**1. Introduction**

#### **1.1 The B-cell response in autoimmune disease<sup>1</sup>**

The pathological effects of autoimmune diseases on the target tissues can be mediated by autoantibodies, cell-mediated immune responses, or both. It is increasingly evident that some autoimmune diseases previously thought to be essentially T-cell-mediated also have a B-cell component, which may involve direct effects of autoantibody secreted by plasma cells, pro- or anti-inflammatory cytokines secreted by activated effector or regulatory B-cells, or through the highly efficient antigen presentation function of B-cells enabling them to activate CD4+ T-cells and *vice versa.* The number of autoimmune diseases known to be mediated partly or largely through autoantibodies has increased markedly in recent times. Systemic lupus erythematosus (SLE)1, in which the pathology is mediated via Type II & III hypersensitivity reactions involving anti-DNA autoantibodies, has long been known to fall into this category. Many other autoantibodies are produced by these patients, principally against nuclear antigens, but most are not thought to be involved in pathology. Hashimoto's thyroiditis and Graves' disease patients produce pathogenic autoantibodies against thyroid antigens, the latter being a rare example of an activating autoantibody inducing signalling via the thyroid stimulating hormone receptor. Myasthenia gravis patients produce autoantibodies against the acetylcholine receptor (AChR), present on the motor muscle endplates, thereby inhibiting muscle contraction. Anti-SS-A and anti-SS-B (anti-Ro & anti-La) autoantibodies are implicated in congenital heart block in children born to mothers with Sjögren's Syndrome due to transplacental uptake of IgG autoantibodies; autoantibodies against α-fodrin are also believed to be pathogenic in these patients. Rheumatoid arthritis (RA), one member of the group of systemic rheumatic autoimmune diseases that also includes SLE, psoriatic arthritis and the various forms of myositis, has now gone full cycle in views on its pathological mechanisms.

<sup>1</sup> Abbreviations: AChR, Acetylcholine Receptor; AID, Activation Induced Cytidine Deaminase; AMC, Arthrogryposis Multiplex Congenita; ARS, Anti-amino acyl-tRNA Synthetase; Bmem, Memory B-cell; CDR, Complementarity Determining Region; DM, Dermatomyositis; EOMG, Early Onset Myasthenia Gravis; FDC, Follicular Dendritic Cell; G.C., Germinal Centre; IBM, Inclusion Body Myositis; IM, Inflammatory Myopathies; LOMG, Late Onset Myasthenia Gravis; MAA, Myositis-Associated Autoantibodies; MAb, Monoclonal Antibody; MIR, Main Immunogenic Region; MSA, Myositis Specific Autoantibodies; PM, Polymyositis; RA, Rheumatoid Arthritis; SLE, Systemic Lupus Erythematosus; TAA, Tumour-Associated Antigen; TIL, Tumour-Infiltrating Lymphocytes; Tfh, Follicular T helper cell; UNG, Uracil Nucleotidyl Glycosylase.

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 397

single strand breaks which are repaired by error prone DNA polymerases (Di Noia & Neuberger, 2007). The mutations are targeted mainly to the complementarity determining regions (CDRs) which are intimately involved in binding to the epitope and therefore determine specificity and affinity of the antibody. Combined with the rapid proliferation, this results in clones of B-cells expressing receptors with a variety of affinities for the antigen, some high, some low; some will have lost the ability to bind to the antigen

Several clones of proliferating, mutating B-cells are usually present within each germinal centre. These cells differentiate into centrocytes expressing mutated antigen receptors and migrate into the light zone. The centrocytes move through the light zone, acquire antigen for a second time from immune complexes on the follicular dendritic cells, which they internalise, process and present to follicular helper T-cells (Tfh-cells) (Patakas *et al.*, 2011), thereby receiving survival signals, probably via costimulatory molecule interactions including CD40/CD154 and CD80/CD28 binding. These signals, together with Tfh-cell cytokines (IL-4 and IFNγ) and AID deamination of cytidines, promote induction of class switch recombination (Patakas *et al.*, 2011). Some of these centrocytes differentiate directly into plasmablasts and antibody-secreting plasma cells; others differentiate into Ig class switched memory B-cells, both of which migrate out of the follicle. Competition for limiting availability of antigen results in selection of B-cells expressing high affinity antigen receptors; recent evidence has shown that a broad range of mutations is involved in selection, not only for high affinity receptors but also for stability and expression of the Bcell receptor (Weiser *et al.*, 2011). Cells expressing antigen receptors with low affinity are unable to compete for survival signals and the default response is that they die by apoptosis and are engulfed by macrophages, in which their degenerating nuclei are visible as tingible bodies. Most of this information is derived from studies in mice, in which the germinal centres reach maximum size about two weeks after immunisation and then gradually decline in the absence of further immunisation, disappearing after several weeks. Although the cell composition and structure of secondary follicles appear similar in Man, the kinetics

