**4. Tissue-infiltrating B-cells in inflammatory myopathies**

### **4.1 Introduction**

The inflammatory myopathies (IM), collectively called myositis, are classified into three principal subsets, Dermatomyositis (DM), Polymyositis (PM) and Inclusion Body Myositis (IBM) (Bohan & Peter, 1975a; Bohan & Peter, 1975b; Dalakas & Hohlfeld, 2003). Each of these disorders is characterised by moderate to severe muscle weakness and muscle fatigue with inflammatory mononuclear cell infiltration within the muscle, but each disorder has distinct clinical and pathological features. IM can be associated with various autoimmune and connective tissue disorders as well as malignancies, the latter being associated with up to 45% of adult DM patients.

DM, the most common of the inflammatory myopathies, is a multi-organ disease not only affecting skeletal muscle but, often, the skin as well as other tissues and is more commonly found in women than men; it also accounts for up to c.85% of all juvenile IM (Rider, 2007). DM is characterised by a heliotrope rash on the upper eyelid, face or upper trunk accompanying, or more commonly preceding, proximal muscle weakness. Muscle inflammation is predominantly perivascular and/or perimysial or in the interfascicular septae and around, rather than within, the muscle fascicles. Perivascular atrophy is a characteristic feature of DM patients, often in groups at the periphery of the fascicle. In DM, muscle lymphocytic infiltrates consist largely of B-cells and CD4+ T-cells (Arahata & Engel, 1984; Engel & Arahata, 1984) suggesting that DM may be a humorally mediated immune response.

PM and IBM, though separate disorders, are both characterised by scattered necrotic and regenerating muscle fibres and endomysial inflammation with invasion and destruction of non-necrotic muscle fibres. PM generally becomes evident in adulthood and is best defined as a subacute myopathy that evolves over weeks to months and presents with symmetrical weakness of the proximal muscles. Its clinical onset is hard to define with no early recognition signs such as the rash observed in DM. PM is uncommon as a stand-alone disorder and more commonly associates with other autoimmune and connective tissue disorders.

Onset of IBM is usually after the age of 50 and occurs more frequently in men. Muscle weakness can be both proximal and distal and is often asymmetrical. Despite similarities with PM, distinctive features of IBM include: rimmed vacuoles; groups of atrophic fibres; increased lymphocytic invasion of non-necrotic fibres; less frequent myofibre necrosis; and a more slowly progressing clinical course with patients being unresponsive to treatment. In both disorders, inflammatory infiltrates typically consist of CD8+ T-cells and macrophages (Arahata & Engel, 1984; Engel & Arahata, 1984) which invade MHC Class 1 antigenexpressing muscle fibres, a feature absent in normal muscle tissue, leading to fibre necrosis. The muscle fibre invading CD8+ T-cells can be clonally expanded in both PM and IBM (Dalakas, 2004; Fyhr *et al.*, 1997; Hofbauer *et al.*, 2003; Mantegazza *et al.*, 1993; Seitz *et al.*, 2006), which persists over time (Amemiya, Granger, & Dalakas, 2000).

#### **4.1.1 Autoantibodies associated with myositis**

As with most autoimmune disorders, different autoantibody specificities have been described in DM and PM; autoantibodies are generally absent from IBM although they have been detected in a small number of cases (Dalakas *et al.*, 1997). They can either be myositisspecific (MSAs) or myositis-associated autoantibodies (MAAs), which can also be associated with other autoimmune diseases. Most bind to protein or ribonucleoprotein complexes involved in protein synthesis, translocation or elongation; MAA target antigens are primarily located in the nucleoplasm or nucleolus. The most prevalent MSAs are directed against amino-acyl-tRNA-synthetases (ARS), and are associated with a distinctive clinical phenotype, anti-synthetase syndrome, characterised by myositis, Raynaud's phenomenon and interstitial lung disease, with a higher mortality. Anti-Jo-1 (anti-histidyl-tRNA synthetase) antibodies are the most prevalent in myositis patients (20-30% of patients), while the other anti-ARS antibodies are only present in 1-3% of IM patients, and are a diagnostic and prognostic marker for disease severity (Mielnik *et al.*, 2006; Zampieri *et al.*, 2005).

#### **4.1.2 Muscle-infiltrating B-cells in myositis**

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

The inflammatory myopathies (IM), collectively called myositis, are classified into three principal subsets, Dermatomyositis (DM), Polymyositis (PM) and Inclusion Body Myositis (IBM) (Bohan & Peter, 1975a; Bohan & Peter, 1975b; Dalakas & Hohlfeld, 2003). Each of these disorders is characterised by moderate to severe muscle weakness and muscle fatigue with inflammatory mononuclear cell infiltration within the muscle, but each disorder has distinct clinical and pathological features. IM can be associated with various autoimmune and connective tissue disorders as well as malignancies, the latter being associated with up to

DM, the most common of the inflammatory myopathies, is a multi-organ disease not only affecting skeletal muscle but, often, the skin as well as other tissues and is more commonly found in women than men; it also accounts for up to c.85% of all juvenile IM (Rider, 2007). DM is characterised by a heliotrope rash on the upper eyelid, face or upper trunk accompanying, or more commonly preceding, proximal muscle weakness. Muscle inflammation is predominantly perivascular and/or perimysial or in the interfascicular septae and around, rather than within, the muscle fascicles. Perivascular atrophy is a characteristic feature of DM patients, often in groups at the periphery of the fascicle. In DM, muscle lymphocytic infiltrates consist largely of B-cells and CD4+ T-cells (Arahata & Engel, 1984; Engel & Arahata, 1984) suggesting that DM may be a humorally mediated immune

