**3. The ectopic germinal centre response in myasthenia gravis**

## **3.1 Pathology of myasthenia gravis**

Myasthenia gravis is an organ-specific autoimmune disease characterised by weakness of striated muscles and thymic hyperplasia (Vincent, 2002). Patients are generally divided into subgroups with early-onset (EOMG, pre-40 years) or late onset (LOMG, post-40 years) forms of the disease, or with thymoma in about 10% of patients. It is a classic autoantibodymediated autoimmune disease, caused by autoantibodies directed against the postsynaptic nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Many thymoma patients and some late onset patients also have serum antibodies against striated muscle antigens, interferon-α and IL-12. Loss of functional AChRs leads to muscle weakness, usually first evident in weakness of eye movement. This can progress to other striated muscles of the body, causing problems with breathing due to effects on the diaphragm, swallowing difficulties and paralysis. These effects can be life-threatening if untreated. Evidence that the effects are mediated by autoantibodies against the AChR include induction of similar symptoms by: their transfer from mother to baby *in utero*; passive transfer from patients to mice; immunisation of animals with AChR; and marked improvement of symptoms in patients after removal of circulating IgG antibodies by plasmapheresis. Several pathogenic mechanisms are involved (Vincent, 2002; Drachman, 1994): (i) Cross-linking of the receptor by autoantibodies causes loss of AChR by antigenic modulation, leading to internalisation and degradation of the receptors; (ii) The majority of anti-AChR antibodies are of the IgG1 and IgG3 subclasses, which are particularly efficient at complement activation, resulting in lysis and damage to the muscle membrane; (iii) Less commonly, some antibodies cause direct inhibition of the ion channel function of the AChR; (iv) Antibody-dependent cell-mediated cytotoxicity has also been implicated, although there is little direct evidence for this mechanism. The IgG autoantibodies can cross the placenta of pregnant mothers with myasthenia gravis by an active transport mechanism involving the neonatal Fc receptor, FcRn, resulting in transient symptoms of myasthenia gravis in the newborn infant. The symptoms gradually ameliorate as the maternal antibodies are catabolised and replaced by the infant's own antibodies. More rarely, the autoantibodies produced by multiparous mothers can induce severe, often fatal, developmental abnormalities, termed arthrogryposis multiplex congenita, due to paralysis of fetal muscles *in utero* (see section 3.4.5).

#### **3.2 Structure and epitopes of the acetylcholine receptor**

The AChR is a pentameric transmembrane glycoprotein found almost exclusively at the muscle endplate, comprising two α polypeptide subunits, one β, one δ and, in the adult, one

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 405

demonstrated by the observation that some antibodies bind better to the MIR of the fetal AChR than the adult form, even though the γ and ε subunits do not contribute directly to the MIR (Fostieri, Beeson, & Tzartos, 2000). Titres of MIR antibodies vary considerably between patients and other antibodies may play an equally important role in some individuals. Some patients also produce autoantibodies against the acetylcholine binding sites (that also bind α-bungarotoxin); these are the blocking antibodies described above.

Early onset myasthenia gravis is associated with thymic hyperplasia characterized by secondary lymphoid organ-like structures in the medulla of >90% of patients. These include T-cell areas containing AChR-specific helper T-cells and large numbers of germinal centres with clearly defined mantle, dark and light zones. Plasmablasts and plasma cells secreting autoantibodies against AChR are also detectable within and around the germinal centres (Hill *et al.*, 2008; SHIONO *et al.*, 2003). Approximately 20% of germinal centres contain plasmablasts positive for antibodies against AChR and AChR is trapped on follicular dendritic cells in c.50% of thymic germinal centres (SHIONO *et al.*, 2003). Anti-AChRsecreting hybridomas and AChR-specific Fabs have been cloned from thymic B-cells and thymectomy results in a reduction in the serum anti-AChR titre and reduced clinical symptoms in some patients, although the benefits of thymectomy have never been rigorously proved (Cardona *et al.*, 1994; Farrar *et al.*, 1997; Graus *et al.*, 1997). The relative contribution of the thymus to production of anti-AChR autoantibodies compared with the secondary lymphoid organs is unknown, but it appears to play a significant role. Therefore, using the methods described in section 2, we tested the hypothesis that thymic germinal centres are sites of ongoing autoimmune responses driven by autoantigen, i.e. sites of activated B-cells, clonally proliferating, somatically mutating their expressed Ig V-genes and

