Background
Chronic inflammatory demyelinating polyneuropathy (CIDP) is the most common autoimmune peripheral neuropathy but remains a rare disease with a prevalence of 0.8-8.9 cases per 100.000 [
1,
2]. The disorder causes severe disability in more than 50% of the patients in a chronic-progressive course [
1]. Diagnosis can be difficult given the heterogeneity of CIDP phenotypes. About 50% of the patients suffer from so-called atypical variants including
Distal Acquired Demyelinating Polyneuropathy (DADS) in 25-35% of the cases,
Multifocal Acquired Demyelinating Sensory And Motor Polyneuropathy (MADSAM) in 15% and rare variants such as pure sensory CIDP (10-13%), pure motor CIDP (<10%) and focal CIDP (2%) [
3]. These CIDP subtypes are likely to differ with respect to underlying pathomechanisms and may necessitate different treatment approaches.
Despite recent progress, the underlying immunopathogenetic mechanisms remain poorly understood [
4]. Both humoral as well as cellular immune responses are likely to play a role in the induction of autoimmune neuroinflammation, which leads to demyelination and axonal degeneration [
4‐
7].
Peripheral myelin antigens are promising auto-antigens in CIDP pathogenesis. Recently, we demonstrated higher frequencies of auto-reactive IFN-γ responses directed against the peripheral myelin antigens PMP-22 and P2 in treatment naïve patients who responded subsequently well to intravenous immunoglobulin (IVIG) treatment. Clinical improvement under IVIG-treatment correlated with the reduction of antigen-specific responses against PMP-22 and P2 [
8].
Experimental studies in the EAN model of Guillain-Barré-Syndrom (GBS) support a pathogenic role of another compact myelin P0. Immunization with P0 180-199 is capable to induce EAN in wildtype-, IFN-γ
knockout and TNF-α
knockout mice [
9‐
11]. However, an evaluation in CIDP patients remains to be done.
Myelin basic protein (MBP) is a major constituent of the myelin sheath in the central and peripheral nervous system [
12]. Whereas it has been established as an immunodominant auto-antigen for demyelination in the immunopathogenesis of Multiple Sclerosis (MS) its auto-reactive potential in CIDP remains elusive [
13].
T cells can be differentiated into CD45RA+ CCR7+ naïve, CD45RA- CCR7- effector memory (TEM), CD45RA- CCR7+ central memory (TCM) and CD45RA+ CCR7-terminally differentiated effector memory (TEMRA) T cells [
14]. Especially CD4+ T cells play a major role in CIDP immunopathogenesis [
15‐
17]. In blood and CSF of CIDP patients, significantly elevated frequencies of CD4+ TEM and CD4+ TCM were demonstrated, whereas long-term treated CIDP patients showed significantly reduced CD4+ memory subsets in contrast to untreated CIDP patients [
17‐
19].
Here, we hypothesize that autoreactive myelin-specific T cell responses as well as T cell memory subsets differ between atypical and typical manifestations of CIDP.
Methods
Patients
We evaluated 26 CIDP patients using clinical and immunological (enzyme-linked immunospot assay ELISPOT, fluorescence-activated cell sorting FACS) examinations in comparison to 28 healthy, age-matched controls. CIDP patients who met the diagnostic criteria of European Federation of Neurological Sciences (EFNS) 2010 were divided into “typical” vs. “atypical” according to EFNS 2010 [
20]. Therapy response was defined as an improvement of ≥2 in Medical Research Council (MRC) sum score in 2 different muscle groups, an improvement of ≥1 in Inflammatory Neuropathy Cause and Treatment (INCAT) score (excluding changes in arm function from 0 to 1) or alternatively an improvement of ≥50% of the walking distance as described previously [
8]. Patients and controls were recruited in the outpatient clinic of the Department of Neurology, Charité University Medicine Berlin.
Peripheral myelin antigens
ELISPOT assay was performed using peptides of seven peripheral myelin antigens and CEF as positive control for T cell responses (Table
1). CEF is a peptide pool containing 23 MCH class 1 restricted viral antigens [
21]. Peripheral myelin antigens were provided by Dr. R. Volkmer, Institute of Medical Immunology, Charité University Medicine Berlin. CEF was provided by JPT Peptide Technologies GmbH, Berlin.
