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The Journal of Immunology, 2008, 180, 6402-6410
Copyright © 2008 by The American Association of Immunologists, Inc.
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Correlation of Blood T Cell and Antibody Reactivity to Myelin Proteins with HLA Type and Lesion Localization in Multiple Sclerosis1

Judith M. Greer2, Peter A. Csurhes, Diane M. Muller and Michael P. Pender

Neuroimmunology Research Unit, School of Medicine, University of Queensland, Royal Brisbane and Women’s Hospital, Herston, Brisbane, Queensland, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS. The numbers of autoimmune T cells and Abs specific for proteins of CNS myelin are increased in the blood in some patients with MS. The aim of this study was to investigate whether there are correlations between the specificity of the autoimmune responses in the blood, the HLA molecules carried by the patient, and the clinical features of MS, because studies on experimental autoimmune encephalomyelitis, an animal model of MS, indicate that autoimmune responses targeting particular myelin proteins and the genetic background of the animal play a role in determining the pattern of lesion distribution. We tested blood T cell immunoreactivity to myelin proteins in 100 MS patients, 70 healthy controls, and 48 patients with other neurological disorders. Forty MS patients had strongly increased T cell reactivity to one or more myelin Ags. In these 40 patients, the most robust correlation was between CD4+ T cell reactivity to myelin proteolipid protein residues 184–209 (PLP184–209) and development of lesions in the brainstem and cerebellum. Furthermore, carriage of HLA-DR4, -DR7, or -DR13 molecules by MS patients correlated with increased blood T cell immunoreactivity to PLP184–209, as well as the development of lesions in the brainstem and cerebellum. Levels of PLP190–209-specific Abs in the blood also correlated with the presence of cerebellar lesions. These findings show that circulating T cells and Abs reactive against specific myelin Ags can correlate with lesion distribution in MS and suggest that they are of pathogenic relevance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis (MS)3 is a chronic inflammatory demyelinating disease of the CNS and is a common cause of progressive disability (1, 2). Although MS is considered to be an autoimmune disease (1, 2), the generally small percentages of individuals showing increased immunoreactivity in their blood to any one autoantigen have raised doubts that autoreactive cells in the blood are of pathogenic relevance. However, if Ag-specific therapy for MS is to be effective, the specific Ags being targeted in individual patients must be known. What is needed, therefore, is stronger evidence that autoreactive responses to myelin Ags can correlate with some feature of MS.

Studies on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, show that the distribution of lesions in the nervous system varies, depending on the Ag and immunization protocol used to induce EAE, and on the MHC and non-MHC genes carried by the animal (3, 4, 5, 6, 7, 8, 9, 10, 11). In MS, the distribution of demyelinated lesions in the CNS can vary from one patient to another, and there is currently no way of predicting where they will develop. Although many studies have previously looked for myelin-Ag-specific T cell responses in patients with MS (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23), only two studies have attempted to correlate T cell autoreactivity to one (24) or several myelin proteins (25) with the distribution of lesions. One investigated reactivity to myelin proteolipid protein (PLP) epitopes by T cells from 10 patients with monocentric monophasic demyelinating syndromes suggestive of a first attack of MS (24), and the other (25) compared immunoreactivity to PLP, myelin basic protein (MBP), and myelin/oligodendrocyte glycoprotein (MOG) in 10 Japanese patients with the optico-spinal form of MS and 11 patients with conventional MS. In these small groups, no clear-cut correlations between the site of the lesions and the reactivity to myelin Ags could be determined.

In the current study we sought to test the hypothesis that autoreactivity directed against certain myelin Ags in patients carrying particular HLA types will lead to development of lesions predominantly affecting certain areas of the CNS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Human subjects

This study was approved by the Royal Brisbane and Women’s Hospital (RBWH) Human Research Ethics Committee and by the University of Queensland Medical Research Ethics Committee. All subjects gave informed consent. One hundred twelve MS patients who met the 2005 revised McDonald criteria for a diagnosis of MS (26) were recruited through the RBWH MS Clinic. The level of disability was assessed using the Kurtzke Expanded Disability Status Scale (EDSS) (27), and the MS severity score (MSSS) was determined from the EDSS and duration of disease, as previously described (28). Cells from 100 of the patients who had not received any immunosuppressant, immunomodulatory, or corticosteroid therapy in the previous 3 mo were used in the T cell and Ab assays. Blood from 12 patients on immunomodulatory therapy was used only for studies correlating lesion localization and HLA type. The age and sex of the MS patients and the age of onset and duration of MS for the 100 patients used in the T cell assays are shown in Table I. Blood and cerebrospinal fluid (CSF) were also collected from five patients undergoing a first attack of CNS demyelination of the type seen in MS within 1 day of a magnetic resonance imaging (MRI) brain scan. Forty-six patients (26 men; 47.7 ± 15.4 years of age at blood collection) with other CNS disorders (CND patients) were also recruited. These included 13 with cerebrovascular disease, 13 with epilepsy, 6 with Parkinson’s disease, 3 with motor neuron disease, 2 with idiopathic intracranial hypertension, 2 with cerebral aneurysm, 2 with CNS tumors, 2 with spinocerebellar degeneration, and 1 each with cerebellar hematoma, sleeping disorder, and hydrocephalus. None of the CND patients had received immunosuppressant, immunomodulatory, or corticosteroid therapy in the previous 3 mo. One hundred twenty healthy controls (39 men; 36.1 ± 11.2 years of age at blood collection) were recruited from hospital and university staff. Blood from 50 of these was used only for HLA typing. The patients and controls were all of Caucasian origin, except for 1 MS patient and 1 healthy control, who were both of Asian ethnic origin.


