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Keywords:

  • Autoimmunity;
  • Myasthenia gravis;
  • Oligoclonal;
  • T cell receptor

Abstract

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

The weakness in myasthenia gravis (MG) is mediated by T helper cell (Th)-dependent autoantibodies against neuromuscular epitopes. So far, analyzing Th phenotypes or antigen specificities has yielded very few clues to pathogenesis. Here we adopt an alternative antigen-independent approach, analyzing T cell receptor (TCR) Vβ usage/expansions in blood from 118 MG patients. We found major expansions (⩾ five standard deviations above the mean of 118 healthy, individually age- and sex-matched controls) in diverse Vβ in 21 patients (17.6%, p<0.001) among CD4+ T cells, and in 45 patients (38.1%, p<0.001) among CD8+ T cells. In informative probands, the expanded CD4+ cells consistently showed a Th cell phenotype (CD57+CXCR5+) and expressed Th1 cytokines. Furthermore, their expression of markers for activation, lymphocyte trafficking and B cell-activating ability persisted for ⩾3 years. Surprisingly, we noted a selective decline in the expansions/their CD57 positivity while the probands’ MG was improving. CDR3 spectratyping suggested mono- or oligoclonal origins, which were confirmed by the prevalent TCR Vβ CDR3 sequences of Th cells cloned from repeat bleeds. Thus, our data provide evidence for persistent clonally expanded CD4+ B helper T cell populations in the blood of MG patients. These unexpected CD4+ expansions might hold valuable clues to MG immunopathogenesis.

Abbreviations:
AChR:

acetylcholine receptor

EOMG:

early-onset myasthenia gravis

LOMG:

late-onset myasthenia gravis

MG:

myasthenia gravis

TCC:

T cell clone

TOMG:

thymoma-associated myasthenia gravis

TCRBV:

T cell receptor β locus variable chain

Introduction

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

Key stages in the pathogenesis of myasthenia gravis (MG) are still poorly understood. The impact of specific IgG autoantibodies against defined epitopes at the neuromuscular junction 15 and their Th dependence in humans and animals are well known 612. There must be a link between the specific T and B cells 13, but it is still completely unclear. Although the pathogenic autoantibodies recognise the native acetylcholine receptor (AChR) and are highly mutated high-affinity IgG, it is still debatable whether the AChR is targeted directly or only as a late result of molecular mimicry and epitope spreading 14. We hoped to elucidate that link by studying TCR usage in MG, and looking for distinctive immunological characteristics in any expanded populations.

Any attempts to identify the Th involved in pathogenesis need to take account of the heterogeneity of patients with generalized MG. They are usually divided into early-onset (EOMG; onset <50 years), thymoma-associated (TOMG) and late-onset MG (LOMG; onset >50 years) groups 15. By definition, these all have autoantibodies against the AChR, but mounting evidence suggests distinct, perhaps partly genetically determined, etiological routes converging on a common final path 14, 15. There are very few clues to its origins, especially in LOMG, which now seems to be the most prevalent 1618.

Many studies in MG have focused on specific T cell responses against known or previously defined AChR epitopes in the thymus or blood. They can be detected in patients with MG 6, 1929, but also in healthy controls 22, 23. Studies using synthetic peptides suggest that the range of relevant epitopes is strikingly broad 2628, though it may be much narrower for epitopes that are naturally processed from whole AChR 8, 28, 29. Perhaps because other muscle or cytokine autoantigens may also be involved in pathogenesis, such as muscle-specific tyrosine kinase 4, titin, ryanodine receptor 30, IFN-α or IL-12 31, studies on autoantigen-specific Th have so far identified few if any dominant epitopes or clinically useful T cell-based specific immunotherapies 28, 32.

We have adopted a wider-ranging approach (to avoid biases from purely qualitative analyses) by testing for expansions of T cell subsets, defined by TCR Vβ usage, in blood from 118 patients with generalized, anti-AChR-positive EOMG, LOMG or TOMG, and 118 individually age- and sex-matched controls. We have analyzed ex vivo TCR Vβ region usage (covering ∼70% of the repertoire) unbiased by specific epitope selection, and confirmed the clonality of selected expansions. TCR Vβ region analyses have been reported in a number of infectious, neoplastic and autoimmune diseases 3339. In MG, however, the phenotypes of the expanded populations have not been checked, nor has their clonality been confirmed by TCR sequencing. We here report a surprisingly high prevalence of CD4+ as well as CD8+ skewings, using Vβ from multiple families. The most expanded CD4+ cells showed Th1 profiles and B helper phenotype, and proved to be of mono- or oligoclonal origin. Moreover, they also declined selectively during follow-up in parallel with the patients’ MG weakness.

