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Keywords:

  • interleukin-17;
  • multiple sclerosis;
  • T regulatory cells;
  • T helper typ 1 1;
  • T helper type 17

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Relapsing–remitting multiple sclerosis (RRMS) is a complex autoimmune disease of the central nervous system with oscillating phases of relapse and remission. RRMS has been considered to be driven by T helper type 1 (Th1) lymphocytes but new data indicate the involvement of Th17 responses. In the present study, blood samples from patients (= 48) and healthy individuals (= 44) were evaluated for their immunological status. T cells from patients with RRMS expressed high levels of the activation marker CD28 (< 0·05) and secreted both interferon-γ (CD8: < 0·05) and interleukin-17 upon polyclonal mitogen or myelin oligodendrocyte glycoprotein antigen stimulation. However, T cells from patients with RRMS in remission, in contrast to relapse, had poor proliferative capacity (< 0·05) suggesting that they are controlled and kept in anergy. This anergy could be broken with CD28 stimulation that restored the T-cell replication. Furthermore, the patients with RRMS had normal levels of CD4+ Foxp3+ T regulatory cells but the frequency of Foxp3+ cells lacking CD127 (interleukin-7 receptor) was lower in patients with MS (mean 12%) compared to healthy controls (mean 29%). Still, regulatory cells (CD25+ sorted cells) from patients with RRMS displayed no difference in suppressive capacity. In conclusion, patients in relapse/remission demonstrate in vitro T-cell responses that are both Th1 and Th17 that, while in remission, appear to be controlled by tolerogenic mechanisms yet to be investigated.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). The precise pathogenic mechanisms are not clearly understood but in the early stages neural damage is thought to result from T-cell-mediated destruction of myelin-expressing cells.1,2 Multiple sclerosis has previously been described as a T helper type 1 (Th1) cell-mediated disease based on data describing high interleukin-12 (IL-12) cytokine levels.3 However, IL-12 is a proinflammatory cytokine composed of a small subunit, p35, and a larger subunit, p40, and the latter is shared with the heterodimeric cytokine IL-23.4 Hence, in some studies that measured the p40 subunit, IL-23 may have been up-regulated instead. Interleukin-23 is connected to the recently characterized Th17 cell response that is distinct from the more established Th1 and Th2 reactions.5 The CD4+ Th17 cells produce IL-17 instead of interferon-γ (IFN-γ) upon antigen recognition. Data supporting the role of IL-23/Th17 in the development of autoimmune diseases derive from the murine experimental autoimmune encephalomyelitis model6 In humans, data are conflicting and support either Th17 or Th178,9 involvement in MS.

Over the past decade, our understanding of the basic processes that control immune tolerance has increased. The identification of Foxp3-expressing CD4+ CD25+ T regulatory (Treg) cells as an important component of self-tolerance has opened a major area of investigation. Numerous studies have demonstrated the potent influence of Treg cells in suppressing pathological immune responses in autoimmune diseases, transplantation and graft-versus-host disease.10 However, a major obstacle to the study of Treg cells has been the lack of specific cell surface biomarkers that uniquely define Treg cells from other T-cell subsets.11 It was recently demonstrated that the IL-7 receptor (CD127) is down-regulated on the surface of Treg cells whereas it is highly expressed on effector cells.11 Previous studies in patients with relapsing–remitting (RR) MS have yielded conflicting results regarding the level of circulating Treg cells and their biological function in patients compared to healthy controls.12–15 So far the combination of the markers Foxp3 and CD127 has not been reported in patients with MS.

Herein, we have combined functional assays with the analysis of cytokines and cell surface molecules to identify the role of circulating T cells of both effector and regulatory types in patients with RRMS. Our results demonstrate the presence of CNS-directed effector T cells of both Th1 and Th17 types, and that these cells are suppressed by tolerogenic mechanisms during the remission phase.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients