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  		Table I. Clinical features of the 100 MS patients used in the T cell proliferation assaysa

 
Blood and CSF

Fifty milliliters of blood was collected by venepuncture from each subject. Five milliliters was used for HLA typing. PBMC were separated from the blood by centrifugation through Ficoll, and plasma was collected from above the PBMC layer. Up to 10 ml CSF was collected, after informed consent had been obtained, at the time of diagnostic lumbar puncture. Lymphocytes were separated from the CSF by centrifugation.

Ascertainment of lesion localization

Patient records from the time of onset of MS were reviewed, and patients were also assessed clinically at the time of venepuncture. Conventional MRI brain scans (T1, T2, and fluid attenuated inversion recovery (FLAIR)) were available for 100 of the MS patients at one or more time points throughout the disease course. The distribution of MRI lesions was determined by radiologists who were unaware of the results of the T cell assays and HLA typing. Patients were then classified according to whether they had lesions in the cerebral hemispheres, brainstem, cerebellum, optic nerve, spinal cord, and spinal roots, based on clinical and MRI findings. Clinical evidence of cerebral involvement was taken to include cognitive impairment, epileptic seizures, dysphasia, hemiparesis, hemisensory loss, and homonymous visual field defects. Clinical evidence of lesions in the brainstem and/or cerebellum included extraocular weakness (III, IV, or VI nerve palsy), internuclear ophthalmoplegia, facial sensory loss, trigeminal neuralgia, lower motor neuron facial palsy, nystagmus, MS-related deafness, dysarthria, dysphagia, dysphonia, limb ataxia without sensory loss, gait ataxia (without sensory loss and with negative Romberg’s sign), head titubation, or cerebellar postural tremor. Nerve root involvement was detected by the presence of radicular pain, localized muscle atrophy, and absent deep tendon reflexes.

Genomic DNA preparation and HLA typing

Genomic DNA was prepared as previously described (13) or using NucleoSpin Blood XL DNA extraction kits (Clontech). Dynal low-resolution SSP kits (Dynal Biotech) were used to type for HLA-DR and HLA-DQB molecules, to a resolution equivalent to that of serotyped subgroups. For subtyping of subjects carrying DR2, DR4, and DR13, and for DQA typing, Dynal AllSet SSP high-resolution kits (Dynal Biotech) were used.

Assessment of T cell proliferation to peptides

Uptake of tritiated thymidine. T cell proliferation assays were performed using established techniques (13) on fresh PBMC or CSF cells. Human PLP and MBP were extracted from human brain tissue as previously described (13). Peptides (see Fig. 1 for sequences) were synthesized according to the human sequences by Auspep or Mimotopes. All peptides were >95% pure. The PLP and oligodendrocyte-specific protein (OSP) peptides were moderately hydrophobic and were dissolved in 0.2 M acetic acid as 5 mg/ml stock solutions. Stock solutions of the other peptides were made in water. The peptides were diluted in tissue culture medium immediately before adding to the microtiter plates at a final concentration of 5, 10, and 25 µg/ml. Con A (final concentration 2 µg/ml) and tetanus toxoid (10 Lf/ml) were used as positive controls. All assays were done in triplicate. For assay of PBMC, 1.5 x 105 fresh PBMC/well were cultured in U-bottom 96-well plates (Nunc) in the presence or absence of Ag for 6 days, with [3H]thymidine being added during the final 18 h. For assay of cells from CSF, 8 x 103 CSF cells plus 105 irradiated (3000 rad) autologous PBMC were added to each well of the 96-well plate in the presence or absence of Ag for 6 days, with [3H]thymidine being added during the final 18 h. Cells were then harvested onto glass-fiber mats, and the cpm determined in a Betaplate counter (Beckman Coulter). To be considered a true positive response and be included as such in the study, all three wells had to show a similar increase in reactivity. Stimulation indices (SI) were determined by the formula: SI = (Mean cpm of peptide-containing wells)/(Mean cpm of control wells without peptide). The mean SI reported in Figs. 2 and 4 are the means of the maximum SI over the three peptide concentrations tested.


