1. Introduction {#sec1}
===============

The human nicotinic acetylcholine receptor (AChR) composed of five subunits (2 *α*, 1 *β*, 1 *δ*, and either 1 *γ* or 1 *ε* subunit) is well characterized as the target antigen in myasthenia gravis (MG) \[[@B1]\]. The *γ* subunit of the fetal receptor is replaced by an *ε* subunit in adult muscle; both subunits share about 53% homology at the amino acid level \[[@B2]\]. Pathogenic antibodies are predominantly directed against the *α* subunit of the AChR. Both antibody responses as well as B-lymphocyte activity have been investigated extensively in MG and are of great diagnostic and prognostic value.

Immunoglobulin G (IgG) autoantibody production is T helper cell-dependent. Although MG is considered a prototypic paradigm for an antibody-driven autoimmune disorder, the pathogenetic importance of T-helper cells is well appreciated. Several studies have been performed comparing T-cell responses involving the *α* subunit versus the developmentally regulated *ε* subunit using recombinant fragments and purified polypeptides of the human AChR \[[@B2]--[@B8]\]. The *ε* subunit is of particular interest as its expression in the adult muscle differs from the fetal *γ* subunit, a fact that may contribute to the escape of clonal deletion and the development of autoreactive T lymphocytes in MG, especially in the myasthenic thymus \[[@B9]\]. In accordance with this hypothesis, two reports describe that, in comparison to healthy subjects, only MG patients responded to synthetic peptides of the *ε* subunit by T-cell proliferation \[[@B2], [@B10]\]. Consistently, in MG patients with thymomas the *ε* subunit is preferentially expressed \[[@B11]\].

We used an enzyme-linked immunospot (ELISPOT) assay to determine T-cell responses against recombinant fragments and synthetic peptides of the human AChR. In accordance with previous observations we found prominent T-cell responses against the *ε* subunit while no significant differences were notable against alpha subunit epitopes.

2. Patients and Methods {#sec2}
=======================

2.1. Patients and Controls {#sec2.1}
--------------------------

Peripheral blood lymphocytes (PBL) were obtained with informed consent from patients with generalized or ocular MG (n=15) or healthy donors (HD, n=9). PBL were isolated by density centrifugation and were either frozen immediately and thawed for analysis or used directly.

2.2. Synthesis of Recombinant Fragments {#sec2.2}
---------------------------------------

Human *α* and *ε* subunit polypeptides were synthesized by PCR on cDNA prepared from total RNA of human calf muscle as described elsewhere \[[@B12]\]. Recombinant protein fragments were kindly provided by Wolfgang Wienhold and Arthur Melms \[[@B13]\]. Fragments were expressed in*E. coli* and purified by SDS/PAGE with a standard protocol \[[@B5], [@B12]\]. Alpha 1-103 and Alpha 1-209 are fragments of the extracellular domain, Alpha 327-298 of the intracellular domain of the *α* subunit \[[@B1]\]. Epsilon 1-221 is a fragment of the extracellular domain of the *ε* subunit.

2.3. Synthesis of Peptides {#sec2.3}
--------------------------

Peptides were synthesized by solid-phase Fmoc-chemistry on an automated peptide synthesizer for multiple peptide synthesis as described previously \[[@B14]\]. Epsilon 116-130, IDGQFGVAYDANVLV, is an HLA-DR3-binding peptide. Epsilon 201-215, ENGEWAIDFCPGVIR, contains a dominant epitope restricted by HLA-DR52a \[[@B7]\]. Epsilon 236-250, IRRKPLFYVINIIVP, contains a dominant T-cell epitope that is not HLA-DR3-restricted. As a specificity control peptide, we used the class II-associated invariant chain peptides, CLIP 97-120, LPKPPKPVSKMRMATPLLMQALPM, and CLIP 105-117, SKMRMATPLLMQA. The tetanus toxoid peptide TT 1272-1284 is a promiscuitive HLA-DR3/DR52a binder.

2.4. ELISPOT Assay {#sec2.4}
------------------

We measured frequencies of interferon (IFN)-*γ*-secreting T-cells using an ELISPOT (enzyme-linked immunospot) assay as described previously \[[@B15]\]. Microtiter filter plates (Millipore) were coated overnight with an anti-human IFN-*γ* monoclonal antibody (mAb) (clone 1-D1K; 10 *μ*g/ml, Mabtech, Sweden). After washing and blocking the plates with culture medium (RPMI 1640 supplemented with 5% fetal bovine serum and antibiotics, all from Gibco), fresh or freshly thawed PBL from MG patients and HD were incubated for 20 h in duplicate in the presence or absence of human AChR antigens or controls (1 *μ*g/ml). Concanavalin A (5 *μ*g/ml; Sigma) was used as positive control. Using biotinylated anti-human IFN-*γ* mAb (clone 7-B61; Mabtech), streptavidin-alkaline phosphatase (Mabtech), and BCIP/NBT as substrate (Sigma), antigen-specific IFN-*γ* secreting T lymphocytes were visualized and counted on a dissecting microscope. Results are calculated and assigned as the numbers of IFN-*γ*-secreting events among 10^6^ PBL minus the corresponding numbers of events per 10^6^ PBL without antigen.