Peripheral blood samples were obtained from 48 patients (38 women) with MS (diagnosis according to the criteria of Poser et al.16 or McDonald et al.,17 depending on the time of diagnosis) with a relapsing–remitting course (i.e. RRMS), median age 35 years (range 24–63; mean 38), median MS duration 5 years (1–29; mean 7·4) (Table 1). Thirty-three patients were on disease-modifying treatment – interferon-β (= 24), glatiramer acetate (= 3), intravenous immunoglobulin (= 8) or mitoxantrone (= 1) at the time of blood sampling; whereas 12 patients had no treatment. Ten patients had undergone a clinical relapse 2–6 weeks before blood sampling; all the others were in clinical remission at the time of blood sampling. Median neurological functional impairment according to the Expanded Disability Status Scale18 (EDSS) was 1·5 (range 0–3·5), i.e. most patients well preserved function. Control samples were obtained from 44 healthy controls. This study was approved by the local ethics committee at Uppsala University (Dnr 2006:089) and informed consent was obtained from all study subjects.

Table 1.   Description of study subjects
Pt noSex (M/F)Age (years)EDSSMS duration (years)Remission/RelapseCurrent treatmentTreatment/DrugIncluded in figure:
  1. Pt no, patient number; EDSS, Extended Disability Status Scale (Kurtzke18); GA, glatiramer acetate; IFN, interferon-β; IvIg, intravenous immunoglobulin; mtx, mitoxantrone.

 1M281·08RemissionGACopaxone1,2a–d
 2F482·029RemissionIFNAvonex1,2a–d
 3F401·05RemissionIFNBetaferon1,2a–d
 4F312·58Relapse1,2a–d
 5F291·03RemissionIFNBetaferon1,2a–d
 6M250·02Remission1,2a–d
 7F522·011RemissionIFNRebif1,2a–d
 8M632·010Remission1,2a–d
 9M340·05RemissionIFNBetaferon1,2a–d
10F563·59RelapseIFNBetaferon1,2a–d
11M271·51RemissionIFNBetaferon1,3a–b
12F370·05RelapseIFNAvonex1,3a–b
13F353·014RemissionMtxMitoxantrone1,3a–d
14F463·514RemissionIvIgGammaglobulin1,3a–d
15F240·03RemissionIvIgGammaglobulin1,3a–d
16F622·05Remission1,3a–d
17F562·010RemissionIvIgGammaglobulin1,3a–d
18F271·01RemissionIFNBetaferon1,3a–d
19F563·59Remission1,3a–d
20M442·04Remission1,3a–d
21F432·029Remission1,3a–d
22M291·04RemissionIFNBetaferon1,3a–d
23M271·53RemissionIFNBetaferon1,3a–d
24M312·08RemissionGACopaxone4a–b
25F360·03Remission4a–b
26F502·020RemissionIFNRebif4a–b
27F281·09RemissionIvIgGammaglobulin4a–b
28F330·01Remission4a
29F330·05RelapseIFNBetaferon4a
30F531·53RemissionIFNBetaferon4a
31F241·08Remission4a
32F240·05Remission4a,6a–b
33F330·02RemissionIvIgGammaglobulin4a,c,6a–b
34F260·01RemissionIFNAvonex4a,c,6a–b
35F463·520RemissionIFNRebif4c,6a–b
36F273·02RemissionIFNAvonex5d,6a–b
37M500·09RemissionIvIgGammaglobulin5d,6a–b
38F270·02RemissionIvIgGammaglobulin5d,6a–b
39F241·01Remission6a–b
40F473·519RelapseIFNRebif4a,6a–b
41F451·517RemissionGACopaxone6a–b
42F531·05RelapseIFNAvonex4a
43F352·07RelapseIFNBetaferon4c
44F253·51RelapseIFNBetaferon4c
45F463·56RelapseIFNBetaferon4c
46F4636RemissionIFNBetaferon4c
47F2922RemissionIFNAvonex4c
48F4212RemissionIvIgGammaglobulin4c

Cell culture reagents

Cells were cultured in RPMI-1640 medium supplemented with 1% sodium pyruvate, 1% non-essential amino acids, 10% fetal bovine serum and 1% penicillin/streptomycin. Medium and supplements were purchased from Invitrogen, Paisley, UK.