Results

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

Increased frequency of TCR skewings in MG patients

The 118 healthy controls were individually matched with the patients for gender (68% female) and age (±2 years). Table 1 shows their demographics and those of the 118 patients in the three main groups with generalized MG 15. In the controls, the means of the CD4+ and CD8+ Vβ subpopulations (Table 2A) are essentially similar to those published previously 38. We arbitrarily designate skewings of ⩾2 SD above the mean of the controls as ‘moderate’, ≥5 SD as ‘major’, and ≥8 SD as ‘exceptional’ expansions. In one particularly striking example in a late-onset male (MG-A2; see also Table 2B, 3 and 4), 45.3% of the blood CD4+ cells were TCR Vβ13.6+ (Fig. 1). This value was >48 SD above the control mean.

Table 1. Clinical characteristics of the 118 MG patients and 118 healthy controls
Early onset (EOMG)a)Late onset (LOMG)b)Thymoma (TOMG)c)all MG patientsHealthy controlsh)
  1. a) Onset up to age 50, no thymoma.

  2. b) Onset age 51 or later, no thymoma.

  3. c) Thymoma, independent of age.

  4. d) MMF: mycophenolate mofetil.

  5. e) Nine with autoimmune thyroid disease, five with rheumatoid arthritis, two with bronchial asthma, two with systemic lupus erythematosus and one each with polymyositis, pernicious anemia or ankylosing spondylitis.

  6. f) Six with autoimmune thyroid disease, two with rheumatoid arthritis and one each with idiopathic thrombocytopenic purpura or scleroderma.

  7. g) Bronchial asthma.

  8. h) 1:1-matched for age and gender.

  9. i) n.a.: not applicable.

n534718118118
Female, n (%)47 (89%)22 (47%)11 (61%)80 (68%)80 (68%)
Age at onset(medians and range in years)28(14–46)62(51–85)39(16–58)42(14–85)n.a.i)
Disease duration(medians and range in years)16(1–49)3(1–22)3.5(1–31)5(1–49)n.a.i)
Age at study entry(medians and range in years)45(20–71)66(53–88)44(17–69)58.5(17–88)58.5(19–86)
Steroids11 (21%)22 (47%)7 (39%)40 (34%)n.a.i)
Azathioprine or MMFd)32 (60%)38 (81%)17 (94%)87 (74%)n.a.i)
Thymectomy36 (68%)11 (23%)18 (100%)65 (55%)n.a.i)
Myasthenic crisis13 (25%)5 (11%)3 (17%)21 (18%)n.a.i)
Associated autoimmuneDiseases21e) (40%)10f) (21%)1g) (6%)32 (27%)n.a.i)
Table 2. TCR Vβ usage indicating normal values from 118 healthy controls (A) and all 21 patients with CD4+ (Th) skewings of ⩾5 SD (B)
A
Healthy controls(n=118)TCR Vβ chain
  1. a) aza: azathioprine; py: pyridostigmine; cs: corticosteroids; thx: thymectomy; n.a.: not available.

1.12.13.15.15.25.36.77.18.19.111.112.213.113.614.116.117.118.120.121.322.123.1
CD4+ (Th)mean (%)3.29.84.36.91.21.04.11.74.75.20.71.93.72.02.61.05.61.32.83.44.10.5
SD (%)0.81.82.11.51.00.31.80.61.02.20.40.50.70.90.60.41.10.61.61.11.10.2
CD8+mean (%)4.76.64.12.90.81.01.73.33.72.60.61.63.21.55.61.15.00.41.82.63.21.6
SD (%)2.63.83.11.70.50.71.92.02.22.10.51.81.81.13.60.92.40.91.51.32.11.5
B
Patientcodecurrenttreatmenta)MGsubgroup, ageNumber of SD above the mean of the controls of expanded TCR Vβ chains
1.12.13.15.15.25.36.77.18.19.111.112.213.113.614.116.117.118.120.121.322.123.1
MG-3aza, pyLOMG, 676
MG-8aza, pyLOMG, 8811
MG-A2aza, pyLOMG, 7448
GP-A16aza, pyEOMG, 6210
GP-A3cs, aza, pyEOMG, 498
GP-A8cs, aza, pyLOMG, 74516
Ox-2thxTOMG, 516
MG-D5pyLOMG, 7613
GP-D4cs, aza, py, thxLOMG, 609
GP-D3aza, pyLOMG, 716
GP-B16cs, aza, pyLOMG, 62810
GP-C3aza, thxTOMG, 6820
GP-C5cs, aza, py, thxEOMG, 415
GP-B24aza, thxTOMG, 605
MG-SIpy, thxLOMG, 779
MG-SCHpyLOMG, 82518
RSS-A15aza, thxEOMG, 547
RSS-B4pyLOMG, 638
FFM-A6n.a.EOMG, 6610
MR-KLPyLOMG, 865
Ox-1ThxTOMG, 2095
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Figure 1. Flow cytometric analysis of a specific Vβ T cell expansion ex vivo. (A) Data from a 74-year-old male MG patient (MG-A2) in whom 45.3% of CD4+ T cells were Vβ13.6+. (B) His complete PBL TCR profile with all 22 Vβ antibodies tested.