Figure 1
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  		FIGURE 1. Proliferative responses to myelin Ags. The percentages of MS patients and CND patients responding to each myelin Ag with a stimulation index greater than or equal to the mean + 2 SD of the healthy control group are shown. *, p = 0.02 or **, p = 0.006 compared with healthy control group by {chi}2 analysis with Yates’ correction, as required. The mean background cpm ± SD for these assays was 1913 ± 968, with a range of 603-4321 cpm for MS patients, 372-3671 cpm for healthy controls, and 412-4142 cpm for CND patients. The mean SI ± SD for healthy controls in response to each Ag was: h-PLP, 1.44 ± 0.76; PLP41–58, 1.54 ± 0.91; PLP184–199, 1.42 ± 0.65; PLP190–209, 1.36 ± 0.58; PLP262–276, 1.44 ± 0.71; h-MBP, 2.25 ± 2.01; MBP82–100, 1.89 ± 1.76; MBP151–171, 1.41 ± 0.67; MOG30–44, 1.83 ± 0.72; MOG41–55, 1.52 ± 1.12; MOG72–86, 1.25 ± 0.52; MOG121–135, 1.32 ± 0.64; MOBP28–46, 1.53 ± 0.65; MOBP37–55, 1.33 ± 0.36; OSP42–52, 1.30 ± 0.47; OSP52–71, 1.21 ± 0.48; OSP66–83, 1.20 ± 0.58; OSP102–121, 1.42 ± 0.71; OSP184–196, 1.22 ± 0.48; OSP202–218, 1.40 ± 0.50.

 

Figure 2
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  		FIGURE 2. Representative CFSE analysis of the T cell responses to PLP184–199 and PLP190–209 in a MS patient. The peptide-responsive dividing cells have decreased CFSE fluorescence, shown in the box on the left hand side of each plot, whereas the nonresponding cells are in the right-hand box. In this patient, and in 7 other of the 10 MS patients tested, the reactive cells were CD4+ T cells, not CD8+ T cells.

 

Figure 4
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  		FIGURE 4. Increased levels of reactivity to PLP184–209 are seen with increasing MSSS (28 ) in patients with brainstem/cerebellar lesions, but not in patients who do not have lesions in these areas. The MSSS was determined from the Kurtzke Expanded Disability Status Scale score and the duration of MS, as previously described (28 ), and reactivity to PLP184–209 (mean SI ± SE) is shown for patients falling within each 2 point range of this score. There were 11 patients in the 0–1.99 range, 7 in the 2.00–3.99 range, 24 in the 4.00–5.99 range, 21 in the 6.00–7.99 range, and 20 in the 8.00–9.99 range. The dotted line represents the mean SI of pooled healthy control and CND groups (SI = 1.4 ± 0.05). *, p < 0.001 compared with the pooled control group using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.

 
CFSE assays. Freshly collected PBMC were labeled with CFSE, as previously described (29), and incubated with 10 µg PLP184–199 + 10 µg PLP190–209 for 10 days in phenol red-free X-vivo 15 serum-free medium (Cambrex). Cells were then washed, stained with PerCP-labeled anti-CD4 or anti-CD8 Abs (BD Biosciences), and analyzed by flow cytometry, with gating on the lymphocyte population.

ELISA

Plasma was collected from each patient and stored at –70°C until required. The total IgG concentration in each sample was determined using Bindarid radial immunodiffusion (RID) kits (The Binding Site). For use, each plasma sample was adjusted to 100 µg/ml total IgG. PLP184–199 and PLP190–209 in 0.2 M bicarbonate buffer (0.5 µg/well) were coated onto Nunc Polysorb ELISA plates, plates were blocked with 2% skimmed milk in PBS-Tween 20, and 100 µl of each diluted plasma sample was added in duplicate to the plates and incubated overnight at room temperature in a humidified chamber. Each plate also contained four dilutions of a high-titer-positive control reference serum. Anti-human IgG-alkaline phosphatase (Sigma-Aldrich) was used as secondary Ab and was detected with p-nitrophenyl phosphate (Sigma-Aldrich). Plates were read at 405 nm. To prevent plate-to-plate variation, the four dilutions of the positive control serum were plotted against absorbance, the second highest dilution was allocated the value of 1, and the relative absorbance of the test samples was determined with reference to the control serum.

Assessment of effect of PLP190–209-specific T cells and Abs in C3H/HeJ mice

All animal experiments were approved by the Animal Ethics Committee of University of Queensland. PLP190–209-specific CD4+ T cell lines were generated by removing the draining lymph nodes from female 8–10-wk-old C3H/HeJ mice (Animal Resources Centre, Canning Vale, WA, Australia) immunized 10 days earlier with 50 µg PLP190–209 in CFA, and culturing peptide-reactive lymph node cells. The lines were stimulated in vitro every 10–14 days with peptide and irradiated splenocytes as APCs for five rounds. The resultant lines were >99% CD4+ T cells, as determined by flow cytometry. PLP190–209-specific Abs were obtained by immunoaffinity purification of Ab from the sera of the C3H/HeJ mice immunized with PLP190–209. The immunosorbent was prepared by coupling PLP190–209 to cyanogen bromide-activated Sepharose 4B (Amersham Biosciences), as per the manufacturer’s instructions. For induction of EAE with T cell lines, activated T cell blasts were enriched by centrifugation through Ficoll 3 days after activation with PLP190–209 and irradiated splenocytes and washed; 5 x 106 T cell blasts were injected i.v. into naive mice. Five of the mice also received 500 µg affinity-purified PLP190–209-specific Ab i.v. 1 day after the T cells and then again 7 days later. Mice were assessed for development of EAE, and they were perfused with modified Karnovsky’s fixative (7) within 2 days of onset of neurological signs. Brains were embedded in HistoResin (Leica Instruments) and 1-µm sections were cut every 10 µm through the brainstem and cerebellum. These were stained with cresyl violet to assess inflammation (7). Whole cross-sections were photographed on a Zeiss Axiophot microscope. The percentage area of each cerebellum covered by lesions was determined using NIH Image software.