Detailed studies of the kinetics and cellular interactions within germinal centres using multiphoton microscopy of living tissue in combination with B & T-lymphocytes expressing defined antigen receptors from transgenic animals have revealed much more dynamic activity than was previously suspected. It is now recognised that there is less distinction between the dark and light zones than suggested by static immunohistological examination, and there is continual recycling of B-cells both between and within the two zones, although there is net migration from the dark zone to the light zone (Beltman *et al.*, 2011)(Figure 1B). Centrocytes move rapidly through the network of follicular dendritic cell processes, apparently sampling the immune complexes attached to their membranes and some of these cells return to the dark zone for further rounds of proliferation and somatic hypermutation. Migration of B-cells between the zones is controlled by chemokines, possibly secreted by stromal cells within the germinal centre. Tfh-cells are present mainly in the light zone and recent data suggest that affinity selection of B-cells may involve competition for signals from cognate Tfh-cells via peptide/MHC Class II binding as well as, or instead of, competition for antigen on the surface of follicular dendritic cells (Victora *et al.*, 2010). Anti-self B-cells that have escaped negative selection in the bone marrow, or have arisen in the germinal centre due to somatic hypermutation, are either eliminated at this stage, suppressed by regulatory T-cells, or alter their antigen specificity by receptor revision, a process similar to V-gene rearrangement in

altogether and rare B-cells may cross-react with a self-antigen.

and some of the detailed cellular interactions may differ.

Initially thought to be caused by the anti-IgG Fc antibody (rheumatoid factor), although approximately 25% of RA patients are rheumatoid factor negative, the evidence then swung in favour of a cell mediated autoimmune response involving effector T-cells and cytokines, principally TNFα. Although these are clearly involved in joint pathology, autoantibodies against cyclic citrullinated proteins are a much better diagnostic marker for RA than rheumatoid factor and there is some limited evidence that they may be pathogenic. It is also now recognised that B-cells play an important role in the pathogenic autoimmune response, as clearly demonstrated by the marked clinical improvement in patients treated with Rituximab®, an anti-CD20 chimaeric (human/mouse) monoclonal antibody that suppresses Bcell responses. Other autoimmune diseases with B-cell involvement include autoimmune haemolytic anaemia, idiopathic thrombocytopaenia, Type I diabetes, and some subtypes of myositis, although the situation is often confused by the presence of non-pathogenic autoantibodies.

#### **1.2 The germinal centre response to foreign antigens**

Germinal centres (g.c.) are the main sites of generation of high affinity, antibody-secreting plasma cells and Ig class-switched memory B-cells during T-cell-dependent immune responses, extensively reviewed by others (Allen, Okada, & Cyster, 2007; Brink, 2007; Hauser, Shlomchik, & Haberman, 2007; Klein & Dalla-Favera, 2008; Leavy, 2010; Minton, 2011). Here we shall summarise briefly the principal features of the g.c. response. The response is initiated by B-cells binding to their cognate antigen on the surface of antigen presenting cells, such as dendritic cells, in a secondary lymphoid organ (lymph node, spleen, Peyer's patches or human tonsil). The antigen becomes internalised, degraded into peptides which are expressed on the cell surface bound to MHC Class II and presented to a helper T (Th) cell that provides costimulatory activation signals, including binding of the Bcell surface molecule CD40 to its ligand, CD154, on the T-cell membrane.