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Overall, the MG patients showed a total of 139 such skewings among their CD4+ and 156 among the CD8+ T cells (Fig. 2A, B). By contrast, the controls also showed 46 CD4+ and 75 CD8+ skewings, but these were never exceptional (p<0.0001). Surprisingly, among the CD4+ cells, we found 125 moderate or major skewings in 61 MG patients versus 46 in 41 controls (p<0.001), and 14 exceptional skewings in 14 MG patients versus 0 in the control group (p<0.001). Overall, a total of 15 of the 22 Vβ families showed major or exceptional skewings in 21 patients (Table 2B). The apparent over-representation of Vβ13.1 did not reach statistical significance (X2=2.47; uncorrected p>0.05, not significant).

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Figure 2. Summary of TCR Vβ skewings in MG, shown as the number of SD above the mean of the controls in all four panels. Both CD4+ (A) and CD8+ (B) populations show significantly more expansions in patients than in individually age-matched healthy controls. They show no significant differences between the MG subgroups (C), though they increase slightly with age, as in the controls. (D) shows the CD4+ analysis.

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The ratio of skewings (MG patients versus controls) was higher among CD4+ (3:1) than CD8+ cells (2 :1) (Fig. 2A, B). Analysis for confounding factors (Fig. 2C, D) did not show a correlation between the number of Th skewings, or multiple intra-individual skewings, and clinical subgroup, immunosuppressive treatment, or thymectomy; ∼30% of the patients with major or exceptional skewings had not received any immunosuppressive drug treatment (Table 2). We next checked for correlations with age. Whereas there are several reports of CD8+ skewings among healthy individuals 38, 40, 41, particularly in the elderly, they are highly unusual among CD4+ T cells but seem particularly relevant in MG in view of its Th-dependent antibody-mediated pathogenesis. In the present series, expansions were already evident before age 50 in some MG patients, but they were more prominent at higher ages. In addition, their extent also correlated weakly with age (Fig. 2D; r=0.28, r2=0.08; p<0.001), as in the controls (Fig. 2D; r=0.38, r2=0.14; p<0.009, not significant after Bonferroni correction).

Serial studies in patients with exceptional expansions

To check for their persistence, phenotype and clonality, we next focused on five of the 14 MG patients with the most readily identifiable exceptional expansions (bold in Table 2), re-bleeding them annually for 3 years.

Th1 profiles of exceptionally Vβ-skewed CD4+ T cells

The IFN-γ expression in the expanded Th cells indicates a pronounced Th1 bias relative to the same patient's non-expanded Th Vβ subsets combined (“B” in Table 3; Fig. 3). There was a similar but weaker trend for TNF-α, but it was in the opposite direction for IL-4, and was more variable for IL-2 (Table 3). Interestingly, the four Th expansions that proved to be clonal (Table 35) showed more extreme IFN-γ/IL-4 ratios than the polyclonally expanded Vβ22.1+ cells in patient MG-SI.

Table 3. Ex vivo flow cytometric cytokine analysis of expanded CD4+ cellsa)
PatientcodeTCR VβExpansion(%)Controls mean ± SD (%)Th subsetb)IFN-γ(%)TNF-α(%)IL-2(%)IL-4(%)RatioIFN-γ/IL-4
  1. a) Note that the IFN-γ/IL-4 ratios show a clear Th1 polarization of the skewed Th subpopulations.

  2. b)A: % of TCR Vβx expanded Th cells expressing the indicated cytokine; B: % of the other Th cells (i.e. expressing other TCR Vβ) expressing the indicated cytokine.

MG-A213.645.32.0±0.9A74.993.112.51.744.1
all other VβB12.483.268.54.13.0
MG-D520.123.62.8±1.6A64.469.040.00.792
all other VβB16.638.522.30.627.7
MG-SCH13.116.83.7±0.7A49.555.33.61.827.5
all other VβB27.250.834.63.09.1
MG-812.27.61.9±0.5A79.487.352.32.334.5
all other VβB25.481.431.76.24.1
MG-SI22.114.74.1±1.1A38.844.816.98.44.6
all other VβB22.439.529.911.61.9
A/B (%)mean ± SD332±179125±3186±8165±326±5
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Figure 3. Ex vivo intracellular flow cytometric detection of cytokines in the expanded Th cells of patient MG-A2. The cells displayed a clear Th1 profile (see also Table 2).

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Table 4. Prevalence of CD4+ expansions among T cells randomly cloned from index patientsa)
Patient codeHLAclass IITCR VβxEx vivoexpansion(%)Total number of cell clones(n)TCC CD4+(n)TCC Vβx(n)TCC Vβx(%)
  1. a) Shown are the total and relative numbers of TCC after single-cell cloning by limiting dilution. The relative proportions of Vβx-specific expansions are mirrored in the numbers of Vβx-specific TCC except for MG-A2. Given are also these patients’ HLA class II alleles (nomenclature according to 74). For HLA-DRB1 and HLA-DQB1 both alleles are listed.

