**5. The ectopic germinal centre response in breast cancer**

In autoimmune diseases there is a failure of immunological tolerance resulting in an immune response to self-antigens, causing pathological damage to the target organ and tissues, the nature of the target tissue depending on the specificity of the response. In malignancy the immune response, if it occurs, is similar in that it is essentially directed against self, i.e. tumour-associated, antigens. These antigens may be mutated, altered by metabolic processes, or merely aberrantly or over-expressed on the tumour. The problem, however, is converse to autoimmune disease in that, whereas in autoimmune disease the aim is to suppress the immune response, preferably specifically against the target organ, in cancer the hope is that it will be possible to boost the immune response which is often too weak to overcome the rapidly growing tumour.

Several autoimmune disorders sometimes associate with certain tumours, most often small cell lung cancer, breast or ovarian carcinomas, ovarian teratomas, neuroblastomas and lymphomas (with Sjögren's syndrome), reviewed by Lang & Vincent and Rosenfeld *et al.*  (Lang & Vincent, 2009; Rosenfeld & Dalmau, 2010). In the examples studied, the target autoantigen(s) are expressed on the tumour which seems to autoimmunise against them. Indeed, if the tumour is removed, autoantibody levels often decline (Chalk *et al.*, 1990). In many syndromes, the autoimmune disorder serves as a valuable early warning of the associated tumour, and may even slow its growth (Maddison & Lang, 2008).

#### **5.1 Pathology of breast cancer**

Breast cancer is the second most common malignancy in women, accounting for 31% of all types of cancer, with a lifetime incidence in the U.K. of 1/8 in women and c.1/1000 in men. Despite advances in screening, diagnosis and therapy, 12,000 women die of breast cancer each year in the U.K. and the global incidence in females is 23%, but there are marked variations between different regions, it being the highest in Western Europe, Australia, New Zealand and North America. The incidence is relatively low in Asian and African countries (figures from Cancer Research UK). There are several different histopathological types of breast cancer, of which the major types are the ductal and lobular carcinomas, either of which can be *in situ* or invasive, the *in situ* type being considered a possible precursor of invasive carcinoma. Ductal and lobular carcinomas *in situ* are confined to the mammary ducts and lobules and have a very high cure rate, approaching 100%. Invasive carcinomas account for the majority of breast cancers and have a much poorer prognosis. Malignant cell growth appears to start in the ducts and lobules and then invades the surrounding tissues and ultimately metastasises to other tissues and organs. A less common type is medullary carcinoma, comprising only c.1 – 5% of breast cancers; this typically has heavy infiltrates of B-lymphocytes and a significantly better prognosis than the invasive ductal and lobular types. Length of disease free survival in breast cancer is unpredictable, with relapse occurring up to ten years post treatment and even beyond; it has been postulated that this may be due to host factors, including the nature and extent of the immune response.

#### **5.2 The immune response to breast cancer**

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

As previously described in other autoimmune diseases, a role for B-cells in IM is implied by the clinical improvement in patients administered Rituximab® therapy, including improvements in muscle strength. Some patients relapsed as their B-cell pools repopulated and depletion of autoantibody titres was variable (Chung, Genovese, & Fiorentino, 2007; Cooper et al., 2007; Levine, 2005). The potential of B-cells as therapeutic targets is further supported by the elevations in serum levels and gene expression of B-cell activating factor (BAFF) in IM patients, a cytokine crucial for B-cell maturation and survival, which is also thought to play a role in autoantibody production (Krystufkova *et al.*, 2008; Salajegheh *et al.*,

Despite all the evidence described here implicating B-cells and loss of B-cell tolerance in the IM, numerous questions still remain to be resolved, including identification of the stimulating antigens and epitopes, sequence characteristics and pathogenicity of highaffinity, antigen-specific antibodies produced *in situ*, and the factors regulating and controlling these autoimmune reactions. The resolution of these questions will enhance our understanding of the immune pathology of IM and facilitate the diagnosis, treatment and

In autoimmune diseases there is a failure of immunological tolerance resulting in an immune response to self-antigens, causing pathological damage to the target organ and tissues, the nature of the target tissue depending on the specificity of the response. In malignancy the immune response, if it occurs, is similar in that it is essentially directed against self, i.e. tumour-associated, antigens. These antigens may be mutated, altered by metabolic processes, or merely aberrantly or over-expressed on the tumour. The problem, however, is converse to autoimmune disease in that, whereas in autoimmune disease the aim is to suppress the immune response, preferably specifically against the target organ, in cancer the hope is that it will be possible to boost the immune response which is often too

Several autoimmune disorders sometimes associate with certain tumours, most often small cell lung cancer, breast or ovarian carcinomas, ovarian teratomas, neuroblastomas and lymphomas (with Sjögren's syndrome), reviewed by Lang & Vincent and Rosenfeld *et al.*  (Lang & Vincent, 2009; Rosenfeld & Dalmau, 2010). In the examples studied, the target autoantigen(s) are expressed on the tumour which seems to autoimmunise against them. Indeed, if the tumour is removed, autoantibody levels often decline (Chalk *et al.*, 1990). In many syndromes, the autoimmune disorder serves as a valuable early warning of the

Breast cancer is the second most common malignancy in women, accounting for 31% of all types of cancer, with a lifetime incidence in the U.K. of 1/8 in women and c.1/1000 in men. Despite advances in screening, diagnosis and therapy, 12,000 women die of breast cancer each year in the U.K. and the global incidence in females is 23%, but there are marked variations between different regions, it being the highest in Western Europe, Australia, New Zealand and North America. The incidence is relatively low in Asian and African countries (figures from Cancer Research UK). There are several different histopathological types of

associated tumour, and may even slow its growth (Maddison & Lang, 2008).