Statistical analyses

Statistical analyses were done using GraphPad Prism version 4.00 (GraphPad Software). T cell and Ab data from the patients and controls were initially tested to determine whether they were normally distributed within each group. This was not found to be the case, and thus the Kruskal-Wallis test for nonparametric data was used to compare all groups together. If this gave a p of <0.05, then Dunn’s multiple comparison test was used to compare pairs of groups. The frequencies of HLA phenotypes and genotypes in patients and controls were compared using {chi}2 analysis with 2 x 2 contingency tables. Yates’ correction was applied when the number of positive samples in any test was <5. p values were corrected for multiple comparisons using the method of Bonferroni according to the formula Pc = 1 – (1 – P)n, where P is the uncorrected p value, Pc is the corrected p value, and n is the number of comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
T cell proliferation to myelin Ags

Initially, T cell proliferation to 20 myelin Ags (human PLP and MBP and 18 peptides derived from the human sequences of PLP, MBP, MOG, myelin oligodendrocyte basic protein (MOBP), and OSP; see Fig. 1 for details) and the positive control Ags, tetanus toxoid and Con A, was assessed in fresh PBMC from 100 MS patients, 46 CND patients, and 70 healthy controls. For MOBP, MOG, and OSP peptides, only 18 CND patients were tested. None of the MS or CND patients had received immunosuppressants or immunomodulatory agents for at least 3 mo before blood collection. There were no significant differences among the three groups for mean responses to tetanus toxoid or Con A. The percentages of individuals with SI greater than or equal to the mean + 2 SD in the healthy control group are shown for each myelin Ag in Fig. 1. The only significant differences between the percentages of MS patients and healthy controls responding to the Ags were for PLP184–199 (p = 0.02) and PLP190–209 (p = 0.006).

To determine the phenotype of the cells proliferating in response to PLP184–199 and/or PLP190–209, CFSE assays were used to analyze T cell proliferation in 10 MS patients who were selected on the basis of having responded to the PLP peptides. In 8 of the 10 MS patients, the cells responding to PLP184–199 and PLP190–209 were predominantly CD4+ T cells (Fig. 2). In the other two patients, proliferating cells comprised both CD4+ T cells and CD8+ T cells.

In total, 40 individuals with MS had SI at least 2 SD above the mean of the healthy subjects for one or more myelin proteins. The clinical notes and MRI data at the time of venepuncture of these 40 MS patients were reviewed to determine the distribution of lesions at that time point. Reactivity to each myelin protein was analyzed according to whether the patients had lesions in the cerebral hemispheres, brainstem, cerebellum, optic nerve, spinal cord, and nerve roots (Table II). The most striking observation was that patients who showed increased reactivity to PLP184–199 and/or PLP190–209 were more likely to have lesions in the cerebellum or brainstem (p = 0.02), and to carry DR4, DR7, or DR13, but not DR3 (p = 0.01), than were patients who did not show increased T cell responses to these PLP peptides. An increased number of patients responding to the PLP peptides also had lesions in the optic nerve compared with patients not showing increased responses to PLP, but this did not reach statistical significance.


Figure 8
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  		FIGURE 8. Proliferative responses in the 40 patients showing strongly increased reactivity to one or more myelin Ags and correlation with lesion location

a For proliferative responses: +, reactivity of >2 SD above mean of healthy control; ++, reactivity of >3 SD above mean of healthy control; +++, reactivity of >4 SD above mean of healthy control.

b For lesion localization: +, mild to moderate involvement; ++, extensive involvement; *, plus spinal nerve root involvement; shading indicates cognitive impairment or other clinical evidence of cerebral involvement.

 
Immunoreactivity to PLP and correlation with lesion localization

We sought to confirm these associations in the larger group of 100 MS patients. The MS patient group was subdivided on the basis of clinical or MRI evidence of lesions in the brainstem and/or cerebellum before or at the time of venepuncture. The mean SI for all peptides were determined and compared between MS patients with lesions in these areas, MS patients without lesions in these areas, healthy controls, and CND patients. There was a significantly increased proliferative response to PLP184–199 and PLP190–209 in the group of MS patients with lesions in these areas (n = 51), compared with MS patients without lesions (n = 49), CND patients, and healthy controls (Fig. 3), but not for any of the other 18 myelin Ags, Con A, or tetanus toxoid (not shown).