MG-A2

DRB1*09,*11

DRB3, DRB4

DQB1*03,*03

13.645.323014164.3
MG-D5

DRB1*16,*03

DRB3, DRB5

DQB1*05,*02

20.121.62421242318.5
MG-SCH

DRB1*15,*07

DRB4, DRB5

DQB1*06,*02

13.115.8196911213.2
MG-8

DRB1*13,*04

DRB3, DRB4

DQB1*06,*03

12.26.03398144.9
MG-SI

DRB1*04,*01

DRB4

DQB1*05,*03

22.112.74641131210.6
Table 5. Clonal origin of expanded CD4+ cells, evident in Vβ sequences of T cells cloned randomly from index patientsa)
Patientn/nTCR Vβ (mAb)TRBV(gene)TRBV(sequence)NDN(sequence)TRBJ(gene)TRBJ(sequence)
  1. a) The sequences highlighted in grey were identical in CDR3 length whether they derived from TCC or CDR3 spectratype peaks ex vivo. Note the strong similarity of NDN in MG-SCH, suggesting a positively selected antigen-binding motif. (…) indicates identical sequence as in the line above.

MG-A26/613.66–6LAAPSQTSVYFCASTPTGG1–1NTEAFFGQGTRLTVV
MG-D512/142030LLLSDSGFYLCAGTGDI1–1TEAFFGQGTRLTVV
2/1430(…)SGN1–5SNQPQHFGDGTRLSIL
MG-SCH5/2113.16–5SAAPSQTSVYFCASNLQG1–1STEAFFGQGTRLTVV
4/216–5(...)SYQG1–5NQPQHFGDGTRLSIL
6/216–5(...)SYSG??
1/216–5(...)SGQLI1–2GYTFGSGTRLTVV
1/216–5(...)SGRSQ2–7GYEQYFGPGTRLSVL
1/216–5(...)STGQL1–1NTEAFFGQGTRLTVV
1/216–5(...)SSKPLA2–4TKNIQYFGAGTRLSVL
1/216–5(...)SGADSG1–2NYGYTFGSGTRLTVV
1/216–5(...)SYSRG2–7TYEQYFGPGTRLSVL
MG-84/81210–3ATSSQTSVYFCAITRQG2–2GTGELFFGEGSRLTVL
1/810–3(...)ISGVD1–6NSPLHFGNGTRLTVT
1/810–3(...)NGWG2–3STDTQYFGPGTRLTVL
1/810–3(...)ISG2–3(…)
1/810–3(...)TRGAD1–1TEAFFGQGTRLTVV
Immunophenotypes of Vβ CD4+ expansions and their stability

We focused on markers for activation, lymphocyte trafficking and B cell-activating ability (Fig. 4, 5). The expansions showed largely homogeneous labelling for CD49d, CD49e (VLA integrin α chains), CD95 (Fas), CD11a (LFA-1) and CD40L (Fig. 4A), even when polyclonal (as in MG-SI). By contrast, other activation markers varied much more, i.e. their ‘memory’ (CD45RACD45RO+CD28+) versus ‘naive’ (CD45RA+ CD45ROCD28+) or ‘effector’ (CD45RA+CD45RO CD28) phenotypes, but also CD57. None of the expanded cell populations expressed CD25 (not shown). Notably, these patterns were each stable in every sample for up to 3 years (Fig. 5). Surprisingly, among the expanded Th cells positive for CD45RA, a higher proportion co-expressed CD62L in three non-thymectomized LOMG cases (MG-A2, MG-SCH, MG-D5) (not shown), a phenotype typical of recent thymic emigrants 42, though not uniquely so.

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Figure 4. Immunophenotype of the expanded TCR Vβx+ CD4+ Th cells (A, C; solid bars) in the five index patients; also the prevalence of the indicated markers in the non-expanded T cells among the 21 patients with expansions ⩾5 SD above the mean of the controls (A, C; hatched bars). Note the relatively high SD of CD45RA, CD45RO, CD28, CD57 and CD62L among the expanded Th populations, suggesting considerable heterogeneity; also the overall CD57+CXCR5+ phenotype of expanded Th cells (B; right) in comparison to all other Vβ (B; left) in the five index patients sampled at month 36, the same trend being evident in the eight available patients with moderate expansions (C).

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Figure 5. Persistent individual immunophenotypes of expanded CD4+ T cells over 3 years. (A-H) Shown are the FACS-derived frequencies of Vβx+ Th cells expressing the indicated CD molecule in the index patients.

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Markers of B helper T cells

These five index exceptional CD4+ expansions showed a largely CD57+CXCR5+ phenotype (Fig. 4B); it was evident in 71.5±15.5% of the expanded Th versus only 5.4±2.2% of the non-expanded CD4+ cells (p<0.008). Moreover, we observed a similar phenotype in early samples of eight other available but less extreme (major) expansions (p<0.012; Fig. 4C). These CD4+CD57+ CXCR5+ T cells reportedly correspond to light-zone-specific T cells in the lymph node follicles, which provide the help required to activate B cells via CD40L/CD40 to differentiate into memory or antibody-producing plasma cells 43.