**5. The ectopic germinal centre response in breast cancer** 

**4.3 Conclusions** 

management of these diseases.

weak to overcome the rapidly growing tumour.

**5.1 Pathology of breast cancer** 

2010).

Most breast cancers contain infiltrates of lymphoid cells with large numbers of T-cells, including CD4+ and CD8+ T-cells, and variable numbers of B-cells, natural killer cells and macrophages. The degree of infiltration varies between different types of breast cancer with extensive lymphoid cell infiltrates in ductal carcinoma *in situ* and some invasive ductal and lobular carcinomas (Ben Hur *et al.*, 2002). Most studies have focused on the role of cytotoxic T-cells in tumour immunity, with variable success in attempting to suppress tumour growth by boosting the T-cell response to tumour-associated antigens. Relatively few studies have addressed the role of B-cells and humoral immunity in response to cancers, including breast cancer, despite the observation that c.40% of ductal breast carcinomas have significant B-cell infiltration.

There is increasing evidence that B-cells play important dual opposing roles in the immune response to tumours; on the one hand as antigen presenting cells and producers of cytotoxic antibodies effective at killing tumour cells by antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell lysis, and as tumour antigen-presenting cells capable of very efficient T-cell activation; on the other hand as promoters of inflammation aiding tumour progression (de Visser, Korets, & Coussens, 2005; de Visser, Eichten, & Coussens, 2006). These seemingly contradictory effects may be due to the difference between a specific, high affinity immune response to antigen versus a low affinity, polyclonal response, or even suppression of the cytotoxic immune response via regulatory B-cells (Mauri, 2010). The importance of antibodies in eliminating tumours is clearly demonstrated by the results of treatment of breast cancer patients with humanised monoclonal antibodies (MAbs) specific for the epidermal growth factor receptor HER-2 (trastuzumab/herceptin and pertuzumab). Not only is herceptin effective in slowing down the progression of established metastatic disease, it has also recently been demonstrated to prevent the emergence of metastases when given as an adjuvant treatment (Hortobagyi, 2005). Pertuzumab has also yielded promising results in clinical trials (Bianco, 2004). Synergistic effects between herceptin and pertuzumab suggest promising new approaches to therapy using cocktails of antibodies (Nahta, Hung, & Esteva, 2004) and elucidation of the molecular structure of the herceptin Fab/HER-2 complex (Cho *et al.*, 2003) allows rational design of therapeutic anti-HER-2 antibodies. MAbs specific for other tumour-associated antigens (TAAs) are needed to work synergistically with trastuzumab and to treat patients who do not overexpress HER-2.

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 419

from the sentinel node to the tumour; this could have been due to insufficient sample sizes

Fig. 10. Examples of proliferating, hypermutating B-cell clones from the breast tumours of four

The best matching germline VH-gene is shown at the origin of each tree. The founder rearranged VH-genes of three of these B-cell clones already appear to be mutated, implying that they originated from memory Bcells, whereas clone A1 appears to have been founded by a naïve B-cell. Genealogical trees were constructed and mutations numbered as described in the legend to Figure 5. Reproduced from Nzula *et al.* 

In order to identify the antigens driving the immune response within the tumour, we reconstructed the antigen receptors expressed by germinal centre B-cells as scFv antibodies

(Nzula, Going & Stott, 2003a; Simsa *et al.*, 2005).

ductal carcinoma patients.

**5.5 Cloning and characterisation of scFv antibodies** 

*(*2003).

Several molecules have been identified that are either over-expressed, mutated, or structurally modified on tumour cells and are therefore potential targets for immunotherapy, including HER-1, HER-2, MUC-1 and p53 (Taylor-Papadimitriou *et al.*, 2002). Some TAAs appear to overcome tolerance and induce a natural immune response as a result of mutation or altered expression; humoral immune responses to these antigens in breast cancer patients are associated with better early disease stage-specific survival (Angelopoulou *et al.*, 2000;Visco *et al.*, 2000; von Mensdorff-Pouilly *et al.*, 2000) and anti-MUC-1 antibodies are cytotoxic to tumour cells (Snijdewint *et al.*, 2001). TAA-specific tumour infiltrating (TIL) B-lymphocytes and recombinant antibodies have been isolated from both tumour (Kotlan *et al.*, 2000; Kotlan *et al.*, 2005) and lymph nodes (Petrarca *et al.*, 1999; Rothe *et al.*, 2004) of medullary and ductal carcinoma patients, showing that they are responding specifically to the tumour. Evasion of the immune response by the tumour can be overcome by passive immunotherapy or active immunisation regimes. B-cells actively responding in the draining lymph node and tumour are therefore ideal sources to study the immune response to the tumour and provide the most relevant source of potentially therapeutic antibodies.