Figure 3
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  		FIGURE 3. T cell responses to PLP184–199 and PLP190–209 are increased in patients who had lesions in the brainstem and/or cerebellum. Blood samples from MS patients who had lesions in the brainstem and/or cerebellum before or at the time of blood collection (BS/Cb lesions), MS patients without lesions in these areas before or at the time of blood collection (no BS/Cb lesions), healthy controls (HC), and CND patients were tested for T cell reactivity to PLP184–199 and PLP190–209 in proliferation assays. The mean SI ± SEM against the PLP peptides were significantly increased in the MS patients with BS/Cb lesions compared with the other groups (*, p < 0.01; **, p < 0.001, as determined by the Kruskal-Wallis test followed by Dunn’s multiple comparison test).

 
Does the increased reactivity to PLP184–209 correlate with the severity of MS

Because lesions in the brainstem and cerebellum may correlate with the burden of disease and duration of MS, we looked at T cell responses to PLP184–209 as a function of the MSSS (28), which is determined from the Kurtzke Expanded Disability Status Scale score and the duration of MS. Overall, there were no differences with respect to the MSSS between MS patients with brainstem and/or cerebellar lesions and MS patients without brainstem or cerebellar lesions (Table I). Whereas there was increased reactivity to PLP184–209 in patients who had an MSSS of ≥2.00 and who had lesions in the brainstem or cerebellum, there was no similar increase in PLP reactivity in patients who did not have lesions in these areas, even in those with a high MSSS (Fig. 4). This finding, together with the lack of increased responses to other myelin Ags in the group with brainstem/cerebellar lesions compared with the group without lesions in these areas (Table II), indicates that the observed differences in responsiveness to PLP184–209 are not due to the former group having a greater overall burden of disease.

We also looked for increased immunoreactivity to PLP184–209 and development of lesions in the brainstem/cerebellum in the early stages of MS. Both PBMC and CSF cells were available within 1 day of a MRI brain scan for five patients undergoing the first attack of CNS demyelination. One of these patients presented with a brainstem lesion and had only one other lesion on MRI scans of the brain and spinal cord. Cells from both the blood and the CSF of this patient showed strongly increased T cell reactivity against both PLP184–199 and PLP190–209 (Fig. 5). None of the other four patients showed any evidence of brainstem or cerebellar lesions or increased reactivity to PLP184–199 or PLP190–209. None of the five patients had increased blood or CSF T cell reactivity to MBP82–100 or MOG41–55 (not shown).


Figure 5
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  		FIGURE 5. T cell responses to PLP184–209 in five patients presenting with a first attack of CNS demyelination. PBMC and CSF cells were collected within 1 day of a MRI brain scan and were tested for proliferation in response to PLP184–199, PLP190–209, MBP82–100, and MOG41–55. Patient no. 3 was the only one of the five to have lesions in the brainstem and/or cerebellum (lesion in the pons) and also the only patient with increased T cell reactivity to the PLP peptides in either the blood or CSF; this patient had increased T cell reactivity to both PLP184–199 and PLP190–209 in the blood (SI >6) and the CSF (SI >4), compared with background proliferation in the absence of Ag (SI = 1). There was no increased reactivity to MBP82–100 or MOG41–55 in any of the blood or CSF samples (not shown).

 
Carriage of HLA-DR4, -DR7, -DR13, or -DQ3 correlates with an increased risk of developing lesions in the brainstem and/or cerebellum

Most Caucasian MS patients carry DRB1*1501 (DR15) (Table III); however, we have previously found that the strongest T cell reactivity against PLP184–209 occurs in patients carrying DR4 or DR7 (13). PLP180–199 and PLP190–209 bind with moderately high affinity to HLA-DRB1*1501 molecules, and PLP180–199 has also been shown to bind with high affinity to DR4, but only poorly to DR3 (30). Analysis of HLA type in MS patients (including an additional 12 patients on immunomodulatory therapies and not included in the T cell assays) and 120 healthy Caucasian controls showed increases in the percentages of individuals carrying DR4, DR7, and DR13 in the subgroup of MS patients who developed brainstem/cerebellar lesions, compared with those who did not develop lesions in these areas (Table III). In contrast, the percentage of patients carrying DR3 was significantly increased in patients who did not develop brainstem or cerebellar lesions compared with those who did develop lesions in these areas (Pc = 0.004).


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  		Table III. HLA-DR and HLA-DQ typing in MS patients and healthy controls

 
The HLA types can be subgrouped based on the sequence of the HLA molecule. Several positions within the HLA sequences are important in determining whether a particular Ag can bind to that HLA molecule. Typing to the molecular level indicated that the frequency of carriage of those HLA-DR4, -DR7, and -DR13 alleles encoding HLA-DR molecules with either arginine, alanine, lysine, or glutamine, but not a glutamic acid, at positions 71 or 74 of the β-chain (which form the P4 pocket of the peptide-binding cleft of the HLA molecule) (i.e., DRB1*0401, DRB1*0404, DRB1*1303, DRB1*1312, and all DRB1*07) was significantly increased in MS patients with brainstem and/or cerebellar lesions (72%) compared with MS patients without lesions in these areas (17.1%) (Pc = 0.0000002).