This interaction takes place at or near the interface between the B-cell follicle and the T-cell area and some activated B-cells proliferate outside the follicle and differentiate into short-lived plasma cells secreting IgM antibodies. Others migrate into the B-cell follicle where they proliferate and differentiate into centroblasts expressing low levels of surface Ig. This region develops into a germinal centre with a dark zone of densely packed, proliferating centroblasts, and a light zone of more loosely packed B-cells (centrocytes) interspersed with the processes of follicular dendritic cells (FDC, Figure 1A). These have distinct stromal origins, unlike the bone marrow derived, extra-follicular dendritic cells; almost uniquely, their C' and Fc receptors trap immune complexes and retain antigens in their native state for months.

The pre-existing IgM+,IgD+ follicle B-cells are pushed out to form a mantle zone around the developing germinal centre, the whole structure being termed a secondary follicle. Proliferation of the dark zone centroblasts is extremely rapid, with cell cycle times estimated at between 6 and 12 hours. These proliferating clones of B-cells switch on the molecular machinery required for somatic hypermutation of their rearranged, expressed, Ig V-genes, including expression of activation-induced cytidine deaminase (AID). This induces mutations specifically targeted to the Ig V-genes at a frequency of 1 per 1000 base pairs per cell division, although much lower levels of mutation can also occur in some other, non-Ig genes such as Bcl-2 & Bcl-6. AID deaminates cytidine to uracil at C/G base pairs, introducing mismatches in the DNA that can be replaced by T/A base pairs. Uracil nucleotidyl glycosylase (UNG) can remove the uracil leading to insertion of any of the four bases at the abasic site; mismatch repair enzymes also recognise the mismatch and induce

Initially thought to be caused by the anti-IgG Fc antibody (rheumatoid factor), although approximately 25% of RA patients are rheumatoid factor negative, the evidence then swung in favour of a cell mediated autoimmune response involving effector T-cells and cytokines, principally TNFα. Although these are clearly involved in joint pathology, autoantibodies against cyclic citrullinated proteins are a much better diagnostic marker for RA than rheumatoid factor and there is some limited evidence that they may be pathogenic. It is also now recognised that B-cells play an important role in the pathogenic autoimmune response, as clearly demonstrated by the marked clinical improvement in patients treated with Rituximab®, an anti-CD20 chimaeric (human/mouse) monoclonal antibody that suppresses Bcell responses. Other autoimmune diseases with B-cell involvement include autoimmune haemolytic anaemia, idiopathic thrombocytopaenia, Type I diabetes, and some subtypes of myositis, although the situation is often confused by the presence of non-pathogenic

Germinal centres (g.c.) are the main sites of generation of high affinity, antibody-secreting plasma cells and Ig class-switched memory B-cells during T-cell-dependent immune responses, extensively reviewed by others (Allen, Okada, & Cyster, 2007; Brink, 2007; Hauser, Shlomchik, & Haberman, 2007; Klein & Dalla-Favera, 2008; Leavy, 2010; Minton, 2011). Here we shall summarise briefly the principal features of the g.c. response. The response is initiated by B-cells binding to their cognate antigen on the surface of antigen presenting cells, such as dendritic cells, in a secondary lymphoid organ (lymph node, spleen, Peyer's patches or human tonsil). The antigen becomes internalised, degraded into peptides which are expressed on the cell surface bound to MHC Class II and presented to a helper T (Th) cell that provides costimulatory activation signals, including binding of the B-

This interaction takes place at or near the interface between the B-cell follicle and the T-cell area and some activated B-cells proliferate outside the follicle and differentiate into short-lived plasma cells secreting IgM antibodies. Others migrate into the B-cell follicle where they proliferate and differentiate into centroblasts expressing low levels of surface Ig. This region develops into a germinal centre with a dark zone of densely packed, proliferating centroblasts, and a light zone of more loosely packed B-cells (centrocytes) interspersed with the processes of follicular dendritic cells (FDC, Figure 1A). These have distinct stromal origins, unlike the bone marrow derived, extra-follicular dendritic cells; almost uniquely, their C' and Fc receptors trap