#### **5.3 Ductal carcinoma infiltrating lymphocytes are clustered into germinal centres**

We and others found infiltrating lymphocytes in ductal carcinomas were aggregated into clusters containing T-cells, B-cells and follicular dendritic cells with plasmablasts/plasma cells in and around the aggregates (Coronella *et al.*, 2002; Nzula, Going, & Stott, 2003a). These cell clusters appeared to be similar to those seen in the target tissues of autoimmune diseases except that there was no mantle zone (also absent in the salivary glands of patients with Sjögren's syndrome (Stott *et al.*, 1998)), so we examined whether they were responding as germinal centres.

#### **5.4 The Ig V-gene repertoire and clonal proliferation of B-cells in ductal carcinoma**

We cloned and sequenced 401 rearranged Ig VH-genes from microdissected tumourinfiltrating B-cell foci of 7 patients with invasive ductal carcinoma and 271 VH-genes from paired sentinel lymph nodes of 3 of the patients. 15 sets of VH-genes from clonally related Bcells within individual foci were identified by their shared VH, D, JH and CDR3 sequences, showing that proliferating, mutating B-cell clones were present in lymphoid foci and that these foci were undergoing a germinal centre response within the tumour, similar to the ectopic germinal centres we have observed in the target tissues of autoimmune diseases (Fig. 10). There was preferential usage of certain VH, D & JH exons, indicating selection of Bcells expressing antigen receptors encoded by these gene combinations. VH & VL-genes from proliferating B-cell clones contained numerous mutations, demonstrating that the somatic hypermutation machinery was switched on within the cell cluster, again characteristic of a germinal centre response. Analysis of the pattern of mutations showed that the B-cell clones are undergoing an antigen-driven response accompanied by selection of specific mutations and affinity maturation *in situ*. Clone founder cells were of both naïve and memory B-cell type, showing that a secondary response involving memory B-cells was taking place, but also new B-cells that had not previously encountered antigen moved into cell clusters and were stimulated by antigen. We also cloned rearranged VH-genes from microdissected germinal centres in the paired sentinel lymph node and identified proliferating, hypermutating B-cell clones there too. These also revealed selection for particular VH & JHgenes showing selection by antigen for B-cells expressing these genes during the immune response but we did not find evidence that members of the same B-cell clones had migrated

Several molecules have been identified that are either over-expressed, mutated, or structurally modified on tumour cells and are therefore potential targets for immunotherapy, including HER-1, HER-2, MUC-1 and p53 (Taylor-Papadimitriou *et al.*, 2002). Some TAAs appear to overcome tolerance and induce a natural immune response as a result of mutation or altered expression; humoral immune responses to these antigens in breast cancer patients are associated with better early disease stage-specific survival (Angelopoulou *et al.*, 2000;Visco *et al.*, 2000; von Mensdorff-Pouilly *et al.*, 2000) and anti-MUC-1 antibodies are cytotoxic to tumour cells (Snijdewint *et al.*, 2001). TAA-specific tumour infiltrating (TIL) B-lymphocytes and recombinant antibodies have been isolated from both tumour (Kotlan *et al.*, 2000; Kotlan *et al.*, 2005) and lymph nodes (Petrarca *et al.*, 1999; Rothe *et al.*, 2004) of medullary and ductal carcinoma patients, showing that they are responding specifically to the tumour. Evasion of the immune response by the tumour can be overcome by passive immunotherapy or active immunisation regimes. B-cells actively responding in the draining lymph node and tumour are therefore ideal sources to study the immune response to the tumour and provide the most

**5.3 Ductal carcinoma infiltrating lymphocytes are clustered into germinal centres**  We and others found infiltrating lymphocytes in ductal carcinomas were aggregated into clusters containing T-cells, B-cells and follicular dendritic cells with plasmablasts/plasma cells in and around the aggregates (Coronella *et al.*, 2002; Nzula, Going, & Stott, 2003a). These cell clusters appeared to be similar to those seen in the target tissues of autoimmune diseases except that there was no mantle zone (also absent in the salivary glands of patients with Sjögren's syndrome (Stott *et al.*, 1998)), so we examined whether they were responding

**5.4 The Ig V-gene repertoire and clonal proliferation of B-cells in ductal carcinoma**  We cloned and sequenced 401 rearranged Ig VH-genes from microdissected tumourinfiltrating B-cell foci of 7 patients with invasive ductal carcinoma and 271 VH-genes from paired sentinel lymph nodes of 3 of the patients. 15 sets of VH-genes from clonally related Bcells within individual foci were identified by their shared VH, D, JH and CDR3 sequences, showing that proliferating, mutating B-cell clones were present in lymphoid foci and that these foci were undergoing a germinal centre response within the tumour, similar to the ectopic germinal centres we have observed in the target tissues of autoimmune diseases (Fig. 10). There was preferential usage of certain VH, D & JH exons, indicating selection of Bcells expressing antigen receptors encoded by these gene combinations. VH & VL-genes from proliferating B-cell clones contained numerous mutations, demonstrating that the somatic hypermutation machinery was switched on within the cell cluster, again characteristic of a germinal centre response. Analysis of the pattern of mutations showed that the B-cell clones are undergoing an antigen-driven response accompanied by selection of specific mutations and affinity maturation *in situ*. Clone founder cells were of both naïve and memory B-cell type, showing that a secondary response involving memory B-cells was taking place, but also new B-cells that had not previously encountered antigen moved into cell clusters and were stimulated by antigen. We also cloned rearranged VH-genes from microdissected germinal centres in the paired sentinel lymph node and identified proliferating, hypermutating B-cell clones there too. These also revealed selection for particular VH & JHgenes showing selection by antigen for B-cells expressing these genes during the immune response but we did not find evidence that members of the same B-cell clones had migrated