Clonality of expanded CD4+ Vβ populations

We checked the clonality of the respective expansions qualitatively in further PBL samples from the five index patients, by ex vivo TCR CDR3 spectratyping. CD4+ and CD8+ cells were separated from fresh PBL samples by MACS. Subsequent CDR3 length analyses revealed clear-cut differences between CD4+ and CD8+ cells in four of the five patients (Fig. 6), their CD4+ fractions each showing a single dominant CDR3 peak of 355–479 bp, suggesting a mono- or oligoclonal origin, whereas the CD8+ cells showed peaks with diverse lengths.

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Figure 6. Clonality of the expanded CD4+ T cells, as shown by TCR CDR3 spectratyping of Th cells ex vivo and Vβ sequences from cloned Vβx CD4+ cells from the same donor. As an internal control, the identical Vβx subsets of the CD8+ T cells show diverse CDR3 lengths. By contrast, the CD4+ cell spectratypes provide clear evidence for mono- (or oligo-) clonal origins of the respective expansions. Furthermore, the identity between CDR3 lengths derived from PBL and from cDNA sequencing from TCC strongly suggests that the clones were derived from the same expanded T cells. The filled peaks represent the CDR3 PCR products; the open graphs are the length standards. n.d., not determined.

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We also cloned T cells randomly from these patients’ PBL by limiting dilution 44, and obtained an average of 252±61 clones of which 110±28 were CD4+ (see Table 4). In the same four patients, the percentage of the resulting CD4+ T cell clones (TCC) with the respective expanded Vβ correlated well with the frequency of the original expansion (see Table 4). Patient MG-A2 (see also Fig. 1) was an interesting exception: his (extremly) expanded population did not express CD28, which probably prevented its stimulation in vitro by the CD3/CD28 expander beads. In the other four patients, nucleotide sequencing (and translation) of CDR3 regions from a total of 49 TCC-derived cDNA clones showed predominant (or co-dominant in MG-SCH) clonal sequences (Table 5) whose CDR3 lengths and sequences corresponded exactly to those identified by spectratyping in the same patient. For example, 12 of 14 CD4+ Vβ20 clones from MG-D5 had the same CDR3 sequence and used the TRBJ 1-1 joining region (Table 5). In patient MG-SCH, the NDN sequences appear similar in 15 of 21 TRBV 6-5 clones (despite belonging to three distinct families), suggesting a conserved antigen-binding motif.

Clinical correlations during follow-up

Despite their overall phenotypic stability (Fig. 5), the expanded Th cells showed a selective, about twofold decline in both their frequencies and their CD57 positivity during 3-year follow-up in three patients, which occurred while the MG weakness was also decreasing (spontaneously in two cases). There was no substantial phenotypic change in the fourth, whose MG had deteriorated slightly by month 36 (Fig. 7).

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Figure 7. Changes (% of initial value) selectively in the frequencies of the expanded CD4/Vβx-double-positive out of total CD4+ cells, in the CD57 positivity of the expansions and in the MG severity in the four patients (A-D) with proven (oligo)clonal expansions during 3-year follow-up. The MG severity was assessed by a single neurologist using the scale of Besinger et al.71. NIT: no immunosuppressive treatment; IAT: initiation of azathioprine treatment (2 months before first bleed), MC: myasthenic crisis. NC: MG-A2 had already taken azathioprine for >2 years when first sampled, and the dose did not change.

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Discussion

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

In MG, the characterization of disease-relevant T cells has long been a subject of debate. In particular, T cell clonality has not been a major focus in the past. In this study, we document exceptional clonal expansions in CD4+ T cells using a variety of TCR Vβ chains in a substantial proportion of MG patients. Their prominence and prevalence here were quite unexpected because, in general, such expansions are much rarer among CD4+ than CD8+ T cells, as also are antigen-specific Th cells 45.

Indeed, expansions have rarely been noted before in other autoimmune diseases 38, 4649. Their phenotypic similarity, whether they were exceptional or major, strongly implies that these expansions represent the ‘tip of an iceberg’, i.e. they may be more widespread in other MG patients but at lower frequencies that do not reach statistical significance 39. Interestingly, the great majority of the expanded Th cells consistently showed a similar CD4+CD57+CXCR5+ helper phenotype, and were negative for CD25 – totally unlike regulatory T cells. Importantly, their expression of almost all the markers tested was stable for up to 3 years, but their frequency and CD57 positivity declined in parallel with clinical improvement in three index patients. These findings must hold novel clues to pathogenesis, clues that are otherwise particularly scarce in LOMG, an increasing majority of MG patients.