The pre-existing IgM+,IgD+ follicle B-cells are pushed out to form a mantle zone around the developing germinal centre, the whole structure being termed a secondary follicle. Proliferation of the dark zone centroblasts is extremely rapid, with cell cycle times estimated at between 6 and 12 hours. These proliferating clones of B-cells switch on the molecular machinery required for somatic hypermutation of their rearranged, expressed, Ig V-genes, including expression of activation-induced cytidine deaminase (AID). This induces mutations specifically targeted to the Ig V-genes at a frequency of 1 per 1000 base pairs per cell division, although much lower levels of mutation can also occur in some other, non-Ig genes such as Bcl-2 & Bcl-6. AID deaminates cytidine to uracil at C/G base pairs, introducing mismatches in the DNA that can be replaced by T/A base pairs. Uracil nucleotidyl glycosylase (UNG) can remove the uracil leading to insertion of any of the four bases at the abasic site; mismatch repair enzymes also recognise the mismatch and induce

autoantibodies.

**1.2 The germinal centre response to foreign antigens** 

cell surface molecule CD40 to its ligand, CD154, on the T-cell membrane.

immune complexes and retain antigens in their native state for months.

single strand breaks which are repaired by error prone DNA polymerases (Di Noia & Neuberger, 2007). The mutations are targeted mainly to the complementarity determining regions (CDRs) which are intimately involved in binding to the epitope and therefore determine specificity and affinity of the antibody. Combined with the rapid proliferation, this results in clones of B-cells expressing receptors with a variety of affinities for the antigen, some high, some low; some will have lost the ability to bind to the antigen altogether and rare B-cells may cross-react with a self-antigen.

Several clones of proliferating, mutating B-cells are usually present within each germinal centre. These cells differentiate into centrocytes expressing mutated antigen receptors and migrate into the light zone. The centrocytes move through the light zone, acquire antigen for a second time from immune complexes on the follicular dendritic cells, which they internalise, process and present to follicular helper T-cells (Tfh-cells) (Patakas *et al.*, 2011), thereby receiving survival signals, probably via costimulatory molecule interactions including CD40/CD154 and CD80/CD28 binding. These signals, together with Tfh-cell cytokines (IL-4 and IFNγ) and AID deamination of cytidines, promote induction of class switch recombination (Patakas *et al.*, 2011). Some of these centrocytes differentiate directly into plasmablasts and antibody-secreting plasma cells; others differentiate into Ig class switched memory B-cells, both of which migrate out of the follicle. Competition for limiting availability of antigen results in selection of B-cells expressing high affinity antigen receptors; recent evidence has shown that a broad range of mutations is involved in selection, not only for high affinity receptors but also for stability and expression of the Bcell receptor (Weiser *et al.*, 2011). Cells expressing antigen receptors with low affinity are unable to compete for survival signals and the default response is that they die by apoptosis and are engulfed by macrophages, in which their degenerating nuclei are visible as tingible bodies. Most of this information is derived from studies in mice, in which the germinal centres reach maximum size about two weeks after immunisation and then gradually decline in the absence of further immunisation, disappearing after several weeks. Although the cell composition and structure of secondary follicles appear similar in Man, the kinetics and some of the detailed cellular interactions may differ.

Detailed studies of the kinetics and cellular interactions within germinal centres using multiphoton microscopy of living tissue in combination with B & T-lymphocytes expressing defined antigen receptors from transgenic animals have revealed much more dynamic activity than was previously suspected. It is now recognised that there is less distinction between the dark and light zones than suggested by static immunohistological examination, and there is continual recycling of B-cells both between and within the two zones, although there is net migration from the dark zone to the light zone (Beltman *et al.*, 2011)(Figure 1B). Centrocytes move rapidly through the network of follicular dendritic cell processes, apparently sampling the immune complexes attached to their membranes and some of these cells return to the dark zone for further rounds of proliferation and somatic hypermutation. Migration of B-cells between the zones is controlled by chemokines, possibly secreted by stromal cells within the germinal centre. Tfh-cells are present mainly in the light zone and recent data suggest that affinity selection of B-cells may involve competition for signals from cognate Tfh-cells via peptide/MHC Class II binding as well as, or instead of, competition for antigen on the surface of follicular dendritic cells (Victora *et al.*, 2010). Anti-self B-cells that have escaped negative selection in the bone marrow, or have arisen in the germinal centre due to somatic hypermutation, are either eliminated at this stage, suppressed by regulatory T-cells, or alter their antigen specificity by receptor revision, a process similar to V-gene rearrangement in