relevant source of potentially therapeutic antibodies.

as germinal centres.

from the sentinel node to the tumour; this could have been due to insufficient sample sizes (Nzula, Going & Stott, 2003a; Simsa *et al.*, 2005).

Fig. 10. Examples of proliferating, hypermutating B-cell clones from the breast tumours of four ductal carcinoma patients.

The best matching germline VH-gene is shown at the origin of each tree. The founder rearranged VH-genes of three of these B-cell clones already appear to be mutated, implying that they originated from memory Bcells, whereas clone A1 appears to have been founded by a naïve B-cell. Genealogical trees were constructed and mutations numbered as described in the legend to Figure 5. Reproduced from Nzula *et al. (*2003).

#### **5.5 Cloning and characterisation of scFv antibodies**

In order to identify the antigens driving the immune response within the tumour, we reconstructed the antigen receptors expressed by germinal centre B-cells as scFv antibodies

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 421

A

B Fig. 11. Exponential enrichment of scFv-phage after panning and elution on a breast tumour

A. VH/Vκ and VH/Vλ scFv mini-libraries, using VH-genes from B-cell clone D4, panned on pooled heterologous tumour extract The eluate from each panning was then subjected to further cycles of panning and elution; B. Global VH/Vκ and VH/Vλ scFv-phage libraries from a human breast tumour,

panned on the same heterologous tumour extract as in A.

extract

by cloning VH and VL-genes into phagemid (pHEN2), as described in section 2.3, to generate scFv-phage libraries expressing randomly assorted combinations of VH and VL-genes. These "mini-libraries" were made from the B-cells in individual germinal centres within the tumour and are therefore much smaller and more restricted than the very large libraries normally made by random combination of large pools of VH and VL-genes. Two scFv-phage mini-libraries were constructed from a germinal centre incorporating all the VH-genes pooled from the largest proliferating B-cell clone (D4 in Fig. 10) linked randomly to either the rearranged Vκ-genes or the rearranged Vλ-genes amplified from the same germinal centre.

Tumour-binding scFv were selected from the mini-libraries by three or four cycles of panning and elution on a heterologous tumour homogenate pooled from breast tumours of 5 patients. During the panning cycles we observed exponential enrichment of the Vλ minilibrary, but not the Vκ mini-library, indicating that scFv within the Vλ mini-library bind specifically to tumour-associated antigens (Fig. 11A). This is consistent with the scFvlibraries being derived from the same B-cell clone, since a single B-cell clone uses either a κ or λ light chain, not both. 13 scFv-phages binding to the tumour extract were cloned and their specificity for tumour tissue confirmed by ELISA. 7 scFv-phage clones that bound to the tumour extract were identified for further characterisation. All 7 used the same combination of VH3-23 with exons D1-26 & JH2, expressed by B-cell clone D4, and the light chain gene Vλ1c with Jλ3b.

We also constructed two scFv-phage libraries from DNA extracted from a whole sample of tumour tissue, as described for the mini-libraries. The 2 libraries were panned on the same heterologous tumour homogenate as used with the mini-libraries. After 4 cycles of panning we observed an enrichment of several logs for both libraries, indicating the presence of tumour-specific antibodies (Fig. 11B). The enrichment of both global libraries shows that tumour-specific B-cells derived from independent B-cell clones were present in the tumour, as expected. 19 scFv-phages were cloned from the 2 global libraries and their specificity for tumour tissue confirmed by ELISA using the same tumour homogenate as source of antigen.

#### **5.6 Identification of the specificity of proliferating B-cells**

Since the scFvs from the VH/Vλ mini-library were derived from proliferating B-cell clone D4, their sequences and antigen specificities reveal the nature of the genes and antigen receptor specificities of the original germinal centre B-cells. We therefore sequenced the scFv clones that showed the strongest binding to the tumour extract and performed a Blast search of the Genbank gene database. The VHDJH heavy chain gene used by all members of B-cell clone D4 exhibited 89% homology with a human anti-HER3 MAb (AF048774) and the VλJλ light chain gene, also used by the same B-cells, matched a human anti-EGFR antibody (DQ666353.1) with 96% homology. These, and scFvs from the global libraries, were tested for binding to recombinant antigens from the epidermal growth factor receptor family: HER-2, HER-3 and HER-4, kindly provided by Genentech (San Francisco, USA) and Pharmexa A/S (Hørsholm, Denmark) by ELISA. Six scFvs from B-cell clone D4 and one from the global tumour library bound to recombinant HER-2, HER-3 & HER-4, indicating that they recognised a shared epitope expressed by all three members of this EGFR family of receptors (Fig. 12). Specificity for HER-2, HER-3 & HER-4 was confirmed using soluble scFv produced in the non-suppressor strain of *E.coli*.