To date, the most thorough flow cytometric or PCR-based studies on TCR usage in MG 10, 34, 35 either did not check for long-term persistence or clonality of expansions, or reported biases in usage of certain Vβ (in Japanese patients) that did not reach significance 39. By contrast, in three times as many patients and controls, and with twice as many Vβ antibodies, we found expansions in 15 of the 22 Vβ, and we confirmed their clonality and 3-year persistence in a quarter of the patients with exceptional expansions. The apparent Vβ13.1 preference is probably a chance finding. Collectively, our data argue against the recurring use of particular Vβ in different MG patients, or even of conserved CDR3, which is sometimes seen in virus-specific CD8+ T cells after infections 50. Nevertheless, they might also be provoking the anti-TCR Vβ antibodies that have been reported in MG patients 51.

Interestingly, overexpansions of CD4+ T cells were uncommon among our thymoma patients. On the other hand, there is strong evidence that thymomas export some of the T cells they generate, and that some of the emigrated Th cells subsequently initiate autoimmune responses in the periphery 5254. The rarity of expansions in thymoma patients suggests that frequencies of such pathogenic Th cells must be low. Moreover, the expansions in some non-thymectomized patients included a surprisingly high proportion of apparently naive CD45RA+CD62L+ CD4+ T cells. That might hint at a recent thymic origin 42 and/or even at prior rejection of an occult thymoma, which could explain the very similar anti-titin, anti-RyR and anti-IFN autoantibodies in most TOMG and some LOMG patients (discussed in 55).

In our large panel of Middle European MG patients, approximately one in five had major or exceptional CD4+ expansions. These were frequent also in the CD8+ cells, where expansions are well known, especially in elderly humans 40, 41, in whom minor CD4+ T cell clones have also been found among the apparently polyclonal CD45RO+ memory cells 41. In MG, by contrast, their evident clonality (Table 5; Fig. 6) in the most expanded CD4+ cell populations, their long-term phenotypic stability (over at least 3 years), their overall B helper T cell phenotype (CD4+CD57+CXCR5+), and their occurrence in all three patient subgroups, without any obvious influence of immunosuppressive treatment, might implicate them in MG pathogenesis (see below). However, chronic viral infection (especially CMV) can lead to clonal expansions, too, though with a different (CD28) phenotype 56. We therefore checked for CMV serostatus (data not shown); some of the major as well as exceptional expansions were clearly in CMV-negative subjects (B. Tackenberg et al., in preparation).

All our five index patients’ expansions showed a Th1-polarized cytokine pattern. Interestingly, in MG, autoantibody responses are believed to be Th1 cytokine-dependent 12, 57, 58. Moreover, the expanded cells are in principle capable of interacting with B cells via their CXCR5 and/or CD40L. They all also express CD95, another activation marker 59, but are heterogeneous in their CD28 and CD62L expression. As discussed below, we suspect they are a sign of some – possibly microbial – stimulus to oligoclonal Th1 responses that may both initiate epitope spreading and create a pro-inflammatory Th1 ‘climate’.

In animals, IFN-γ is necessary for development of antibody-mediated experimental MG 58, 60, and our AChR-specific MG patient's Th clones produced Th1 or Th0 cytokines 61, 62. Remarkably, CD57 and CXCR5 were co-expressed on 71.5–48.5% of the exceptional or major Th expansions versus only 5.4–8.2% of the unexpanded Th cells. Such co-expression is typical of follicular T cells in the light zones of lymph nodes 43. Indeed, CXCR5 is critically involved in follicular localization of Th cells 63. Th2, but probably also Th1, T cells of this (CD40L+) phenotype usually provide help to (CD40+) germinal center B cells 43, favouring their differentiation into memory or plasma cells 64 by inducing activation-induced cytidine deaminase expression, Ig class switching and antibody production 43. While recent reports suggest that the CD57 on this Th subset is not essential for their B helper activity, this function is nevertheless restricted to CD57+CXCR5+ Th cells (which are also ICOS+) 43, 6466 Obviously, their counterparts in humans are less easy to define: if they are, indeed, providing help for B cells, their specificity demands further investigation, e.g. with peptide libraries 67, 68.

The clonally expanded Th cells in MG could be playing two possible roles: (1) acting as agents provocateurs by reacting to mimetic microbial antigens; (2) directly targeting the AChR. While one might expect role (2) to have been noted previously, (1) could easily have been overlooked. The high affinity of specific AChR autoantibodies in MG, of course, implies that the human AChR is the ultimate autoantigenic target 14, 69. However, molecular mimicry could be crucial at much earlier inductive stages. Indeed, based on findings in rabbits, Vincent et al.14, 70 proposed an alternative two-step scenario: in the first step, thymic or peripheral presentation of microbial antigens (e.g. CMV or herpes simplex virus) leads to the production of low-affinity AChR-specific antibodies. In the second, these attack muscle endplates, or AChR+ thymic myoid cells 13, and initiate epitope spreading that culminates in pathogenic autoantibodies against the native AChR. Importantly, such agents provocateurs need not necessarily show clear anti-AChR reactivity. We suggest that the gradual disappearance of the mimetic provoking factor(s) could account for both the decline in the expansions during follow-up and the parallel amelioration of the MG, an unexpected clue that demands further pursuit.

