs. WT splenocytes that was noted at 14dpi was more pronounced during chronic disease. In addition, spleen cells from B cell−/− mice showed significantly higher proliferation to PLP_186–206 than did WT mice (Figure 3B). Furthermore, B cell−/− splenocytes produced significantly more IFNγ in response to the overlapping PLP_185–206 (2–3 fold more) and PLP_185–199 (3–4 fold more) than did WT mice (Figure 5A). IFNγ in response to the immunizing PLP_180–199 was similar in the two strains (Figure 5A). The significantly increased production of TNFα by WT mice in response to all 3 peptides that was noted at 14dpi was no longer evident, with similar production noted in both strains Figure 5B). On the other hand, characterization of interleukin-10 (IL-10) demonstrated higher levels of this protective cytokine produced by primed WT than primed B cell−/− splenocytes in response to the immunizing peptide PLP_180–199 (Figure 5C). Neither strain produced significant amounts of IL-10 in response to PLP_185–206 or PLP_185–199 (data not shown).

Figure 5. Cytokine production by spleen cells isolated from PLP_180–199 immunized WT and B cell−/− mice during chronic EAE.

WT and B cell−/− BALB/c mice were immunized with PLP_180–199 and spleens were removed during chronic disease. (A) IFNγ, (B) TNFα and (C) IL-10 production was measured by ELISA. Results expressed as cytokine produced in the presence of peptide minus cytokine produced in presence of media alone. Data are representative of 6 mice analyzed in 2 separate experiments. Peak cytokine production by each group in response to each peptide was compared by unpaired t-test, and significant results are noted. N.D.: Not detected.

4. Discussion

Clinical findings from MS patients support a role for B cells and/or antibody in disease pathogenesis [1, 3, 6, 22–24]. However, evidence supporting a role of B cells in EAE, the primary animal model for MS, has not been universally observed. Results differ depending on the mouse strain and antigen used for disease induction [12, 14–16, 25].

Historically, BALB/c mice were considered resistant to EAE induction [17, 18]. We recently characterized the encephalitogenicity of myelin proteolipid protein (PLP) in the BALB/cJ and BALB/cBy substrains, identifying PLP_180–199 and PLP_185–206 as encephalitogenic epitopes [19]. This allowed us to investigate the role of B cells in the BALB/c model using the JhD B cell-deficient strain. Immunization of WT and B cell−/− mice on the BALB/c background with either PLP_180–199 or PLP_185–206 demonstrated a more severe disease course in the absence of B cells. This correlated with evidence of epitope spreading in B cell−/− mice.

Our previous work suggested that the core PLP_185–199 common to the encephalitogenic PLP_180–199 and PLP_185–206 peptides was not encephalitogenic [19]. We show here that this peptide can be encephalitogenic in WT and B cell−/− mice, but disease incidence is low, of moderate severity, and much delayed in onset in both strains when mice are immunized with a dose similar to that used for the longer overlapping peptides. The encephalitogenicity of the PLP_185–199 peptide was increased by immunization with a larger dose of peptide. Thus, the PLP_185–199 core epitope shared by the encephalitogenic PLP_180–199 and PLP_185–206 peptides is a weaker epitope for EAE induction than the overlapping peptides, and may be the minimal encephalitogenic epitope in BALB/c mice.

Characterization of cell proliferation and cytokine production by lymphoid cells during acute EAE revealed greater proliferation to the core PLP_185–199 peptide by cells isolated from B cell−/− mice compared to WT mice. This difference in the epitope response broadened to include the PLP_185–206 epitope during chronic EAE. Analysis of cytokine production showed a similar pattern. Early in the disease process, both strains produced similar levels of IFNγ in response to all 3 peptides. Paradoxically, lymphoid cells from WT mice produced significantly more TNFα during acute disease, with very little of this proinflammatory cytokine detected in B cell−/− cultures. However, during chronic disease significantly higher production of IFNγ in response to PLP_185–199 and PLP_185–206 was evident in PLP_180–199-immunized B cell−/− mice compared to WT mice. The in vivo processing of PLP180–199 to additional epitopes shared with PLP_185–206 or PLP_185–199 in the absence of B cells could influence the induction of the pathogenic T cell population and the breadth of autoimmune responses which contribute to EAE induction.