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

by cloning VH and VL-genes into phagemid (pHEN2), as described in section 2.3, to generate scFv-phage libraries expressing randomly assorted combinations of VH and VL-genes. These "mini-libraries" were made from the B-cells in individual germinal centres within the tumour and are therefore much smaller and more restricted than the very large libraries normally made by random combination of large pools of VH and VL-genes. Two scFv-phage mini-libraries were constructed from a germinal centre incorporating all the VH-genes pooled from the largest proliferating B-cell clone (D4 in Fig. 10) linked randomly to either the rearranged Vκ-genes or the rearranged Vλ-genes amplified from the same germinal

Tumour-binding scFv were selected from the mini-libraries by three or four cycles of panning and elution on a heterologous tumour homogenate pooled from breast tumours of 5 patients. During the panning cycles we observed exponential enrichment of the Vλ minilibrary, but not the Vκ mini-library, indicating that scFv within the Vλ mini-library bind specifically to tumour-associated antigens (Fig. 11A). This is consistent with the scFvlibraries being derived from the same B-cell clone, since a single B-cell clone uses either a κ or λ light chain, not both. 13 scFv-phages binding to the tumour extract were cloned and their specificity for tumour tissue confirmed by ELISA. 7 scFv-phage clones that bound to the tumour extract were identified for further characterisation. All 7 used the same combination of VH3-23 with exons D1-26 & JH2, expressed by B-cell clone D4, and the light

We also constructed two scFv-phage libraries from DNA extracted from a whole sample of tumour tissue, as described for the mini-libraries. The 2 libraries were panned on the same heterologous tumour homogenate as used with the mini-libraries. After 4 cycles of panning we observed an enrichment of several logs for both libraries, indicating the presence of tumour-specific antibodies (Fig. 11B). The enrichment of both global libraries shows that tumour-specific B-cells derived from independent B-cell clones were present in the tumour, as expected. 19 scFv-phages were cloned from the 2 global libraries and their specificity for tumour tissue confirmed by ELISA using the same tumour homogenate as

Since the scFvs from the VH/Vλ mini-library were derived from proliferating B-cell clone D4, their sequences and antigen specificities reveal the nature of the genes and antigen receptor specificities of the original germinal centre B-cells. We therefore sequenced the scFv clones that showed the strongest binding to the tumour extract and performed a Blast search of the Genbank gene database. The VHDJH heavy chain gene used by all members of B-cell clone D4 exhibited 89% homology with a human anti-HER3 MAb (AF048774) and the VλJλ light chain gene, also used by the same B-cells, matched a human anti-EGFR antibody (DQ666353.1) with 96% homology. These, and scFvs from the global libraries, were tested for binding to recombinant antigens from the epidermal growth factor receptor family: HER-2, HER-3 and HER-4, kindly provided by Genentech (San Francisco, USA) and Pharmexa A/S (Hørsholm, Denmark) by ELISA. Six scFvs from B-cell clone D4 and one from the global tumour library bound to recombinant HER-2, HER-3 & HER-4, indicating that they recognised a shared epitope expressed by all three members of this EGFR family of receptors (Fig. 12). Specificity for HER-2, HER-3 & HER-4 was confirmed using soluble scFv

**5.6 Identification of the specificity of proliferating B-cells** 

produced in the non-suppressor strain of *E.coli*.

centre.

chain gene Vλ1c with Jλ3b.

source of antigen.

A

Fig. 11. Exponential enrichment of scFv-phage after panning and elution on a breast tumour extract

A. VH/Vκ and VH/Vλ scFv mini-libraries, using VH-genes from B-cell clone D4, panned on pooled heterologous tumour extract The eluate from each panning was then subjected to further cycles of panning and elution; B. Global VH/Vκ and VH/Vλ scFv-phage libraries from a human breast tumour, panned on the same heterologous tumour extract as in A.

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 423

It has become increasingly clear that infiltrating B- and T-lymphocytes organise themselves into ectopic lymphoid follicles and germinal centres within tissues undergoing inflammatory processes. This has been observed in several autoimmune diseases (Table 1), usually within the target organ or tissue, myasthenia gravis being the exception to the rule for reasons discussed in section 3.5. However, ectopic g.c.s are not restricted to autoimmune diseases but can also develop in other chronic inflammatory diseases, such as Crohn's disease and ulcerative colitis; at sites of infection such as the liver during chronic hepatitis C virus infection and the skin of oncocerciasis patients (Brattig *et al.*, 2010); and in neoplasias, including lymphoma of the mucosal-associated lymphoid tissue associated with Sjögren's

syndrome (Bombardieri *et al.*, 2007a) and, as shown here, in breast cancer (Table 1).

formation may be secondary to inflammation (Aloisi & Pujol-Borrell, 2006).

germinal centre reaction (Adams *et al.*, 2003).