Lymphocyte separation and flow cytometric analysis

Peripheral blood from patients with MS was collected in heparin-coated 10-ml tubes (Becton Dickinson, San Diego, CA). The blood samples were centrifuged to collect plasma. Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Paque gradient centrifugation (Amersham Bioscience, Uppsala, Sweden) within 16 hr after collection. The blood was stored in accordance with the guidelines of the Uppsala University Hospital Blood Central and the PBMCs were incubated 10 min at 4° with monoclonal antibodies specific for CD3, CD4, CD8, CD28, CD25 or CD127 that were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) for surface staining (BD Biosciences, San Diego, CA). Cells were washed with phosphate-buffered saline (PBS) and resuspended in 1% paraformaldehyde in PBS. For intracellular staining of Foxp3 (eBioscience, San Diego, CA; clone 259D and PCH101) cell surface staining was first completed whereupon cells were permeabilized before adding Foxp3-FITC for 30 min at 4°. Samples were analysed at the same time and with the same settings for patients and healthy controls on a FACScalibur or FACScanton (BD Biosciences).

Analysis of cytokines in peripheral blood

The collected plasma was analysed for protein levels of IL-12 and IL-10 using a Cytometric Bead Analysis Inflammation Kit according to the company protocol (BD Biosciences). Plasma samples were also analysed for protein levels of IL-23 and IL-17 using enzyme-linked immunosorbent assay kits (eBioscience) according to company protocols.

Stimulation of T cells

Interferon-γ production upon stimulation was measured by intracellular cytokine staining after blocking of cellular secretion. The PBMCs were cultured overnight and stimulated with 1 μg/ml OKT-3 Orthoclone (anti-CD3) antibody (Cilag Ag Int, Zug, Switzerland) or 1 μg of a 15-mer peptide mix of 11 overlapping amino acids from myelin oligodendrocyte glycoprotein (MOG) 1–125 designed to react with many different human leucocyte antigen haplotypes (JPT Peptide Technologies, Berlin, Germany) per 1 million cells followed by 4 hr incubation at 37°. Stimulated cells were treated with brefeldin A (BD Biosciences) according to the company protocol and incubated at 37° for 5 hr. Cells were permeabilized before adding APC-, FITC- and PE-conjugated monoclonal antibodies specific for surface expression of CD3, CD4 and CD8 and the intracellular cytokines IL-17 and IFN-γ (BD Biosciences). Cells were washed with PBS centrifugation and the cells were resuspended in 1% paraformaldehyde in PBS. Samples were analysed by flow cytometry using FACScalibur (BD Biosciences).

Proliferation assay

The PBMCs or CD25+/− T-cell populations separated by magnetic-activated cell sorting beads (MACS) (Miltenyi Biotech, Gergisch Gladbach, Germany) were cultured in different ratios with 50 U/ml recombinant human IL-2 (R&D Systems Inc, Minneapolis, MN), and OKT-3 Orthoclone (anti-CD3) antibodies (1 μg/ml) (Cilag Ag Int) with or without anti-CD28 antibodies (BD Pharmingen, San Diego, CA) in 200 μl RPMI-1640 medium with the addition of 20 μl Alamar Blue™ according to the manufacturer’s protocol (Biosource International, Camarillo, CA). Stimulator cells were irradiated at 25 Gy. Inhibition of the CD25+ lymphocyte fraction from patients was assayed by labelling healthy donor lymphocytes with carboxy-fluorescein diacetate succinimidyl ester (CFSE) according to the company protocol (Molecular Probes, Eugene, OR). The CD25+ patient lymphocyte fraction was irradiated at 25 Gy and added to the stimulated healthy donor lymphocytes (1·5 μg/ml OKT3 and 50 U/ml IL-2) in different ratios in a 96-well plate. Cells were cultured for 5 days with fresh media and additional IL-2 on day 3. Cell proliferation was evaluated by fluorescence-activated cell sorting (FACSCalibur, BD Biosciences).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The systemic cytokine milieu is similar in MS patients and healthy individuals

Patients with MS, independent of the disease phase, had similar levels of Th1- (IL-12) and Th17- (IL-17 and IL-23) related plasma cytokines to healthy controls except for the Th2/Th3/Tr1-related cytokine IL-10, which was significantly lower (< 0·05) in patients with MS; although with individual values greatly overlapping (Fig. 1). Individuals among the patients with MS who exhibited high cytokine values were represented by the same patients in the different cytokine analysis. High or low IL-10 was not correlated with disease phase or current therapy (data not shown).

image

Figure 1.  Similar systemic cytokine levels in patients with multiple sclerosis (MS) and healthy controls (HC). Detection of the T helper type 17 (Th17)-related cytokines interleukin-17 (IL-17) and (IL-23) and the Th1 and Th2 cytokines IL-12 and IL-10, respectively, in plasma from patients with MS and healthy controls. Levels of cytokines were analysed using cytometric bead array and enzyme-linked immunosorbent assay. Twenty-three patients with MS were compared to 20 healthy controls. The patients displayed significantly lower levels of IL-10 compared to healthy controls (< 0·05; Mann–Whitney test).