PMP-22 32–51 | NGHATDLWQNCSTSSSGNVH |
PMP-22 51–64 | HHCFSSSPNEWLQS |
PMP-22120–133 | RHPEWHLNSDYSYG |
P2 14–25 | ENFDDYMKALGV |
P2 61–70 | EISFKLGQEF |
P0 180-199 | ASKRGRQTPVLYAMLDHSRS |
MBP 82-100 | DENPVVHFFKNIVTPRTPP |
CEF | peptide pool |
Cryopreservation of Peripheral Blood Monocytes (PBMC)
To evaluate T cell responses efficiently we preserved PBMC in liquid nitrogen over a maximum of 6 months. Blood was sampled in CPT tubes for ELISPOT and in EDTA tubes for flow cytometry. PBMC were isolated within 2 h after venipuncture by 1500 g centrifugation for 20 min. After washing, we diluted the PBMC at a concentration of 2x107cells/ml in freezing medium A (60% FCS; 40% RPMI, Biochrom, Berlin, Germany) at 4 °C. The same volume of freezing medium B (20% DMSO, 80% FCS) at 4 °C was added before cell suspensions were transferred into cryovials (Sarstedt, Nürnbrecht, Germany) and set in one at 4 °C prechilled Nalgene Cryogenic Freezing Container (Fisher Scientific, Hannover, Germany) which was placed in −80 °C overnight. After 12-24 h, cryovials were transferred into liquid nitrogen tanks for storage until ELISPOT.
Thawed cell suspensions were transferred into a 15 ml tube containing 10 ml of ice cold PBS. After two washing steps, cells were pipetted in complete RPMI medium (93% RPMI-1640. 5% heat-inactivated FCS, 1% L-glutamin, 1% penicillin-streptomycin) and counted manually using Trypan blue-staining and light microscopy.
ELISPOT
IFN-γ ELISPOT assay in this study was performed on human PBMC as previously described [
8]. We plated 4×10
5cells/well in triplicates for each antigen and positive (CEF) or negative control (medium). CEF, a peptide pool containing viral antigens functioning as a positive control for T cell responses, was added at 9 μg/ml [
21]. The peripheral myelin antigens PMP-22 32-51, PMP-22 51-64, PMP-22 120-133, P2 14-25, P2 61-70 were used at 40 μg/ml and P0 180-199, MBP 82-100 were used at 20 μg/ml. Spot counts were analyzed via ELISPOT Reader Immunospot (CTL Analyzers, Cleveland, Ohio, USA) and custom software. Spot forming units (SFU) for each antigen were subtracted by SFU of spontaneous IFN-γ secretion (usually <5) and then calculated for a cell amount of 10
6 cells.
FACS
Flow cytometry analyses were performed on lymphocyte- and T cell-subpopulations in EDTA whole blood within 12 h after venipuncture.
Flow cytometric analysis was performed as we described recently [
17]. Briefly, mouse anti-human fluorescently labelled monoclonal antibodies allowed to quantifying the frequencies of lymphocyte and T cell subpopulations. The following antibodies were used: CD3 Allophycocyanine-Alexa Fluor 750 (APC-A750), CD4 energy coupled dye (ECD), CD8 APC, CD14 Fluorescein isothiocyanate (FITC), CD16 Phycoerythrine (PE), CD19 PE-Cy5.5, CD56 PE, CD45RA Pacific-Blue (PB), CD45 Krome-Orange (KrO) (all by Beckman Coulter) and CCR7 Phycoerythrine (PE) (R&D Systems). Stained samples were evaluated on a ten-colour Navios flow cytometer and were analyzed using Navios Software (Beckman Coulter).
Statistics
All statistical tests were performed using GraphPadPrism 6.0 software. The study was assessed as an exploratory analysis. Typical or atypical CIDP measurements were compared with healthy, age-matched controls (HC) using Kruskal-Wallis test followed by post-hoc unpaired t-test or Mann-Whitney-test when p < 0.05. For group differences with regard to sex, prior treatment, disease activity and therapy response, Fisher’s exact test was used. For age and INCAT score, unpaired t-test was used. For time since diagnosis, Mann-Whitney-test was used. Level of significance was defined as p < 0.05 for all comparative tests.
Discussion
In the present study, typical CIDP differed from the group of atypical variants. Here, we found a stronger activated immune system in patients suffering from atypical variants of CIDP defined by a trend towards increased peripheral myelin antigen-specific (PMP-22, P0 180-199, MBP 82-100) T cell responses associated with a specific altered CD4+ memory compartment of increased CD4+ TEM and CD4+ TCM counts in the blood. Further we detected elevated T cell responses against antigens P0 180-199 and MBP 82-100 in CIDP patients which have not described before.