2.5. Statistical Analysis {#sec2.5}
-------------------------

Student\'s*t*-test was performed for statistical analysis. A p value of \< 0.05 was accepted to be significant.

3. Results and Discussion {#sec3}
=========================

The T-cell responses were heterogeneous throughout MG patients. While single individuals did not show any detectable IFN-*γ* secreting T-cells after stimulation with AChR fragments ([Table 1](#tab1){ref-type="table"}), others exhibited marked responses (Figures [1](#fig1){ref-type="fig"} and [2](#fig2){ref-type="fig"}). The responses did not correlate with the AChR-antibody status. For example, patient MG-8 was AChR-antibody negative but exhibited a T-cell response to the AChR protein fragments. Accordingly, some HD presumably AChR-antibody negative gave positive T-cell responses. Analyzing the mean responses of all donors, the mean of detectable (positive) responses only, or the median positive responses only, recombinant fragments of both the *α* and the *ε* subunit induced a higher T-cell response in MG than in HD ([Table 1](#tab1){ref-type="table"}). However, statistical analysis could only determine a trend and not statistical significance.

The fragment Epsilon 1-221 showed the highest response. Synthetic peptides of the *ε* subunit induced a lower response. The most remarkable difference between MG and HD was observed with Epsilon 201-215 containing a dominant T-cell epitope ([Table 1](#tab1){ref-type="table"}) \[[@B7]\]. Consistent with this finding, Ragheb and colleagues have demonstrated proliferative T-cell responses upon stimulation with synthetic peptides of the *ε* subunit in up to 15% of MG patients including Epsilon 194-209 \[[@B2]\].

Correlations between AChR-specific T-cell responses and paraclinical data (sex, age, or anti-AChR antibody serum titer) were not observed (see [Table 1](#tab1){ref-type="table"}). In an animal model of MG, experimental autoimmune myasthenia gravis (EAMG), Gaertner et al. investigated the pathogenicity of T-cell determinants of the *ε* subunit \[[@B8]\]. Although IFN-*γ* secretion by T-cells reactive to *ε* subunit peptides was observed, these cells failed to induce EAMG upon transfer. Hence, these T lymphocytes were demonstrated to be nonpathogenic. It remains to be determined if this observation reflects a peculiarity of the EAMG model or if nonpathogenic, *ε* subunit-specific T lymphocytes are present in MG patients. If so, it is speculated that they may contribute to the integrity of the neuromuscular junction \[[@B8]\].

We conclude that the IFN-*γ* ELISPOT method may provide a valuable tool to measure AChR-specific T-cell responses in MG. In MG patients who tested positive, T lymphocytes specific for epitopes of the AChR *ε* subunit may be a target for therapeutical intervention in MG.

We thank Bettina Heiling for excellent technical assistance. We thank Wolfgang Wienhold and Arthur Melms for providing reagents.

Data Availability
=================

Data supporting the results of this study can be provided by the corresponding author.

Conflicts of Interest
=====================

The authors declare that they have no conflicts of interest.

![Frequency of IFN-*γ*-secreting events per 10^6^ PBL in single donors. ELISPOT assay was performed as described in the text. Results are assigned as the numbers of IFN-*γ*-secreting events among 10^6^ PBL minus the corresponding numbers of events per 10^6^ PBL without antigen. Negative results (spot number without antigen exceeding spot number with antigen) were defined zero. Grey bars, mean response ± SD; black bars, mean positive response ± SD; crosses, median positive response; MG, myasthenia gravis patients; HD, healthy donors. Note the high standard deviations due to heterogeneous responses of single individuals (see [Table 1](#tab1){ref-type="table"}). Note the different Y axis scales using four different recombinant fragments. (a) fragment Alpha 1-103; (b) Alpha 1-209; (c) Alpha 327-398; (d) Epsilon 1-221.](NRI2019-1969068.001){#fig1}

![(a) Mean response, (b) mean positive response, and (c) median response of the numbers of IFN-*γ*-secreting events among 10^6^ PBL minus the corresponding numbers of events per 10^6^ PBL without antigen. Negative results (spot number without antigen exceeding spot number with antigen) were defined zero. MG, myasthenia gravis patients; HD, healthy donors.](NRI2019-1969068.002){#fig2}