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 399

neogenesis appears to be directly related to the extent of infiltration of lymphoid and other immune cells (Aloisi & Pujol-Borrell, 2006). Examples of autoimmune diseases in which germinal centre-like structures have been identified in the target, or disease-related tissues are shown in Table 1. It is now apparent that ectopic germinal centres, also known as tertiary lymphoid organs, can also develop in other chronic inflammatory diseases, such as the gut in Crohn's disease and ulcerative colitis patients, in chronic infections (Aloisi & Pujol-Borrell, 2006) and some types of cancer (Table 1). The questions these observations raise are: 1. How do they develop?; 2. How closely do they resemble germinal centres in secondary lymphoid organs?; 3. Are the B-cells within them undergoing a germinal centre response, as described in section 1.2 above?; 4. Are they generating plasma cells secreting pathogenic autoantibodies?; 5.

> **Antigen(s) Recognised by GC B-cells**

Thyroperoxidase

Thyroperoxidase

IgG Fc, Cyclic citrullinated protein/peptide

Lungs ? (Wallace *et al.*, 1996)

Lymphoma ? (Bombardieri *et al.*, 2007a)

receptor family

Stomach Bacterial antigens (Genta, Hamner, & Graham, 1993)

Myasthenia gravis Thymus Acetylcholine receptor (Yoshitake *et al.*, 1994)

Uveoretinits Choroid of the eye ? (Liversidge *et al.*, 1993) Autoimmune hepatitis Liver ? (Mosnier *et al.*, 1993)

Crohn's disease Gastrointestinal tract ? (Kaiserling, 2001) Ulcerative colitis Descending colon ? (Kaiserling, 2001)

Chronic hepatitis C infection Liver ? (Mosnier *et al.*, 1993)

Oncocerciasis Skin ? (Brattig *et al.*, 2010)

Medullary breast carcinoma Breast tumour Ganglioside (Coronella *et al.*, 2001)

Table 1. Diseases in which ectopic germinal centres have been observed.

Multiple sclerosis Meninges (?) ? (Prineas, 1979) (Serafini *et al.*, 2004)

Sjögren's syndrome Salivary glands SS-A (Ro), SS-B (La) (Stott *et al.*, 1998)

**Reference** 

(Knecht, Saremaslani, & Hedinger, 1981) (Armengol *et al.*, 2001)

(Armengol *et al.*, 2001)

(SHIONO *et al.*, 2003)

(Gerhard *et al.*, 2002)

(Stolte & Eidt, 1989)

(Coronella *et al.*, 2002) (Nzula, Going, & Stott, 2003a) (Simsa *et al.*, 2005) and section 5.

(Kotlan *et al.*, 2005)

? (Ghosh *et al.*, 2005)

? (Canete *et al.*, 2007)

(Manzo & Pitzalis, 2007) (Humby *et al.*, 2009)

What role do they play in the pathogenesis of autoimmune disease?

**Germinal Centres** 

Hashimoto's thyroiditis Thyroid Thyroglobulin,

Graves' disease Thyroid Thyroglobulin,

of joints

of joints

Chronic Lyme disease Synovial membranes of

joints

Ductal breast carcinoma Breast tumour Epidermal growth factor

**Autoimmune Diseases Organ containing** 

Rheumatoid arthritis Synovial membranes

Psoriatic arthritis Synovial membranes

Cryptogenic fibrosing

**Chronic Inflammatory** 

**Infectious Diseases** 

*Helicobacter pylori* or *Campylobacter* gastritis

Lymphoma of MALT associated with Sjögren's

Syndrome

alveolitis

**Diseases** 

 **Cancers** 

developing B-cells. This involves re-expression of RAG1 and RAG2 and rearrangement of an upstream light chain V-gene to an unused J exon (Nemazee, 2006). Despite the absence of D exons in the rearranged heavy chain locus, we have shown that an upstream heavy chain Vgene can also replace all or part of a rearranged VH-gene, thereby altering the specificity of the receptor away from self antigen (Darlow & Stott, 2005). The architecture, cellular components and processes occurring in a typical germinal centre are summarised in Figure 1.

Fig. 1. Diagrammatic representation of a germinal centre in a lymph node.