We confirmed or previous findings that changes of the T memory compartment is a common finding especially in untreated patients [
8,
17], which is in contrast to Sanvito and colleagues who showed no differences in T cell subpopulation [
23]. In the present study, we detected elevated TEM and TCM primarily in atypical CIDP patients. Clinical experience and studies suggest that typical CIDP patients respond better to therapy than atypical CIDP variants, especially DADS [
24], which is in line with our presented data showing that 100% (9/9) of typical compared to 47% (8/17) of atypical CIDP patients were therapy-responders. The reason for different treatment responses of CIDP subtypes remains unknown. The higher specific immune responses against myelin-derived peptides in atypical compared to typical variants may be a cause for the lower treatment-responses. Likewise, the increased immune reactivity in atypical CIDP patients could result from insufficient treatment.
Recently, it has been demonstrated that CIDP patients show a diminished pro-regenerative function of Schwann cells leading to the axonal loss and therefore incomplete clinical recovery after treatment which is probably caused by inflammatory mediators [
25]. Thus, differences in immune responses between typical and atypical CIDP we have demonstrated might also influence Schwann cell function resulting in different treatment responses and long-term outcome. The INCAT score was significantly lower in atypical cases. However, there was no difference in the time since diagnosis so that a longer disease course and hence pronounced disability and/or altered immune response is not the cause of this difference. Yet, we included mainly atypical case with mild motoric disability (6 patients with sensory CIDP [35.3%] and 5 patients with DADS [29.4%]) who are less often dependent on walking aids leading to lower INCAT disability scores.
Since we included mainly clinically unstable patients who had partly received treatment before, we are not able to answer this question at present. Based on previous results of reduced CD4+ memory subsets in GS-treated patients [
17], it might be further argued that GS treatment may be efficient for this patient group. In contrast to Sanvito et al. [
26], we identified higher IFN-γ responses to P2 and PMP22 peptides which have been more pronounced in the atypical compared to the typical CIDP subgroup. A higher number and proportion of atypical patients might explain this discrepancy as well as the fact that we included mainly clinically unstable and newly diagnosed patients.
Earlier publications detected P0 IgG-antibodies in CIDP patients and P0 180-199 specific T cell responses in spontaneous autoimmune polyneuropathy-mice [
11]. Although EAN resembles Guillain-Barré-Syndrom (GBS) much more than CIDP, we regarded P0 as possible further candidate autoantigen of compact myelin for CIDP. Here, we detected elevated P0 180-199 specific T cell responses primarily in atypical CIDP.
Up to now, only little is known about the role of MBP 82-100 in the pathogenesis of CIDP even though MBP has been detected as part of the myelin sheath of peripheral nerves. Nevertheless, there is long-standing evidence that MBP 82-100 can induce neuroinflammation in autoimmune diseases [
27]. Glatirameracetat, known antagonist of MBP 82-100 specific T cell receptor and part of MS therapy has been demonstrated to alleviate symptoms also in EAN- mice [
28,
29]. Here, we demonstrated significantly elevated MBP 82-100 specific T cell responses in CIDP patients, again primarily in patients with atypical manifestations.
There is growing evidence for the autoimmune potential of antigens which are derived from non-compact myelin of the nodal/paranodal region such as neurofascin 155 of 186 leading to antibody response in distinct subgroups of CIDP or multifocal motoric neuropathy (MMN) [
30‐
33]. Thus, antigenic targets derived from both compact and non- compact myelin leading to humeral and/or cellular immune response may define underlying immune mechanism of different clinical phenotypes of CIDP.
Several limitations may have affected our results. First, our clinically heterogeneous group of atypical patients was too small to distinguish between subgroups of atypical CIDP, which would be necessary to characterize atypical subtypes and to define specific cut offs for our immunological parameters. Second, differences in gender and INCAT score between typical and atypical CIDP patients might have influenced our immunological findings. Third, we aimed to recruit treatment-naïve patients in active-unstable stages of the disease. However, only 46% of patients (12/26) were treatment-naïve at enrollment. Previous immunosuppressive and –modulating therapy might have influenced our immunological findings.
Acknowledgment
The authors thank Sandra Bauer, Sonya Becker and Maik Stein for technical assistance.