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T cells in MS patients react with IFN-γ or IL-17 production

T cells were evaluated for activation status and subpopulations. The frequency of CD4+ and CD8+ T cells was similar between patients with RRMS and controls with a slight shift in the CD4 : CD8 ratio toward CD4 in patients with RRMS (Fig. 2a,b). T cells from patients with MS expressed significantly higher levels of the activation marker CD28 (CD4+< 0·001; CD8+, < 0·001) (Fig. 2c,d) regardless of whether the patient was in clinical remission, relapsed or on treatment (data not shown). Polyclonal T-cell stimulation (OKT3) generated more IFN-γ-producing CD8+ T cells in patients with MS (0·14–24·5%) than in controls (0–9·8%) (Fig. 3a). Low frequencies of IFN-γ-producing CD4+ T cells were seen in each group after this stimulation. Instead, polyclonal stimulation initiated IL-17 production by CD4+ T cells in a range of 0–1·24% in patients with MS compared to 0–0·2% in controls (Fig. 3b). CD8+ T cells did not respond by IL-17 production to OKT3 stimulation in any group.

image

Figure 2.  Activated T cells in patients with multiple sclerosis (MS). Peripheral blood mononuclear cells from patients with MS and healthy controls (HC) were stained for surface molecules CD3, CD4 and CD28 and analysed by flow cytometry. The peripheral blood mononuclear cells were gated on CD3+ lymphocytes (using forward and side light scatter and CD3 staining) and analysed for surface expression of CD4, CD8 and CD28 in a population of 10 patients with MS and seven healthy controls. (a) Surface expression of CD8+ cells. (b) Surface expression of CD4+ cells. (c) Surface expression of CD8+ CD28+ cells. (d) Surface expression of CD4+ CD28+ cells. Both means (c and d) are significantly different (< 0·001; Mann–Whitney test).

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image

Figure 3.  The T cells of patients with multiple sclerosis (MS) produce both interleukin-17 (IL-17) and interferon-γ (IFN-γ) upon stimulation. Stimulated peripheral blood mononuclear cells from patients with MS and healthy controls (HC) were stained for the surface molecules CD3, CD4, CD8 and the intracellular cytokines IFN-γ and IL-17. (a) Expression of IFN-γ by CD4+ and CD8+ T cells after stimulation with OKT-3 orthoclone (anti-CD3) antibodies in 13 patients with MS and six healthy controls. (b) Expression of IL-17 by CD4+ and CD8+ T cells after stimulation with OKT-3 in 13 patients with MS and six healthy controls. (c) Expression of IFN-γ by CD4+ and CD8+ T cells after stimulation with myelin oligodendrocyte glycoprotein (MOG) 1–125 in 11 patients with MS and 11 healthy controls. Means are significantly different (< 0·05) using Mann–Whitney. (d) Expression of IL-17 by CD4+ and CD8+ T cells after stimulation with MOG 1–125 in 11 patients with MS and 10 healthy controls.

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MOG-reactive T cells are present in both Th1 and Th17 type populations