A: Showing a dark zone containing proliferating clones of mutating centroblasts and a light zone containing centrocytes in contact with follicular dendritic cells and follicular helper T-cells (Tfh cells). Long-lived memory B-cells, plasmablasts and plasma cells secreting antibody molecules migrate out of the g.c. and leave the lymph node via the efferent lymphatic vessel. Apoptotic B-cells, macrophages containing tingible bodies and the mantle zone are not shown.

B: The same germinal centre showing recirculation of B-cells within and between the dark and light zones.

#### **1.3 The ectopic germinal centre response in autoimmune disease**

It has been known for many years that the target tissues of autoimmune diseases contain infiltrating lymphocytes and other immune cells, including T-cells, B-cells, plasma cells, macrophages, dendritic and follicular dendritic cells. In many cases the infiltrating cells organise themselves into structures resembling germinal centres. Some of these have a mantle zone, suggesting that they were formed from a primary follicle whereas, even when absent, it is often possible to distinguish a dark zone, containing few or no CD4+ T-cells or follicular dendritic cells, and a light zone containing both. Autoantigens have been identified on the finger-like processes of follicular dendritic cells (Shiono *et al.*, 2003) and, in some cases, autoantibodies have been identified in g.c. B-cells. Separate T-cell areas containing dendritic cells and, sometimes, high endothelial venules, can also be seen. The stage of lymphoid

developing B-cells. This involves re-expression of RAG1 and RAG2 and rearrangement of an upstream light chain V-gene to an unused J exon (Nemazee, 2006). Despite the absence of D exons in the rearranged heavy chain locus, we have shown that an upstream heavy chain Vgene can also replace all or part of a rearranged VH-gene, thereby altering the specificity of the receptor away from self antigen (Darlow & Stott, 2005). The architecture, cellular components

and processes occurring in a typical germinal centre are summarised in Figure 1.

Fig. 1. Diagrammatic representation of a germinal centre in a lymph node.

**1.3 The ectopic germinal centre response in autoimmune disease** 

tingible bodies and the mantle zone are not shown.

A: Showing a dark zone containing proliferating clones of mutating centroblasts and a light zone containing centrocytes in contact with follicular dendritic cells and follicular helper T-cells (Tfh cells). Long-lived memory B-cells, plasmablasts and plasma cells secreting antibody molecules migrate out of the g.c. and leave the lymph node via the efferent lymphatic vessel. Apoptotic B-cells, macrophages containing

B: The same germinal centre showing recirculation of B-cells within and between the dark and light zones.

It has been known for many years that the target tissues of autoimmune diseases contain infiltrating lymphocytes and other immune cells, including T-cells, B-cells, plasma cells, macrophages, dendritic and follicular dendritic cells. In many cases the infiltrating cells organise themselves into structures resembling germinal centres. Some of these have a mantle zone, suggesting that they were formed from a primary follicle whereas, even when absent, it is often possible to distinguish a dark zone, containing few or no CD4+ T-cells or follicular dendritic cells, and a light zone containing both. Autoantigens have been identified on the finger-like processes of follicular dendritic cells (Shiono *et al.*, 2003) and, in some cases, autoantibodies have been identified in g.c. B-cells. Separate T-cell areas containing dendritic cells and, sometimes, high endothelial venules, can also be seen. The stage of lymphoid neogenesis appears to be directly related to the extent of infiltration of lymphoid and other immune cells (Aloisi & Pujol-Borrell, 2006). Examples of autoimmune diseases in which germinal centre-like structures have been identified in the target, or disease-related tissues are shown in Table 1. It is now apparent that ectopic germinal centres, also known as tertiary lymphoid organs, can also develop in other chronic inflammatory diseases, such as the gut in Crohn's disease and ulcerative colitis patients, in chronic infections (Aloisi & Pujol-Borrell, 2006) and some types of cancer (Table 1). The questions these observations raise are: 1. How do they develop?; 2. How closely do they resemble germinal centres in secondary lymphoid organs?; 3. Are the B-cells within them undergoing a germinal centre response, as described in section 1.2 above?; 4. Are they generating plasma cells secreting pathogenic autoantibodies?; 5. What role do they play in the pathogenesis of autoimmune disease?