MG encompasses heterogeneous immunological responses. The extensively and persistently expanded clonal T cell populations reported here now offer excellent opportunities for further analyses of T cell function and antigen specificity in individual patients, without pre-judging their specificity – which can be hard to define. Indeed, one strength of our study is that the expanded T cells can be cloned directly ex vivo, which opens up new possibilities for defining their specificity, such as use of peptide libraries 67, 68, probably the most advanced method for unassigned T cell clones. Furthermore, future studies on their disposition in the thymus or elsewhere may elucidate their puzzling origins and induction.

Materials and methods

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

MG patients and healthy controls

Between 2000 and 2003, we recruited 118 adult patients consecutively from the authors’ myasthenia clinics and with the help of the German Myasthenia Society (DMG; www.dmg-online.de). They all had clinical, electrophysiological and serological evidence of MG. Only patients with generalized MG and positive titres of AChR antibodies were included. A history of cancer, acute inflammation within the preceding 6 wk or withdrawal of consent led to exclusion from the study. Thymoma was confirmed histologically after surgery in 18 patients. The non-thymectomized EOMG or LOMG cases showed no evidence of retro-sternal masses in computed tomography of the chest. The patients’ clinical data and treatments are summarized in Table 1; they were matched individually with healthy controls (118 medical staff, patients’ spouses) for gender and age (to within 2 years).

For serial analyses, patients with exceptional TCR Vβ expansions were re-bled prospectively once a year for 3 years. Their clinical severities were rated by a single neurologist using a well-established scale 71 of muscle strength tested in the arms and legs, the neck, the face, and in chewing, swallowing and respiration. Scores range from 0 (asymptomatic) to 21 (severe myasthenia/crisis). All patients and controls gave written informed consent. The study was approved by the local Ethics Committee according to the Helsinki declaration.

T cell surface antibody panel

For specific TCR Vβ staining and additional ex vivo immunophenotyping of the expanded CD4+ or CD8+ T cells, we used a panel of mAb that cover 70% of the human TCR Vβ repertoire 56: anti-Vβ1-FITC (clone BL37.2), anti-Vβ2-PE (clone MPB2D5), anti-Vβ3-PE (clone CH92), anti-Vβ5.1-FITC (clone IMMU157), anti-Vβ5.2-FITC (clone 36213), anti-Vβ5.3-PE (clone 3D11), anti-Vβ6.7-FITC (clone OT145), anti-Vβ7-PE (clone ZOE), anti-Vβ8-FITC (clone 56C5.2), anti-Vβ9-PE (clone FIN9), anti-Vβ11-FITC (clone C21), anti-Vβ12-PE (clone VER2.32.1), anti-Vβ13.1-PE (clone IMMU222), anti-Vβ13.6-FITC (clone JU74.3), anti-Vβ14-PE (clone CAS1.1.3), anti-Vβ16-FITC (clone TAMAYA1.2), anti-Vβ17-FITC (clone E17.5F3.15.13), anti-Vβ18-PE (clone BA62.6), anti-Vβ20-PE (clone ELL1.4), anti-Vβ21.3-FITC (clone IG125), anti-Vβ22-FITC (clone IMMU546), anti-Vβ23-PE (clone AF23), anti-CD45RO-FITC (clone UCHL1), anti-CD27-FITC (clone M-T271), anti-CD28-FITC (clone JJ319), anti-CD49d-FITC (clone HP2/1), anti-CD49e-FITC (clone SAM1), anti-CD54-FITC (clone 84H10), anti-CD57-FITC (clone NC1), all from Immunotech, now Beckman Coulter GmbH-Diagnostics (Krefeld, Germany), and anti-CD4-allophycocyanin (clone RPA-T4), anti-CD8-PerCP (clone SK2), anti-CD45RA-FITC (clone ALB11), anti-CD62L-FITC (clone Dreg56), anti-CD95-FITC (clone DX2), anti-CD11a-FITC (clone HI111), anti-CD154-FITC (clone TRAP1), anti-CD69-FITC (clone FN50), anti-CD25-FITC (clone M-A251) and anti-CXCR5-Alexafluor (clone RF8B2) from BD Pharmingen (Heidelberg, Germany). We tested isotype control IgG on PBL plus anti-IgG-FITC (BD Pharmingen)

Staining and four-colour flow cytometry

Using published methods 72, we stained whole EDTA-anticoagulated blood cells diluted 1:1 with ice-cold PBS, in round-bottomed 96-well plates (Nunc, Roskilde, Denmark). The cells were incubated with mAb combinations for 30 min on ice, before lysing erythrocytes twice with PharMingen Lyse (BD Pharmingen), washing twice with PBS/2.5% FCS, transfer to 5 mL Falcon tubes (BD Pharmingen) and analysis with a four-colour flow cytometer (FACSCalibur®; BD Pharmingen). Based on the white blood cell SSC/FSC height scatter, at least 5000 events were acquired in the lymphocyte gate, using BD CellQuest® software.