To determine if circulating T cells had CNS reactivity they were stimulated with a 15-mer MOG (1–125) peptide mix containing 11 overlapping amino acids. MOG expression is confined to the CNS and sequestered at the outermost surface of the myelin sheath19,20 and may be one of the major targets for CNS-directed T cells.21 The MOG peptide mix was designed to target both major histocompatibility complex classes I and II simultaneously as well as different haplotypes to allow investigation of antigen-specific CD4+ and CD8+ T cells in many different individuals. Four of the 11 patients with MS responded with MOG-reactive Th1-type CD4+ T cells producing IFN-γ and seven of them had IFN-γ-producing CD8+ T cells (< 0·05) (Fig. 3c). Six of 12 patients reacted to MOG peptide stimulation by IL-17 production in CD4+ cells (Fig. 3d, left). Two of these patients also had IL-17-producing CD8+ T cells in response to MOG stimulation (Fig. 3d, right). In the control group, T cells from two individuals (out of nine) became activated after stimulation with the MOG peptide mix. One responded with low levels of IFN-γ- and IL-17-producing CD4+ T cells as well as with IFN-γ-producing CD8+ T cells. In the CD8/IL-17 panel (Fig. 3d, right) one additional healthy control responded to the MOG mix with low levels of IL-17-producing CD8+ T cells.

Recent studies classify Th1 and Th17 subsets on the basis of IFN-γ and IL-17 production, respectively. Since some of the MS patients could respond with both IFN-γ and IL-17 we performed double staining to investigate if an individual T cell could express both IFN-γ and IL-17 as seen in mice22 and human patients with Crohn’s disease.23 We analysed only Th1/Th17-responsive patients but could not detect simultaneous expression of these two cytokines in individual cells by flow cytometry (data not shown).

Systemic T cells in patients with remitting MS exhibit proliferative anergy

Even if T cells from patients with MS could react by producing IFN-γ or IL-17 upon stimulation, these cells demonstrated defective proliferation. As demonstrated in Fig. 4a, T cells from patients with MS subjected to polyclonal stimuli (OKT-3 plus IL-2) showed limited proliferation compared to healthy individuals (< 0·05). Patients in relapse had a higher proliferative capacity compared to patients in remission. There were no differences in live : dead cell ratio in the different groups (data not shown). Addition of agonistic anti-CD28 antibody to unsorted T cells was able to break the MS T-cell anergy and the cells immediately reached maximal and similar proliferative capacity compared to T cells from healthy individuals (Fig. 4b). To study functionally active Treg cells in MS, patient CD25+ T cells were irradiated and mixed with stimulated proliferating T cells from healthy donors in a 1 : 1 ratio. As demonstrated in Fig. 4(c), CD25+ T cells from patients with MS in both remission and relapse could inhibit the proliferation of T cells from healthy individuals to a similar degree to control CD25+ T cells. The experiment was repeated using the CFSE-based proliferation assay and these data confirmed the previous results (Fig. 5). Collectively, these experiments demonstrate the presence of CD25+ suppressive cells in patients with MS and that such cells are functionally active.

image

Figure 4.  Proliferative capacity of circulating T cells in patients multiple sclerosis (MS). Peripheral blood mononuclear cells (PBMC) from patients with MS and healthy controls (HC) were stimulated with interleukin-2 (IL-2) and OKT-3 in an Alamar Blue™ assay. (a) Proliferation of unsorted T cells from 10 patients with MS in remission and three patients in relapse compared to 10 unsorted T cells from healthy controls stimulated with OKT-3 and IL-2. Patients with MS in remission and healthy controls differed significantly, < 0·05; Students t-test. (b) T-cell anergy to OKT3 and IL-2 stimulation in four patients with MS was broken by addition of αCD28 antibodies. (c) Functional suppressor assay of regulatory T cells (Treg) from six patients with MS in remission, three patients in relapse and 10 healthy controls. Results are displayed as the suppressive ability of the CD25+ T-cell fraction from each group mixed with stimulated healthy donor PBMC, in per cent. T cells from patients with MS and healthy controls were positively sorted for CD25 using magnetic antibody cell sorting beads and irradiated at 25 Gy. CD25+ cell fractions were then mixed together with stimulated PBMC (see above) from healthy controls in a ratio of 1 : 1 in a 96-well plate. All error bars display SD values in the different populations and groups.