  --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Group                      Donor   Sex  \     Age  \     AChR-Ab  \               Spots per 10^6^ PBL                                                                                              
                                     (f/m)      (years)    (nmol/l)                                                                                                                                  
  -------------------------- ------- ---------- ---------- ------------ ----------- --------------------- ----------- ----------- ----------- ---------- ---------- ---------- ---------- ---------- -----------
  MG                         MG-1    f          70         135.6        1           0                     0           0           0           n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             MG-2    f          62         0.5          11          0                     0           0           0           n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             MG-3    m          48         10.2         2           160                   560         416         680         n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             MG-4    m          66         0.0          10          0                     0           36          0           n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             MG-5    m          50         2.2          5           151                   160         320         409         n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             MG-6    m          64         0.4          12          2                     0           28          0           n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             MG-7    f          66         0.0          38          39                    0           5           0           n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             MG-8    m          71         0.0          2           88                    20          10          158         n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             MG-9    m          58         2.8          13          5                     5           0           13          0          0          0          0          0          0

                             MG-10   f          82         8.1          6           18                    5           0           0           0          3          5          0          0          0

                             MG-11   f          25         76.9         20          18                    0           13          0           0          0          0          0          0          0

                             MG-12   f          36         1.7          39          0                     0           0           0           0          0          0          0          0          0

                             MG-13   f          53         1.5          1           13                    20          20          26          33         20         20         39         0          26

                             MG-14   f          38         46.0         3           0                     0           0           11          43         0          0          0          0          0

                             MG-15   f          32         22.2         134         111                   0           156         0           33         78         0          0          0          0

                                                                                                                                                                                                     

  Mean response                      **54.7**   **20.5**   **19.8**     **40.2**    **51.3**              **66.8**    **86.4**    **15.5**    **14.3**   **3.5**    **5.6**    **0.0**    **3.7**    

  SD                                            **16.4**   **38.5**     **33.9**    **57.5**              **146.5**   **129.7**   **197.0**   **19.6**   **29.0**   **7.3**    **14.7**   **0.0**    **9.8**

  Mean positive response                                   **62.7**     **128.3**   **118.0**             **216.0**   **36.1**    **33.3**    **12.3**   **39.0**   **0.0**    **26.0**              

  SD                                                                                **60.7**              **219.7**   **150.9**   **274.1**   **6.2**    **39.6**   **10.3**                          

  Median positive response                                 **38.0**     **19.8**    **41.5**              **91.8**    **32.5**    **19.5**    **12.3**   **39.0**   **0.0**    **26.0**              

                                                                                                                                                                                                     

  HD                         HD-1    m          47         n.d.         2           0                     5           10          13          n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             HD-2    f          26         n.d.         3           0                     10          0           3           n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             HD-3    f          30         n.d.         1           0                     0           0           0           n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             HD-4    f          32         n.d.         19          56                    0           39          0           n.d.       n.d.       n.d.       n.d.       n.d.       n.d.

                             HD-5    m          79         n.d.         4           5                     3           0           13          0          0          3          0          0          0

                             HD-6    f          64         n.d.         49          0                     43          0           135         50         10         0          35         0          165

                             HD-7    f          53         n.d.         1           4                     11          0           11          11         0          11         0          7          7

                             HD-8    f          48         n.d.         6           0                     0           0           0           0          0          0          0          6          0

                             HD-9    m          60         n.d.         95          0                     0           0           0           0          0          0          0          63         0

                                                                                                                                                                                                     

  Mean response                      **48.8**              **20.0**     **7.1**     **7.9**               **5.5**     **19.3**    **12.1**    **2.0**    **2.6**    **7.0**    **15.2**   **34.4**   

  SD                                            **17.5**                **32.1**    **18.3**              **13.7**    **13.1**    **43.8**    **21.7**   **4.5**    **4.5**    **15.7**   **26.7**   **73.1**

  Mean positive response                                   **21.4**     **14.1**    **24.8**              **34.7**    **30.3**    **10.0**    **6.5**    **35.0**   **25.3**   **86.0**              

  SD                                                                                **29.8**              **16.2**    **20.3**    **56.2**    **27.9**              **5.7**               **32.2**   **111.7**

  Median positive response                                 **5.0**      **10.0**    **24.8**              **12.5**    **30.3**    **10.0**    **6.5**    **35.0**   **7.0**    **86.0**              
  --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

ELISPOT assay was performed as described in the text. Results are assigned as the numbers of IFN-gamma-secreting events among 10^6^ PBL minus the corresponding numbers of events per 10^6^ PBL without antigen. Negative results (spot number without antigen exceeding spot number with antigen) were defined zero. MG, myasthenia gravis patients; HD, healthy donors; n.d., not done.

[^1]: Academic Editor: Herbert Brok