Thymi from 5 EOMG patients were examined by immunohistology. All 5 contained large numbers of germinal centres with typical mantle zones within the thymic medulla, histologically indistinguishable from germinal centres in human tonsil controls. The mantle zones contained densely packed CD20+ B-cells surrounding the germinal centre B-cells (Fig. 3A). These were interspersed with a network of follicular dendritic cells extending throughout the dark and light zones (Fig. 3C) and a crescent of T-cells can be seen at the apex of the light zone (Fig. 3B). Proliferating B-cells were distributed throughout the germinal centre but in larger numbers within the dark zone (Fig. 3D). Autoradiography with 125I-α-bungarotoxin alone, which binds to AChR, diffusely labelled c.50% of germinal centres and appeared to be associated with the follicular dendritic cell processes. No labelling was seen in human tonsils or thymi from two seronegative myasthenia patients and bungarotoxin binding was blocked by the cholinergic drug, carbamyl choline, which is structurally similar to AChR, indicating that the follicular dendritic network contained

In contrast, 125I-α-bungarotoxin-labelled AChR bound to individual cells in 20% of germinal centres, including large numbers of moderately labelled centrocytes in the light zone, smaller numbers in the dark zone, and heavily labelled plasmablasts/plasma cells in and

undergoing affinity maturation, driven by the acetylcholine receptor.

**3.4 The thymic germinal centre response in myasthenia gravis** 

**3.4.1 Germinal centres in the thymus** 

membrane-bound antigen or immune complexes.

around the germinal centres (Fig. 3E & F).

**3.3 Role of the thymus** 

ε subunit; in the fetus there is also one γ subunit, which is gradually replaced by an ε from the third trimester onwards (Fig. 2) (Vincent, 2002). The five subunits are combined into a cylindrical structure with a central cation channel that is closed in the inactive conformation. There are two binding sites for acetylcholine, formed at the interfaces between one α and δ subunit and the second α and ε or γ subunits. Electrical impulses passing down the motor nerve trigger release of acetylcholine molecules at the nerve termini. When these bind to the two receptor binding sites, they cause the central cation channel to open and sodium ions to flood into the muscle resulting in local membrane depolarisation. When this reaches threshold the resulting action potential spreads across the muscle triggering it to contract. Loss of at least 50% of receptors is required to produce overt muscle weakness.

Fig. 2. Diagrammatic representation of the structure of the acetylcholine receptor:

(a) the complete pentameric molecule in the cell membrane; (b) the topology of the subunits, illustrated for the α subunit that contributes to the acetylcholine/α-bungarotoxin binding site, the main immunogenic region (MIR) and the very immunogenic cytoplasmic epitope (VICE); It is doubtful whether the latter plays any significant role in pathogenesis; (c and d) the fetal and adult subtypes of AChR. Reproduced with permission from (Vincent et al., 1997), Plenum Press.

Since the patients' autoantibodies are almost exclusively specific for the complex native conformation of the extracellular AChR subunit domains, and not short peptides or even whole subunit polypeptides, mapping of the autoantibody epitopes has proved to be difficult. The antibodies are mainly IgG1 or IgG3, of high avidity and heterogeneous in their sequences and fine specificity. Disease severity correlates poorly with autoantibody titre, suggesting that pathogenicity may depend upon precise epitope specificity. Many of the antibodies bind to a region of the extracellular domain of the α chain, the main immunogenic region or MIR. Its conformation is affected by the ε ↔ γ interchange, as demonstrated by the observation that some antibodies bind better to the MIR of the fetal AChR than the adult form, even though the γ and ε subunits do not contribute directly to the MIR (Fostieri, Beeson, & Tzartos, 2000). Titres of MIR antibodies vary considerably between patients and other antibodies may play an equally important role in some individuals. Some patients also produce autoantibodies against the acetylcholine binding sites (that also bind α-bungarotoxin); these are the blocking antibodies described above.