There was also a correlation between carriage of HLA-DQ3, which is in linkage dysequilibrium with HLA-DR4 and which can also occur with HLA-DR7, -DR11, and -DR13, and development of lesions in the brainstem and cerebellum (Table III).

The HLA type was predictive of where lesions would occur, with 59 of 68 MS patients (86.8%) who carried DR4, DR7, or DR13 developing brainstem or cerebellar lesions; in contrast, only 14 of 44 individuals (31.8%) who did not carry DR4, DR7, or DR13 developed brainstem or cerebellar lesions (p = 2.5 x 10–9; OR = 14.0 (5.5–36.2)). Thus, carriage of DR4, DR7, or DR13 gives a relative risk of 2.7 for development of brainstem or cerebellar lesions. Forty of 51 individuals (78.4%) who carried DQ3 developed brainstem or cerebellar lesions; in contrast, only 32 of 60 (53.9%) of those who did not carry DQ3 developed brainstem or cerebellar lesions (p = 0.005; OR = 3.2 (1.4–7.4)).

Carriage of HLA-DR4, -DR7, or -DR13 correlates with increased T cell reactivity to PLP184–209

When the T cell reactivity to PLP184–209 was analyzed on the basis of the HLA-DR type, the strongest responses occurred in MS patients carrying DR4, DR7, or DR13, particularly in conjunction with DR15, which is linked to susceptibility to MS (Fig. 6A). Individuals carrying DR15 together with HLA-DR molecules other than DR4, DR7, or DR13 did not show increased T cell reactivity to PLP184–199 or PLP190–209, indicating that the DR15 molecules are not presenting these PLP peptides to the T cells. When MS patients who did not carry DR15 were assessed, there was a similar pattern of differences in PLP reactivity between patients who carried DR4, DR7, or DR13 and patients who carried other HLA types, but these were not significant after correction for multiple comparisons, owing to the relatively small number of MS patients available for analysis who did not carry DR15 (data not shown). Additionally, the proliferative response to PLP184–209 of MS patients carrying HLA-DR4, -DR7, or -DR13 molecules that do not have a glutamic acid residue at β71 or β74 (within the P4 pocket of the Ag-binding cleft) was significantly increased compared with those who carried molecules with a glutamic acid at these positions (mean SI ± SEM of 3.36 ± 0.75 vs 1.94 ± 0.37; p < 0.01).


Figure 6
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  		FIGURE 6. HLA restriction of T cell proliferative responses to PLP184–199 and PLP190–209. A, DR15 MS patients who additionally carry DR4, DR7, or DR13 show increased proliferative responses to PLP184–199 and PLP190–209. The higher of the two SI against PLP184–199 and PLP190–209 from each individual was used to determine the mean. The mean SI values were elevated in patients carrying DR15-DR4, -DR7, -DR13, compared with those carrying DR15-DR3 and with those carrying other HLA molecules in association with DR15 (as determined by the Kruskal-Wallis test followed by Dunn’s multiple comparison test). The "other" category indicates genotypes consisting of HLA-DR1, -DR8, -DR10, -DR11, -DR12, -DR14, or -DR16 in combination with HLA-DR15. These "other" genotypes occurred at low frequency (n < 5 for each) and are therefore grouped together for this analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, The increased response of individuals who carry DR4, DR7, or DR13 was specific for MS patients, because healthy controls carrying these molecules did not show increased proliferative responses to PLP184–199 and PLP190–209. Additionally, both MS patients carrying DQ3 and those not carrying DQ3 showed increased proliferative responses to the PLP peptides, compared with controls. *, p < 0.05 compared with controls.

 
For HLA-DQ, the mean SI for reactivity to PLP184–199 and PLP190–209 was not significantly higher in DQ3+ MS patients than in DQ3– MS patients (SI = 2.7 ± 0.6 vs 2.2 ± 0.2) (Fig. 6B), suggesting that the marked increase in the number of MS patients with brainstem or cerebellar lesions who carry DQ3 and show increased reactivity to PLP184–209 may be due primarily to the strong linkage disequilibrium of DQ3 with all DR4 and some DR13 alleles (and to a lesser extent with DR7). Only 5 of the 100 MS patients in the study carried DQ3 in the absence of either DR4, DR7, or DR13, and the response to PLP184–209 was only marginally increased in those patients. Interestingly, the association of increased T cell reactivity to PLP184–209 with carriage of DR4, DR7, DR13, or DQ3 was not observed in healthy subjects, indicating its clear relationship to MS (Fig. 6B).

Role of PLP184–209-specific Abs in development of brainstem and cerebellar lesions

Abs against myelin components have been suggested to play a role in lesion development in MS. PLP is a integral membrane protein on the surface of oligodendrocytes and is present throughout the myelin lamellae, including on the outmost loop of myelin (31), and the PLP184–209 is located on the extracellular face of PLP (32). Initial testing showed increased levels of PLP184–209-specific Abs in MS patients compared with healthy controls and CND patients (Fig. 7A). When this was stratified according to whether MS patients had brainstem and/or cerebellar lesions, there were no differences between the groups for Abs against PLP184–199, but significantly higher levels of Abs against PLP190–209 were present in patients with cerebellar lesions compared with those without cerebellar lesions (Fig. 7B).