Table 1. Diseases in which ectopic germinal centres have been observed.

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 401

salivary glands, they may easily be overlooked. Tissues containing different types of cells respond in a variety of ways to inflammatory signals and this may also determine whether, and to what extent, lymphoid organ neogenesis occurs. The origin of follicular dendritic cells is unclear but it has been proposed that they develop from precursor cells already present in the tissue, either fibroblasts or fibroblast precursor cells (Park & Choi, 2005). Alternatively, the precursor cells may be induced to migrate into the tissue by the same or similar chemokines as those attracting the B and T-lymphocytes. In several autoimmune diseases (Table 1) and animal models of autoimmune diseases (Astorri *et al.*, 2010; Nacionales *et al.*, 2009), it has been demonstrated that ectopic germinal centres are generating plasma cells secreting pathogenic autoantibodies and, almost certainly, memory B-cells bearing anti-self antigen receptors, implying that they aid the diversification of the autoantibody repertoire and contribute to the maintenance of immune pathology. In addition to autoantibody production, self-reactive B-cells generated in ectopic germinal centres may also contribute to autoimmune pathology by secretion of pro-inflammatory cytokines and activation of pathogenic T-cells by presentation of processed self-antigens. Bcells may contribute in this way to immune pathology in autoimmune diseases generally considered to be principally T-cell mediated, and may be one explanation for the efficacy of

Rituximab therapy for rheumatoid arthritis.

**2.1 Identification and cellular composition of ectopic germinal centres** 

**2.2 Cloning and sequence analysis of rearranged Ig V-genes** 

The methods we used to identify ectopic germinal centres, characterise their cellular composition, analyse the rearranged Ig V-gene sequences expressed by germinal centre Bcells and identify their antibody specificity have been described in detail in previously published papers (Nzula, Going, & Stott, 2003a; Sims *et al.*, 2001). Briefly, sections were cut from snap frozen tissue biopsies and every tenth section stained for B-cells with anti-CD20. Sections containing germinal centre-like structures or B-cell aggregates were further characterised by staining for T-cells (anti-CD3, CD4, CD8), regulatory T-cells (anti-FoxP3), follicular dendritic cells (anti-FDC (DAKO) or anti-CD35), plasma cells (DAKO), macrophages (anti-CD68) and proliferating cells (anti-Ki67). Double immunofluorescent staining with the above cell subset-specific antibodies and Ki67 was used to identify dividing cells. Acetylcholine receptor-specific B-cells in germinal centres from the thymus of myasthenia gravis patients were identified by 125I-α-bungarotoxin-labelled acetylcholine receptor and autoradiography (Shiono *et al.*, 2003; Hill *et al.*, 2008); other autoantibodyproducing cells were identified by immunofluorescence staining with the relevant antigen.

Ectopic germinal centres and B-cell aggregates were excised by microdissection, digested with proteinase K and the released DNA used as a template for amplification of the rearranged Ig V-genes by nested PCR. Details of the method and the primers are described in Sims *et al.* (2001) and Nzula *et al.* (2003). Amplified DNA was purified by agarose gel electrophoresis, ligated into plasmid DNA and cloned in *E. coli*. Cloned plasmid DNA was purified and the Ig V-genes sequenced in both directions using primers complementary to sequences flanking the cloning site. The best matching germline V, D & J sequences were identified initially by comparison with the VBASE directory of human Ig V-genes and later, after VBASE ceased to be updated, using the Immunogenetics (IMGT) Database of Human