#### **3.3 Role of the thymus**

404 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

ε subunit; in the fetus there is also one γ subunit, which is gradually replaced by an ε from the third trimester onwards (Fig. 2) (Vincent, 2002). The five subunits are combined into a cylindrical structure with a central cation channel that is closed in the inactive conformation. There are two binding sites for acetylcholine, formed at the interfaces between one α and δ subunit and the second α and ε or γ subunits. Electrical impulses passing down the motor nerve trigger release of acetylcholine molecules at the nerve termini. When these bind to the two receptor binding sites, they cause the central cation channel to open and sodium ions to flood into the muscle resulting in local membrane depolarisation. When this reaches threshold the resulting action potential spreads across the muscle triggering it to contract.

Loss of at least 50% of receptors is required to produce overt muscle weakness.

Fig. 2. Diagrammatic representation of the structure of the acetylcholine receptor:

AChR. Reproduced with permission from (Vincent et al., 1997), Plenum Press.

(a) the complete pentameric molecule in the cell membrane; (b) the topology of the subunits, illustrated for the α subunit that contributes to the acetylcholine/α-bungarotoxin binding site, the main immunogenic region (MIR) and the very immunogenic cytoplasmic epitope (VICE); It is doubtful whether the latter plays any significant role in pathogenesis; (c and d) the fetal and adult subtypes of

Since the patients' autoantibodies are almost exclusively specific for the complex native conformation of the extracellular AChR subunit domains, and not short peptides or even whole subunit polypeptides, mapping of the autoantibody epitopes has proved to be difficult. The antibodies are mainly IgG1 or IgG3, of high avidity and heterogeneous in their sequences and fine specificity. Disease severity correlates poorly with autoantibody titre, suggesting that pathogenicity may depend upon precise epitope specificity. Many of the antibodies bind to a region of the extracellular domain of the α chain, the main immunogenic region or MIR. Its conformation is affected by the ε ↔ γ interchange, as Early onset myasthenia gravis is associated with thymic hyperplasia characterized by secondary lymphoid organ-like structures in the medulla of >90% of patients. These include T-cell areas containing AChR-specific helper T-cells and large numbers of germinal centres with clearly defined mantle, dark and light zones. Plasmablasts and plasma cells secreting autoantibodies against AChR are also detectable within and around the germinal centres (Hill *et al.*, 2008; SHIONO *et al.*, 2003). Approximately 20% of germinal centres contain plasmablasts positive for antibodies against AChR and AChR is trapped on follicular dendritic cells in c.50% of thymic germinal centres (SHIONO *et al.*, 2003). Anti-AChRsecreting hybridomas and AChR-specific Fabs have been cloned from thymic B-cells and thymectomy results in a reduction in the serum anti-AChR titre and reduced clinical symptoms in some patients, although the benefits of thymectomy have never been rigorously proved (Cardona *et al.*, 1994; Farrar *et al.*, 1997; Graus *et al.*, 1997). The relative contribution of the thymus to production of anti-AChR autoantibodies compared with the secondary lymphoid organs is unknown, but it appears to play a significant role. Therefore, using the methods described in section 2, we tested the hypothesis that thymic germinal centres are sites of ongoing autoimmune responses driven by autoantigen, i.e. sites of activated B-cells, clonally proliferating, somatically mutating their expressed Ig V-genes and undergoing affinity maturation, driven by the acetylcholine receptor.