Figure 7
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  		FIGURE 7. A, Relative absorbance (compared with positive reference serum) of plasma samples (diluted to 100 µg/ml total IgG) from healthy controls, MS patients, and CND patients against plates coated with a mixture of PLP184–199 and PLP190–209 by ELISA. The mean relative absorbance (horizontal line) in MS patients is significantly higher than in healthy controls (p < 0.001) and CND patients (p < 0.01) using Dunn’s test for multiple comparisons of nonparametrically distributed data. B, The mean relative absorbance for Abs against PLP190–209 in MS patients with cerebellar lesions was significantly higher than in healthy controls (p < 0.001) and MS patients without cerebellar lesions (p < 0.05).

 
Because PLP190–209-specific Abs correlated with the presence of cerebellar lesions, we asked whether T cells and Abs were both required for the development of cerebellar lesions. To address this, we used C3H/HeJ mice, which develop predominantly brainstem and cerebellar lesions when immunized with PLP190–209 (5, 7). The encephalitogenic T cells are CD4+ and I-Ak restricted. I-A is the mouse ortholog of HLA-DQ, and the residues making up the Ag-binding pocket of the I-Ak molecule most closely align to HLA-DQ3. Mice were injected either with PLP190–209 in CFA subcutaneously, or with 5 x 106 PLP190–209-specific T cells i.v. One and 3 days later, five of the mice injected with the PLP190–209-specific T cells were injected i.v. with 500 µg immunoaffinity-purified PLP190–209-specific Ab. On the first or second day of neurological signs, mice were perfused and the CNS was removed for histological analysis. All mice developed lesions in the brainstem; however, none of 30 mice injected with T cells alone developed cerebellar lesions (Table IV). In contrast, all 10 mice immunized with PLP190–209 in adjuvant and 3 of 5 mice injected with PLP190–209-specific T cells and Abs developed cerebellar lesions (Table IV).


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  		Table IV. Abs specific for PLP190–209 play a role in lesion distribution in C3H/HeJ mice with EAE

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study demonstrates that autoimmune reactivity detected in the peripheral blood can correlate with the clinical and MRI features of MS, and it provides evidence for a pathogenic role for autoimmunity in MS. We found a highly significant correlation between increased T cell and Ab reactivity to the 184–209 region of PLP, carriage of HLA-DR4, -DR7, or -DR13, and development of brainstem and/or cerebellar lesions. This is striking because, in the C3H/HeJ mouse, PLP190–209-specific autoreactivity causes lesions predominantly targeting the brainstem and cerebellum (5, 7). In the present cross-sectional study, we found that 56% of MS patients with brainstem or cerebellar lesions had increased T cell reactivity to PLP184–209. This is likely to be an underestimate because blood was collected on only one occasion, which was not necessarily during an attack, and because there are major fluctuations in the frequency of circulating T cells reactive to PLP184–209, with surges of increased reactivity sometimes preceding relapses (16). Our findings do not mean that patients not carrying DR4, DR7, or DR13 and/or not showing increased reactivity to PLP184–209 cannot develop brainstem or cerebellar lesions, but the likelihood that lesions will occur in these areas is substantially increased if they do.

Three lines of evidence from human studies support a causal role of autoreactivity to PLP in the targeting of the brainstem and cerebellum in MS. First, we have shown herein that 21 of 22 individuals with strongly increased T cell reactivity to PLP184–209 in the blood had brainstem and/or cerebellar lesions at the time of testing and that elevated levels of T cell reactivity against PLP184–209 were present in both the blood and CSF and correlated with a brainstem lesion during the first attack of CNS demyelination, when only one other lesion could be detected by MRI. Second, in a previous longitudinal study, we investigated the frequency of myelin-specific T cells in the blood during a 12–18-mo period in five patients, and correlated surges in the frequency of these cells with the presence of gadolinium-enhancing MRI brain lesions (16). Two of these patients, who were also included in the present study, had relapses involving the brainstem (patient MS35) or the cerebellum (patient MS48), and in both cases there was a surge in the frequency of T cells reactive to PLP184–199 and/or PLP190–209 just before the onset of these attacks (16). Third, in the study of Tuohy et al. (24), in which lesion localization and PLP reactivity were assessed longitudinally in 10 patients with monocentric monophasic syndromes suggestive of MS, 2 of 3 patients with anterior brainstem syndrome showed increased reactivity to the PLP180–210 region of PLP and 2 of these patients additionally carried HLA-DRB1*04. In the present study we also found that MS patients with cerebellar lesions had higher circulating levels of Abs against PLP190–209 than did healthy controls and MS patients without cerebellar lesions.