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image

Figure 5.  Functional CD25+ suppressor cells in patients with multiple sclerosis (MS). Healthy donor lymphocytes labelled with carboxy-fluorescein diacetate succinimidyl ester (CFSE) were mixed together with the CD25+ cell fractions of lymphocytes from patients or healthy controls (HC) at a 1 : 1 ratio on a 96-well plate. Cells were stimulated as described in Fig. 4(c) and cultured for 5 days; proliferation was determined by fluorescence-activated cell sorting. Cells were gated on the lymphocyte gate and comparable numbers of gated events were counted. Every peak in the histograms corresponds to proliferation because the membrane dye CFSE decreases with every cell division. (a) CFSE-labelled proliferation of unstimulated PBMC from a healthy control. (b) CFSE-labelled proliferation of stimulated PBMC from a healthy control. (c and d) Patient CD25+ lymphocytes were able to suppress allogeneic activated lymphocytes in the same manner as healthy control lymphocytes. One representative experiment out of three patients with MS and three healthy controls is shown.

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Patients with MS exhibit lower levels of CD127 Treg cells

To confirm the presence of circulating Treg cells in patients with MS, T cells were simultaneously labelled with fluorophore-conjugated antibodies detecting CD4, CD25, CD127 and the transcription factor Foxp3, which is considered a hallmark for Treg cells. Regardless of disease phase, patients with MS had normal levels of Foxp3+ T cells as compared to healthy subjects when the anti-Foxp3 259D clone was used (Fig. 6a). The clone PCH101 used in many reports has recently been shown to give false-positive signals when staining previously activated T cells. Here we demonstrate that PCH101 gives a stronger signal in patients with MS than in healthy controls. However, Foxp3 can be transiently expressed in activated T cells. True Treg cells must then be separated from T effector cells by IL-7 receptor (CD127) analysis because this receptor is lacking on Tregs but can be found on T effector cells. Patients with MS had lower levels (mean 12%) of circulating CD127 CD4+ Foxp3+ CD25+ Treg cells than healthy controls (mean 29%) while they had levels of CD127 CD25 Treg cells similar to those of controls (Fig. 6b). Similar observations have been made in other autoimmune diseases, e.g. a study on patients with systemic lupus erythematosus where CD4+ CD25Foxp3+ T cells were more frequent in patients than in healthy controls.24

image

Figure 6.  Lower levels of regulatory T (Treg) cells in patients with multiple sclerosis (MS) than in healthy individuals. Peripheral blood mononuclear cells (PBMCs) from patients with MS and healthy controls (HC) were stained for cell surface expression of CD4, CD25 and CD127. The stained cells were fixed and stained intracellularly for the expression of Foxp3. For analysis, the PBMCs were gated on CD4+ lymphocytes (based on forward and side scatter and CD4 staining) and analysed for Foxp3 expression in different subpopulations of the CD4+ cells. (a) Total expression of Foxp3+ cells in the entire CD4+ population using antibody clone 259D and PCH101 of Foxp3 in 10 patients with MS and 10 healthy controls. Evaluated cells were gated on the lymphocyte population based on a forward and side scatter dot plot and percentages were calculated based on the total CD4+ population. (b) Distribution of Foxp3+ cells in the same individuals in different subpopulations (CD127 CD25+ and CD127 CD25) of CD4+ cells using antibody clone 259D. Representative fluorescence-activated cell sorting plots are shown in the right panel of each figure. All error bars display SD values in the different populations and groups.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It has been debated whether the inflammatory CNS damage in MS is mediated by Th1- or Th17-type CD4+ T cells. The Th1 cells traditionally activate cytotoxic T cells that are capable of direct killing of their target cells. Upon ligation of the T-cell receptor to the major histocompatibility complex antigen plus peptide, these cells rapidly secrete IFN-γ. In contrast, the Th17 cells are part of the proinflammatory responses. Instead of IFN-γ, Th17 cells secrete IL-17 upon T-cell receptor stimulation. It is thought that the Th17 cells exert their main role by stimulation of neutrophil mobilization, thereby placing them at the interface between adaptive and innate immune responses.25,26 Studies have demonstrated the presence of IL-17 in patients with MS 9 and an active role of IL-17 in the pathogenesis of MS has also been suggested.27 Furthermore there are data supporting the possibility that both Th1 cells28 as well as Th17 cells29 can migrate effectively across the blood–brain barrier. Herein we provide evidence for the simultaneous existence of Th1 and Th17 pathways in patients with RRMS because T cells from these patients secrete both IFN-γ and IL-17 when stimulated with specific as well as unspecific stimulators. Patients with the the highest responses to the 15-mer MOG (1–125) mix as well as to the αCD3 antibodies produced both IFN-γ and IL-17 upon stimulation. Also, we did not see any difference in peripheral expression of Th1- or Th17-related cytokines that could distinguish a single Th1- or Th17-type response in patients with RRMS. However, patients with MS had lower levels of the immunosuppressive cytokine IL-10 which may contribute to their inability to maintain tolerance to self-antigens.