Intracellular cytokine analysis

The ex vivo cytokine profiles were assessed by intracellular staining and flow cytometry. PBL were isolated from patients with TCR Vβ expansions of Th cells (on Ficoll, 1077 density; Biochrom, Berlin, Germany), and activated non-specifically with phorbol 12-myristate 13-acetate (5 ng/mL; Sigma-Aldrich, Taufkirchen, Germany) and ionomycin (500 ng/mL; Sigma-Aldrich) for 6 h at 37°C in 5% CO2, before staining as above. Intracellular cytokines were detected with anti-IL-2-FITC (clone MQ1-17H12), anti-TNF-α-FITC (clone MAb11), anti-IFN-γ-FITC (clone 4S.B3) and anti-IL-4-FITC (clone MP4-25D2), using brefeldin A to prevent secretion (all from BD Pharmingen, according to the manufacturer's instructions). Isotype controls were performed routinely. At least 10 000 stained cells were analyzed with a four-colour flow cytometer, as above.

Ex vivo magnetic-activated cell sorting and CDR3 spectratyping

For molecular analysis of expanded CD4+ T cells, freshly isolated PBL were separated into CD3+CD4+ and CD3+CD8+ populations by MACS sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. If the purity was >98%, we then isolated and reverse-transcribed cytosolic RNA from these T cells (RNeasy Mini; Qiagen, Hilden, Germany) before PCR using the respective primers for each human TCR β gene locus variable chain (TCRBV; according to the Lefranc nomenclature, http://imgt.cnusc.fr) and a fluorescent-tagged reverse primer 73. We used a PTC-200 Thermal Cycler (Biozym, Hessisch Oldendorf, Germany) with 31 cycles (initial melting 3 min at 94°C; denaturation 30 s at 94°C; annealing 30 s at 56°C; extension 45 s at 72°C). After visualization of the PCR product on a 2% agarose gel, CDR3 length polymorphism was detected on an ABI Prism 310 Genetic Analyser (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions, and compared with that of the TCC to identify the expanded clonal population.

Single-cell cloning and expansion in long-term cell culture

We raised TCC by limiting dilution according to established methods 44 but stimulating with Dynabeads CD3/CD28 T cell expander (107 beads/mL; Dynal, Oslo, Norway) in RPMI 1640 with 2.5% pooled human inactivated serum, 1% penicillin/streptomycin, 1% L-glutamate (all from Gibco, Karlsruhe, Germany), 1% non-essential amino acids, 1% sodium pyruvate and 1.125% HEPES buffer (all from Sigma-Aldrich). We cultured a mixture of 1000, 3000 or 10 000 PBL plus 50 000 irradiated allogeneic PBL in round-bottomed 96-well plates at 37°C in 5% CO2. Every 7 days, 5 IU per well recombinant human IL-2 (Diaclone, Besançon, France) was added starting at day 2 of culture. After 2-3 wk, visibly growing clones bearing the expanded TCR Vβ chain were identified by qualitative flow cytometry. Cultures >99% positive for the respective TCR Vβ chain were expanded by further CD3/CD28 bead stimulation.

TCRBV cDNA and amino acid sequencing of T cell clones

To determine their sequences, we amplified the expanded TCR Vβ chains, as above, purified PCR products of the same CDR3 length by the QIAquick PCR purification kit (Qiagen) and sequenced them on an ABI Prism 310 Genetic Analyzer using a DNA sequencing kit (both from PE Biosystems, Foster City, CA). Nucleotide sequences were translated into amino acids using Chromas 2.3® software and analyzed according to the Lefranc nomenclature for human TCRBV.

Biostatistical methods

For detecting categorical differences between MG patients and healthy controls, we used the X2-test. We analyzed for confounding effects of age at sampling time, disease duration, gender, immunosuppressive drug treatment, thymectomy, and clinical MG subtype using a two-sided Mann–Whitney test and Spearman's rank correlation coefficient. For all our statistical comparisons, we used SPSS 12.0® software, two-sided tests and a 5% significance level. To correct for multiple testing of 34 hypotheses, the conservative Bonferroni correction was 0.05/34=0.0015. Consequently, p<0.0015 was considered to be significant and 0.05>p>0.0015 as statistical trend.

Acknowledgements

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

This work was supported by the German Myasthenia Society (Deutsche Myasthenie Gesellschaft, DMG e.V.; www.dmg-online.de), the Deutsche Forschungsgemeinschaft (DFG; www.dfg.de), and the Sir Jules Thorn Charitable Trust (www.julesthorntrust.org.uk). We thank Michael Happel and Annette Hehenkamp for organisational and technical help, and the referees for their constructive suggestions.

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