It is unlikely that increased PLP184–209-specific immunoreactivity is merely a marker for brainstem/cerebellar lesions, because this same region of PLP induces lesions in the same region of the CNS in C3H/HeJ mice. We do not know why T cells specific for the PLP184–209 region have a predilection for the brainstem and cerebellum in MS patients and in C3H/HeJ mice. There is no leakiness of the blood-brain barrier (33) or up-regulation of MHC class II, B7-1 (CD80), B7-2 (CD86), or a variety of chemokine receptors in these regions of the CNS in normal mice (D. M. Muller, M. P. Pender, and J. M. Greer, unpublished), indicating that these factors are not responsible. The most likely explanation is that the form or distribution of PLP in the brainstem and cerebellum is different from that in other regions of the CNS, or that other molecules found solely in these areas share some sequence homology with PLP. In support of the former idea, several studies have shown that regional differences in PLP concentrations do occur (34, 35); in support of the latter, a neuronal homolog of PLP known as M6b is expressed strongly in the molecular layer of the cerebellum (36). The current study shows that Ab against PLP190–209 can target lesions to the cerebellum in mice and suggests that it may also do so in patients with MS.

Several common HLA-DR types, including DR4, DR7, and DR15, share largely overlapping peptide-binding repertoires, and these differ considerably from the peptide-binding repertoires of DR3 and DR12 (37). This is in keeping with our findings on the HLA-DR types of individuals responding to PLP184–209. Additionally, carriage of HLA-DR4, -DR7, and -DR13 types that do not have a glutamic acid residue at position 71 or 74 of the β-chain also correlated strongly with development of brainstem and/or cerebellar lesions and T cell reactivity to PLP184–209. We have previously shown that the primary progressive form of MS (PP-MS) is associated with HLA-DR types that do have a glutamic acid residue at position 71 or 74 of the β-chain (38), and that patients with PP-MS have less T cell reactivity to PLP184–209 than do patients with other forms of MS (13). These observations are in keeping with the current findings and suggest that PLP184–209 is not a major target Ag for T cells in most patients with PP-MS.

It is interesting that of the proteins that are inserted only into the CNS myelin membrane, and not into peripheral nervous system myelin (PLP, OSP, MOBP, and MOG), the frequency of increased blood T cell reactivity to each protein among MS patients increased with the abundance of the protein in CNS myelin. Thus, increased T cell reactivity occurred most frequently against PLP (constituting 50% of CNS myelin protein), then against OSP and MOBP (5–10% of CNS myelin protein), and least against MOG (0.05% of CNS myelin protein) (Fig. 1). In contrast, increased T cell reactivity to MBP, which makes up ~30% of the protein of both CNS and peripheral nervous system myelin, was uncommonly observed in our MS patients, as we also found in an earlier study (13).

Although we did not see statistically significant correlations between increased reactivity to other myelin Ags and lesion distribution, it is possible that such correlations do exist. For example, we observed clinical evidence of spinal nerve root involvement in three patients, each of whom showed increased reactivity to MBP. This is notable because the spinal nerve roots in the peripheral nervous system are a major site of demyelination and nerve conduction abnormalities in EAE induced by immunization with MBP (3, 4) or by injection of MBP-specific T cells (39). Additionally, 8 of 11 patients showing increased reactivity against MOG had clinical evidence of cerebral involvement. This is interesting because immunization with MOG has been shown to induce cerebral cortical demyelination in marmosets (40) and rats of certain MHC types (10). Further investigations with larger numbers of patients who exhibit these particular clinical manifestations are needed to determine whether these are robust associations. Additionally, other measures of the pathogenic or protective potential of expanded T cells, such as production of IL-17 or IL-4, may illuminate other associations. It will be important to further define these relationships if Ag-specific therapies for MS are to be successfully developed and used.


   Acknowledgments
 
We thank Dr. Kerryn Green, Dr. Nigel Wolfe, Tim O’Maley, Bernie Gazzard, Kaye Hooper, and Rose Scott for assistance in clinically assessing patients and in the collection of blood samples.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts 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 Grant RG 3190-A-1 from the National Multiple Sclerosis Society and funding from Multiple Sclerosis Australia. Back

2 Address correspondence and reprint requests to Dr. Judith Greer, Neuroimmunology Research Unit, School of Medicine, University of Queensland, Clinical Sciences Building, Royal Brisbane and Women’s Hospital, Herston, Brisbane, Queensland 4029, Australia. E-mail address: j.greer{at}uq.edu.au Back

3 Abbreviations used in this paper: MS, multiple sclerosis; CND, patients with other CNS disorders; CSF, cerebrospinal fluid; EAE, experimental autoimmune encephalomyelitis; EDSS, Kurtzke Expanded Disability Status Scale; MBP, myelin basic protein; MOBP, myelin oligodendrocyte basic protein; MOG, myelin oligodendrocyte glycoprotein; MRI, magnetic resonance imaging; MSSS, MS severity score; OSP, oligodendrocyte-specific protein; PLP, myelin proteolipid protein; SI, stimulation index. Back

Received for publication December 10, 2007. Accepted for publication February 28, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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