How these ectopic g.c.s develop, their role in the pathology of autoimmune diseases, and in combating infections and malignancies is still unclear but evidence is beginning to emerge. Cytokines, chemokines and signalling molecules involved in lymphoid neogenesis in the secondary lymphoid organs also appear to be required for ectopic g.c. formation, including lymphotoxins-α, β and α1β2, TNFα, Grb2, the chemokine receptor CXCR5, its ligand CXCL15, and the B-cell attracting chemokines CXCL13 and CCL21, in this case due to release of these molecules within the inflammatory environment, suggesting that g.c.

When reports first emerged of germinal centre-like structures within the target tissues of autoimmune diseases, there was some scepticism regarding whether these structures were involved in true germinal centre reactions. These doubts have now been dispelled. Identification of dark and light zones and a follicular dendritic cell network in intimate contact with B-cells was highly suggestive of a germinal centre reaction, especially when it was shown that autoantigen was trapped on the follicular dendritic cell processes, e.g. (SHIONO *et al.*, 2003). Studies by us and other researchers have shown that the B-cells within ectopic germinal centres are activated to antigen-driven clonal proliferation, somatic hypermutation and class switching, similar to the response in orthotopic g.c.s responding to foreign antigens. That they switch on the somatic hypermutation machinery has been shown in several autoimmune diseases by sequencing studies of the expressed, rearranged Ig Vgenes cloned from microdissected g.c.s. This has been confirmed in the salivary gland g.c.s of Sjögren's patients and the synovial g.c.s of rheumatoid arthritis patients by identification of activation induced cytidine deaminase (AID), a key enzyme in somatic hypermutation and class switch recombination (Bombardieri *et al.*, 2007b; Humby *et al.*, 2009). Interestingly, expression of AID has recently been observed in hyperplastic fibroblasts of rheumatoid arthritis patients (Igarashi et al., 2010). Expression correlated with mutations in the *p53* gene and was induced by TNFα *in vitro*. AID is known to induce mutations in non-Ig genes at a lower frequency and it was suggested that the mutations of this tumour suppressor gene may be the cause of the fibroblast hyperplasia. Affinity maturation of B-cell receptors during somatic hypermutation has been demonstrated by analysis of replacement mutations, although early analyses failed to take into account the bias towards replacement mutations in the CDRs resulting from targeting of AID to sequence motifs such as RGYW. Final confirmation requires direct affinity measurements of autoantibodies cloned from germinal centre B-cells and/or by 3-D molecular modelling of the antigen-binding site bound to its epitope, as we have shown for anti-hen egg lysozyme antibodies produced in an orthotopic

**6. General conclusions** 

Fig. 12. EGFR family specificity of scFv antibodies cloned from a mammary carcinoma germinal centre.

Individual scFv-phage (80, 96, 14 & 107 & 120) from B-cell clone D4, proliferating and mutating in a breast tumour, and scFv-phage (471), cloned from whole breast tumour tissue, bind to members of the epidermal growth factor receptor family Her-2, Her-3 & Her-4.

### **5.7 Conclusions**

Clusters of B-cells, T-cells and follicular dendritic cells form within human ductal breast carcinomas, resembling the ectopic germinal centres observed in the target tissues of patients with autoimmune diseases. These clusters of lymphoid cells contain clones of proliferating B-cells that are undergoing somatic hypermutation of their rearranged, Ig V- (D)-J-genes and affinity selection of their B-cell receptors driven, in the case described here, by members of the epidermal growth factor receptor family, viz. HER2, HER3 and HER4. The antibodies produced during this response recognise an epitope shared by these 3 cell surface receptors, which are known to be overexpressed on breast carcinoma cells and several other types of carcinoma, including ovarian cancer. It is very probable that other tumour associated antigens are also able to stimulate a local B-cell response within the tumour, e.g. antibodies specific for a ganglioside were cloned from medullary carcinoma Bcells, although it is not clear whether these are involved in attacking the tumour (Kotlan *et al.*, 2005).

Single chain Fv antibodies cloned from tumour germinal centre B-cells can readily be converted to complete antibodies by splicing their V-genes on to Ig constant regions of any desired isotype. These fully human antibodies can be produced in large quantities in a protein expression system and are therefore potential candidates for diagnosis, monitoring and therapy of breast and other types of cancer.

#### **6. General conclusions**

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

Fig. 12. EGFR family specificity of scFv antibodies cloned from a mammary carcinoma

epidermal growth factor receptor family Her-2, Her-3 & Her-4.

and therapy of breast and other types of cancer.