**2. Methods**

It has now been shown by combined immunohistochemistry, identification of antigen specificity of B-cells and plasma cells in and around ectopic germinal centres, and sequence analysis of expressed, rearranged Ig V-genes and their somatic mutations, that germinal centre B-cells in the target tissues of several autoimmune diseases are undergoing clonal expansion, somatic hypermutation and affinity selection, in a similar manner to that seen in the germinal centres of secondary lymphoid organs (Table 1 and section 1.2). This has been demonstrated in Sjögren's syndrome, rheumatoid arthritis, psoriatic arthritis, myasthenia gravis, multiple sclerosis and also in breast cancer. In some of these cases, expression of RAG1 and 2 have been observed (Armengol *et al.*, 2001), indicating that receptor revision also takes place in ectopic germinal centres and therefore the generation and attempted elimination of self-reactive B-cells. The signals involved in tertiary lymphoid organ neogenesis appear to be similar to those in development of secondary lymphoid organs, although the temporal and causal relationship between appearance of these structures in the target tissue and autoimmune pathology-related tissue damage is unclear. One scenario is that an initial event in the tissue, which could, in some cases, include microbial infection, leads to the release of molecules seen by the immune system as "danger signals" (Matzinger, 2007) thereby inducing infiltration of inflammatory cells and subsequent lymphoid neogenesis, causing further tissue damage with concomitant release of selfantigens, more danger signals and a vicious cycle, perpetuating a chronic autoimmune reaction. Alternatively, initial tissue damage may be caused by an autoimmune response commencing in the secondary lymphoid organs, with subsequent events following a similar course to that described above. Lymphotoxins α, β, α1β2 and TNFα have been shown to be required for development of ectopic germinal centres. Growth-factor receptor-bound protein-2 (Grb2) has recently been shown to control orthotopic lymphoid follicle organisation and the germinal centre response by inducing production of lymphotoxin-α via CXCR5 signalling (Jang *et al.*, 2011). These molecules are secreted by infiltrating B and Th1 cells and activated NK cells; on binding to their receptor on stromal cells they induce expression of adhesion molecules and secretion of chemokines which induce further lymphocyte infiltration and segregation into B-cell follicles, formation of a follicular dendritic cell network and T-cell areas. It has also recently been proposed that overexpression of costimulatory molecules on Tfh-cells may contribute to overcoming B-cell tolerance (Patakas *et al.*, 2011). This may be a contributory factor in ectopic as well as orthotopic germinal centres. Primary B-cell follicles are rarely seen in autoimmune disease target tissues but this may be because chronic antigen stimulation has been in progress for a considerable time before biopsies are taken. For example, in type I diabetes mellitus there is evidence that the autoimmune response develops long before overt disease is diagnosed. Whether ectopic germinal centres are initiated by naïve or memory B-cells is unclear but recent evidence shows that at least some B-cell clones arise *de novo* from naïve B-cells (Sims *et al.*, 2001; Nzula, Going, & Stott, 2003b; Nzula, Going, & Stott, 2003a).

The frequency of ectopic germinal centres varies markedly between autoimmune diseases; as one might expect, the highest incidence is in diseases where pathogenic autoantibodies are most strongly implicated. Thus, they have been identified in thyroid tissues of 100% of Hashimoto's thyroiditis patients and 54 – 63% of Graves' disease cases; in rheumatoid arthritis the figure is 25 – 50% but in Sjögren's syndrome it is only 17%, although variations may to some extent reflect differences in the difficulty of finding the germinal centres. In Sjögren's syndrome, the source is usually biopsies of the small labial salivary glands of which there is a large number; as g.c.s are only present in some of the many small labial salivary glands, they may easily be overlooked. Tissues containing different types of cells respond in a variety of ways to inflammatory signals and this may also determine whether, and to what extent, lymphoid organ neogenesis occurs. The origin of follicular dendritic cells is unclear but it has been proposed that they develop from precursor cells already present in the tissue, either fibroblasts or fibroblast precursor cells (Park & Choi, 2005). Alternatively, the precursor cells may be induced to migrate into the tissue by the same or similar chemokines as those attracting the B and T-lymphocytes. In several autoimmune diseases (Table 1) and animal models of autoimmune diseases (Astorri *et al.*, 2010; Nacionales *et al.*, 2009), it has been demonstrated that ectopic germinal centres are generating plasma cells secreting pathogenic autoantibodies and, almost certainly, memory B-cells bearing anti-self antigen receptors, implying that they aid the diversification of the autoantibody repertoire and contribute to the maintenance of immune pathology. In addition to autoantibody production, self-reactive B-cells generated in ectopic germinal centres may also contribute to autoimmune pathology by secretion of pro-inflammatory cytokines and activation of pathogenic T-cells by presentation of processed self-antigens. Bcells may contribute in this way to immune pathology in autoimmune diseases generally considered to be principally T-cell mediated, and may be one explanation for the efficacy of Rituximab therapy for rheumatoid arthritis.