Despite the increased cell surface CD28 and the functional IFN-γ or IL-17 production upon antigen (MOG) encounter or polyclonal stimulation, T cells from patients with MS who were in remission were anergic to proliferation. This proliferation block could be broken by the addition of agonistic anti-CD28 antibodies, which is known to potently break anergy in T cells.30,31 Patients in clinical relapse had better proliferative capacity, indicating that the anergy may have been broken in these patients and that the T cells in such individuals are capable of exerting an active immune response. However, in this study only a few patients met our criteria for relapse (within 2–6 weeks of onset) and our results need to be validated in a larger population. Still, these preliminary data may be of interest for the MS community and may initiate studies on the status and role of T-cell subsets in different types and phases of MS. Studies based on long-term monitoring of selected patients with MS would be beneficial for the understanding of T-cell regulation in autoimmune diseases in general but for MS in particular.

Conflicting data have been published on the Treg cell levels in patients with RRMS. Several studies analysed circulating CD4+ CD25high cells and reported both normal13 and decreased levels32 of this population compared to healthy individuals. The addition of Foxp3 as a marker for Treg cells also indicated lower levels.14 However, the latter study detected a higher proportion of Treg cells in the cerebrospinal fluid in RRMS than in progressive MS as well as other neurological diseases. Furthermore, in a study of patients with MS in a state of relapse it was shown that numbers of CD4+ CD25high cells were increased.33 Since Foxp3 was not used, the CD4+ CD25high population may have included activated CD4 T cells. Most studies demonstrating significantly lower levels of Foxp3 in patients with RRMS have been analysed with the antibody clone PCH101, which has been described as unspecific especially when staining activated T cells.34 In the present study we compared the 259D and the PCH101 Foxp3 clones and found that the latter antibody gave a false-positive signal in patients with MS also. Our results confirm a decrease in Treg (CD4+ Foxp3+ CD25+ CD127) cells in patients with MS. Conflicting data in several reports on Tregs in MS may be the result of different phenotypic criteria and of which clone was used for the Foxp3 staining. In the current study, Treg cells have been defined as CD4+ Foxp3+ CD25+/− CD127 cells, a phenotype that has been demonstrated to pinpoint a more exact Treg population.11

Most studies have indicated that the suppressive function of Treg cells in patients with MS may be impaired.13,14,32 Suppressor function may be difficult to determine because purification of CD25+ cells may include activated effector T cells, which may be increased in patients with MS.14 Unfortunately, there is currently no alternative method to sort live Treg cells. In the current study, crude CD25+ T cells from patients with MS were equally good at suppressing proliferating T cells as corresponding cells from healthy controls. However, the majority of the Foxp3-expressing cells lacked CD25 so there is still a population of possible suppressor cells to be evaluated. Since our functional data on Treg cells were in conflict with those from previous studies we validated our findings by two different methods to measure proliferation, with similar results. Multiple sclerosis is a complex disease and there may be different genetic predispositions that cause multiple immunological patterns. Importantly, the criteria for remission–relapse may differ among studies, which may affect the overall conclusions. A current problem is the definition of clinical remission because the time frame of clinical pathology may not be equal to that of inflammation. Since some plaque formations in the CNS are asymptomatic it is clear that inflammatory relapses occur more frequently than clinical manifestations, which further hampers attempts to immunologically separate patients in clinical remission from those in relapse.

Taken together, our findings demonstrate the simultaneous presence of the Th1 and Th17 effector pathways in patients with RRMS. Furthermore, patients with MS have functionally active Treg cells that may keep their T cells in proliferative anergy. Our data indicate that there may be additional mechanisms to the previously reported defects in Treg cells that explain how CNS autoimmunity is regulated during the oscillating phases of remission and relapse.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was sponsored by grants from the Medical Faculty at Uppsala University, the Göransson Sandviken Fund and the Swedish Research Council.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References