Individual scFv-phage (80, 96, 14 & 107 & 120) from B-cell clone D4, proliferating and mutating in a breast tumour, and scFv-phage (471), cloned from whole breast tumour tissue, bind to members of the

Clusters of B-cells, T-cells and follicular dendritic cells form within human ductal breast carcinomas, resembling the ectopic germinal centres observed in the target tissues of patients with autoimmune diseases. These clusters of lymphoid cells contain clones of proliferating B-cells that are undergoing somatic hypermutation of their rearranged, Ig V- (D)-J-genes and affinity selection of their B-cell receptors driven, in the case described here, by members of the epidermal growth factor receptor family, viz. HER2, HER3 and HER4. The antibodies produced during this response recognise an epitope shared by these 3 cell surface receptors, which are known to be overexpressed on breast carcinoma cells and several other types of carcinoma, including ovarian cancer. It is very probable that other tumour associated antigens are also able to stimulate a local B-cell response within the tumour, e.g. antibodies specific for a ganglioside were cloned from medullary carcinoma Bcells, although it is not clear whether these are involved in attacking the tumour (Kotlan *et* 

Single chain Fv antibodies cloned from tumour germinal centre B-cells can readily be converted to complete antibodies by splicing their V-genes on to Ig constant regions of any desired isotype. These fully human antibodies can be produced in large quantities in a protein expression system and are therefore potential candidates for diagnosis, monitoring

germinal centre.

**5.7 Conclusions** 

*al.*, 2005).

It has become increasingly clear that infiltrating B- and T-lymphocytes organise themselves into ectopic lymphoid follicles and germinal centres within tissues undergoing inflammatory processes. This has been observed in several autoimmune diseases (Table 1), usually within the target organ or tissue, myasthenia gravis being the exception to the rule for reasons discussed in section 3.5. However, ectopic g.c.s are not restricted to autoimmune diseases but can also develop in other chronic inflammatory diseases, such as Crohn's disease and ulcerative colitis; at sites of infection such as the liver during chronic hepatitis C virus infection and the skin of oncocerciasis patients (Brattig *et al.*, 2010); and in neoplasias, including lymphoma of the mucosal-associated lymphoid tissue associated with Sjögren's syndrome (Bombardieri *et al.*, 2007a) and, as shown here, in breast cancer (Table 1).

How these ectopic g.c.s develop, their role in the pathology of autoimmune diseases, and in combating infections and malignancies is still unclear but evidence is beginning to emerge. Cytokines, chemokines and signalling molecules involved in lymphoid neogenesis in the secondary lymphoid organs also appear to be required for ectopic g.c. formation, including lymphotoxins-α, β and α1β2, TNFα, Grb2, the chemokine receptor CXCR5, its ligand CXCL15, and the B-cell attracting chemokines CXCL13 and CCL21, in this case due to release of these molecules within the inflammatory environment, suggesting that g.c. formation may be secondary to inflammation (Aloisi & Pujol-Borrell, 2006).

When reports first emerged of germinal centre-like structures within the target tissues of autoimmune diseases, there was some scepticism regarding whether these structures were involved in true germinal centre reactions. These doubts have now been dispelled. Identification of dark and light zones and a follicular dendritic cell network in intimate contact with B-cells was highly suggestive of a germinal centre reaction, especially when it was shown that autoantigen was trapped on the follicular dendritic cell processes, e.g. (SHIONO *et al.*, 2003). Studies by us and other researchers have shown that the B-cells within ectopic germinal centres are activated to antigen-driven clonal proliferation, somatic hypermutation and class switching, similar to the response in orthotopic g.c.s responding to foreign antigens. That they switch on the somatic hypermutation machinery has been shown in several autoimmune diseases by sequencing studies of the expressed, rearranged Ig Vgenes cloned from microdissected g.c.s. This has been confirmed in the salivary gland g.c.s of Sjögren's patients and the synovial g.c.s of rheumatoid arthritis patients by identification of activation induced cytidine deaminase (AID), a key enzyme in somatic hypermutation and class switch recombination (Bombardieri *et al.*, 2007b; Humby *et al.*, 2009). Interestingly, expression of AID has recently been observed in hyperplastic fibroblasts of rheumatoid arthritis patients (Igarashi et al., 2010). Expression correlated with mutations in the *p53* gene and was induced by TNFα *in vitro*. AID is known to induce mutations in non-Ig genes at a lower frequency and it was suggested that the mutations of this tumour suppressor gene may be the cause of the fibroblast hyperplasia. Affinity maturation of B-cell receptors during somatic hypermutation has been demonstrated by analysis of replacement mutations, although early analyses failed to take into account the bias towards replacement mutations in the CDRs resulting from targeting of AID to sequence motifs such as RGYW. Final confirmation requires direct affinity measurements of autoantibodies cloned from germinal centre B-cells and/or by 3-D molecular modelling of the antigen-binding site bound to its epitope, as we have shown for anti-hen egg lysozyme antibodies produced in an orthotopic germinal centre reaction (Adams *et al.*, 2003).