IL-10 is considered a protective cytokine in the EAE model and multiple sclerosis [26]. WT mice produced significantly more IL-10 in response to PLP_180–199 than did B cell−/− mice. A variety of cells produce IL-10, including T cells, B cells, and macrophages. The source of IL-10 in WT mice was not elucidated, but it is tempting to speculate that B cells may have contributed, as been suggested in EAE induced by myelin oligodendrocyte glycoprotein [27, 28]. The observed difference in the cytokine profile in WT and B cell−/− mice in response to the overlapping epitopes could be responsible for the differences in disease severity noted between these two strains: lack of IL-10 production to temper disease severity coupled with a broader epitope response to PLP in B cell−/− mice could result in the more severe disease noted in the B cell−/− mice.

Activated B cells are very efficient at presenting antigen (Ag) to T cells and focusing the T cell response when both B cell and T cell are specific for the same Ag [29]. Increased IFNγ production to overlapping peptides by the B cell−/− splenocytes suggested that epitope spreading had occurred to a larger extent in B cell−/− mice. In some systems, targeting Ag to B cells has been shown to induce a Th2-like response [30–32], while in others a Th1-like response is induced [33, 34]. Factors governing the role of B cell-mediated antigen presentation in Th1 versus Th2 differentiation include costimulatory molecule expression [35], kinetics of B cell receptor recognition of antigen [36], and adjuvants [32, 33]. Thus, the lower levels of Th1 cytokines and higher levels of IL-10 in WT mice as compared to B cell−/− mice may be due in part to B cell-mediated antigen presentation of the PLP peptides.

The function of antibody in PLP-induced disease in BALB/c mice is unknown. Although it is accepted that antibody plays a role in demyelination [3, 37–40], this does not appear to be a major role in our model. Histopathologic characterization of lesions in WT and B cell−/− mice demonstrated inflammation and demyelination characteristic of EAE in both strains. This is in agreement with MOG-induced disease in C57BL/6 mice with and without B cells [13, 16].

A role for antibody in directing antigen processing and presentation in model systems [41–46], viral infection [47], and autoimmunity [48–50] is well-recognized. Antibodies can potentiate the T cell response to some epitopes while suppressing the response to others [41, 45]. Antibodies specific for glutamic acid decarboxylase 65 (GAD65), considered to be an autoantigen important in Type I diabetes, were found to suppress the generation of GAD65-specific T cell epitopes when both resided within the same peptide, yet enhance T cell epitope generation when the T cell epitope was located distant from the B cell epitope [48]. Similarly, thyroxin-specific autoantibodies were found to inhibit the generation of pathogenic T cell epitopes in a mouse model of thyroiditis [50]. Most commonly, suppression of T cell epitope generation was mediated by Ab of the IgG subclass via Fcγ receptor [41, 48]. A similar mechanism could be responsible for the observations presented here. Initial studies indicate that serum IgG may be responsible for mediating EAE severity in our model (unpublished observation). Indeed, a robust IgG response to the immunizing peptide was detected in WT mice. Thus, in WT mice IgG specific for the immunizing peptide could block the presentation of the overlapping peptides, preventing the spread of the T cell response to these other epitopes. In the absence of Ab in the B cell−/− mice, these epitopes would not be protected, allowing epitope spreading to occur. This model, as well as a role for FcγR, warrants further study.

We previously characterized a pathogenic role for B cells/Ab in rMOG-induced EAE, but not MOG_35–55-induced EAE, in C57BL/6 mice [15, 16]. Studies of peptide-induced EAE in other strains of mice were inconsistent in supporting a role for B cells in disease [12, 14, 25]. Studies in B10.PL mice indicate that B cells are involved in the recovery phase of disease [12, 14]. Data presented here are the first to support a role for B cells in PLP peptide-induced EAE. In this case, B cells temper the disease. The differences in the requirement and roles for B cells in PLP peptide-induced EAE vs. MOG peptide-induced EAE are likely due to differences in the myelin antigen used for immunization as well as genetic differences between mouse strains. Whether B cells will be necessary for EAE induction by whole PLP in BALB/c mice has yet to be investigated. However, whole PLP is difficult to purify in large quantities and these investigations will likely require development of a recombinant form (personal communication, John L. Trotter, M.D.).

We present data here supporting a role for B cells in moderating PLP-induced disease in a BALB/c model of EAE. The mechanism of disease amelioration warrants further investigation, as T cell responses specific for the same PLP peptides used for disease induction in BALB/c mice have also been noted in MS patients [51–53]. Thus, these findings may be applicable to the human disease state and could contribute to the development of effective therapies.