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 425

Allen, C. D. C., Okada, T., & Cyster, J. G. 2007, "Germinal-Center Organization and Cellular

Aloisi, F. & Pujol-Borrell, R. 2006, "Lymphoid neogenesis in chronic inflammatory diseases",

Amemiya, K., Granger, R. P., & Dalakas, M. C. 2000, "Clonal restriction of T-cell receptor

Arahata, K. & Engel, A. G. 1984, "Monoclonal antibody analysis of mononuclear cells in

Armengol, M. P., Juan, M., Lucas-Martin, A., Fernandez-Figueras, M. T., Jaraquemada, D.,

Astorri, E., Bombardieri, M., Gabba, S., Peakman, M., Pozzilli, P., & Pitzalis, C. 2010,

Beltman, J. B., Allen, C. D. C., Cyster, J. G., & de Boer, R. J. 2011, "B cells within germinal

Ben Hur, H., Cohen, O., Schneider, D., Gurevich, P., Halperin, R., Bala, U., Mozes, M., &

Berrih-Aknin, S., Ruhlmann, N., Bismuth, J., Cizeron-Clairac, G., Zelman, E., Shachar, I.,

Bianco, A. R. 2004, "Targeting c-erbB2 and other receptors of the c-erbB family: rationale and

Bohan, A. & Peter, J. B. 1975a, "Polymyositis and dermatomyositis (first of two parts)",

Bohan, A. & Peter, J. B. 1975b, "Polymyositis and dermatomyositis (second of two parts)",

Bombardieri, M., Barone, F., Humby, F., Kelly, S., McGurk, M., Morgan, P., Challacombe, S.,

De Vita, S., Valesini, G., Spencer, J., & Pitzalis, C. 2007a, "Activation-induced cytidine deaminase expression in follicular dendritic cell networks and interfollicular large B cells supports functionality of ectopic lymphoid neogenesis in

clinical applications", *J Chemother.*, vol. 16 Suppl 4, pp. 52-54.

patients with breast cancer.", *Clin.Biochem.*, vol. 33, pp. 53-62.

expression by infiltrating lymphocytes in inclusion body myositis persists over time. Studies in repeated muscle biopsies", *Brain*, vol. 123 ( Pt 10), pp. 2030-2039. Angelopoulou, K., Yu, H., Bharaj, B., Giai, M., & Diamandis, E. P. 2000, "p53 gene mutation,

tumor p53 protein overexpression, and serum p53 autoantibody generation in

myopathies. I: Quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cells",

Gallart, T., & Pujol-Borrell, R. 2001, "Thyroid autoimmune disease: demonstration of thyroid antigen-specific B cells and recombination-activating gene expression in chemokine-containing active intrathyroidal germinal centers", *American Journal of* 

"Evolution of Ectopic Lymphoid Neogenesis and In Situ Autoantibody Production in Autoimmune Nonobese Diabetic Mice: Cellular and Molecular Characterization of Tertiary Lymphoid Structures in Pancreatic Islets", *The Journal of Immunology*, vol.

centers migrate preferentially from dark to light zone", *Proceedings of the National* 

Zusman, I. 2002, "The role of lymphocytes and macrophages in human breast tumorigenesis: an immunohistochemical and morphometric study", *Anticancer Res*,

Dartevelle, P., De Rosbo, N. K., & Le Panse, R. 2009, "CCL21 overexpressed on lymphatic vessels drives thymic hyperplasia in myasthenia", *Ann.Neurol.*, vol. 66,

Dynamics", *Immunity*, vol. 27, pp. 190-202.

*Ann.Neurol.*, vol. 16, no. 2, pp. 193-208.

*Academy of Sciences*, vol. 108, pp. 8755-8760.

*N.Engl.J.Med.*, vol. 292, no. 7, pp. 344-347.

*N.Engl.J.Med.*, vol. 292, no. 8, pp. 403-407.

*Pathology*, vol. 159, pp. 861-873.

185, pp. 3359-3368.

vol. 22, pp. 1231-1238.

pp. 521-531.

*Nature Reviews Immunology*, vol. 6, pp. 205-217.

In several cases it has been shown that the autoantibodies generated in ectopic g.c.s have similar specificities to the autoantibodies found in the blood, notably in Hashimoto's thyroiditis, Sjögren's syndrome and rheumatoid arthritis, suggesting that the g.c.s contribute to pathological mechanisms, although whether they are critical in the early stages of development of the disease, or only contribute to its maintenance once the initial tissue damage has commenced, has yet to be established. Production of cytokines and chemokines at sites of damage that attract lymphocytes and contribute to lymphoid neogenesis suggests that the latter may be the more likely scenario. Nevertheless, a detailed understanding of the mechanisms involved in generation of ectopic g.c. structures and maintenance of production of plasma cells and memory B-cells producing potentially pathogenic antibodies is essential for a full understanding of the pathology of autoimmune disease and holds promise for developing new methods of therapy, based on controlling this response or inducing immunological tolerance to the autoantigens.

Even more work needs to be done to determine the role of ectopic g.c.s in other diseases, including sterile and infectious chronic inflammatory diseases and cancer. What other types of cancer, in addition to breast cancer and lymphoma, induce germinal centre reactions within the tumour and the nature of their response in elimination of cancer cells have yet to be determined. The identification of intra-tumour g.c.s producing antibodies and memory Bcells with specificity for members of the epidermal growth factor receptor family holds out hope that therapeutic vaccines can be developed to boost this response for therapy of breast cancer and, potentially other neoplasias, such as ovarian cancer, in which these molecules are overexpressed. Experimental approaches using mouse models of breast cancer support this optimism (Renard *et al.*, 2003; Renard & Leach, 2007; Mukhopadhyay, MS in preparation). Cloning of antibodies against tumour-associated antigens from intra-tumour g.c.s is also a novel way of producing fully human antibodies for passive immunotherapy.