PM and IBM, though separate disorders, are both characterised by scattered necrotic and regenerating muscle fibres and endomysial inflammation with invasion and destruction of non-necrotic muscle fibres. PM generally becomes evident in adulthood and is best defined as a subacute myopathy that evolves over weeks to months and presents with symmetrical weakness of the proximal muscles. Its clinical onset is hard to define with no early recognition signs such as the rash observed in DM. PM is uncommon as a stand-alone disorder and more commonly associates with other autoimmune and connective tissue

Onset of IBM is usually after the age of 50 and occurs more frequently in men. Muscle weakness can be both proximal and distal and is often asymmetrical. Despite similarities with PM, distinctive features of IBM include: rimmed vacuoles; groups of atrophic fibres; increased lymphocytic invasion of non-necrotic fibres; less frequent myofibre necrosis; and a more slowly progressing clinical course with patients being unresponsive to treatment. In both disorders, inflammatory infiltrates typically consist of CD8+ T-cells and macrophages (Arahata & Engel, 1984; Engel & Arahata, 1984) which invade MHC Class 1 antigenexpressing muscle fibres, a feature absent in normal muscle tissue, leading to fibre necrosis. The muscle fibre invading CD8+ T-cells can be clonally expanded in both PM and IBM (Dalakas, 2004; Fyhr *et al.*, 1997; Hofbauer *et al.*, 2003; Mantegazza *et al.*, 1993; Seitz *et al.*,

As with most autoimmune disorders, different autoantibody specificities have been described in DM and PM; autoantibodies are generally absent from IBM although they have been detected in a small number of cases (Dalakas *et al.*, 1997). They can either be myositisspecific (MSAs) or myositis-associated autoantibodies (MAAs), which can also be associated

2006), which persists over time (Amemiya, Granger, & Dalakas, 2000).

**4.1.1 Autoantibodies associated with myositis** 

**4. Tissue-infiltrating B-cells in inflammatory myopathies** 

**4.1 Introduction** 

45% of adult DM patients.

response.

disorders.

As described above B-cells have been found to be prominent within the muscle infiltrating cell populations of DM patients and are rarely found, or absent, in the inflamed muscle of PM and IBM patients. CD138+ plasma cells have been identified within the infiltrating populations, predominantly in the endomysial areas of muscle tissue of PM and IBM patients (Greenberg *et al.*, 2002; Greenberg *et al.*, 2005). This was confirmed by sequence analysis of immunoglobulin V-genes expressed by laser dissected cells as well as microarray studies which showed an abundance of immunoglobulin transcripts.

The role for B-cells and plasma cells in IM is still currently unresolved, with continuing studies providing further insight into the immune mechanisms. The identification of muscle infiltrating B-cells, plasma cells and autoantibodies suggests that these diseases may be at least partly driven by a loss of B-cell tolerance and, in the case of PM and IBM patients, not solely by the oligoclonal expansion of T-cells. We therefore investigated whether there is clonal expansion of infiltrating, autoantibody producing B-cells *in situ* in IM.

#### **4.2 The muscle infiltrating B-cell response in myositis**

#### **4.2.1 The cellular composition of infiltrating lymphoid cells in myositis**

To determine whether specific, antigen-driven, B-cell adaptive immune responses were occurring *in situ*, we used the methods described in section 2 to study the cellular composition of muscle infiltrating cells in twelve different muscle samples (2 DM, 9 PM, 1 IBM); we also examined their Ig V-gene repertoire and the processes of somatic hypermutation and clonal diversification of the rearranged V-genes. In contrast with other autoimmune diseases (see above), no classical ectopic germinal centre structures were observed within the inflamed muscle; instead, muscle–infiltrating cells were present in cellular aggregations which varied from loose to dense in the appropriate perivascular/perimysial or endomysial locations, as in previous studies. B-cells were a significant component of the inflammatory infiltrate in all samples examined for all three myositis subsets, either as CD20+ B-cells or differentiated plasma cells (Figure 7A-D), although the most significant infiltration of CD20+ B-cells was observed within the muscles of the two DM patients. FDCs were rare, and were seen only in one IBM and three PM samples, and not at all in DM. In addition to these cell phenotypes, CD3+, CD4+, CD8+, CD68+ and FoxP3+ cells were also present. Double immunofluorescence staining of cell phenotypes with the proliferating cell marker Ki67 identified proliferating cells within the infiltrating population. In addition to CD20+ B-cells (Figure 7E & F), proliferating CD3+, CD4+, CD8+ and CD68+ cells were observed, as well as FoxP3+ cells in one DM patient.

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 415

majority of B-lineage cells being CD138+ plasma cells that had class switched to either IgG or IgA. Clonally related sequences were isolated from whole muscle sections from ten of the twelve myositis patients, with up to four different clonal sets isolated from each muscle sample. Further studies also support the absence of classical ectopic germinal centre structures and the clonal expansion and maturation of B-cells within inflamed muscle (Salajegheh *et al.*, 2010). Collectively this and our work strongly suggest the participation of

Fig. 8. Oligoclonal expansion of B-cells and plasma cells in inflammatory myopathies

intermediates whose sequences were not isolated from the muscle-infiltrating population.

Fig. 9. Infiltrating antigen-specific B-cells and plasma cells in inflammatory myopathies

cells within the muscle-infiltrating population of polymyositis patients.

CD20+ B-cells and antigen-specific cells were visualised by Fluorescein-Avidin D (green) and Texas Red-Avidin D (red) respectively. Original magnification was 630X. Arrows identify antigen-specific

Representative examples of clonal genealogical trees constructed from sequences isolated from muscleinfiltrating B-cells and plasma cells in individual patients, representing the minimum number of cell divisions required to generate each daughter cell. Clone A from a DM patient; clone B from a PM patient. The letters in the circles refer to individual sequences isolated from each B-cell clone. Genealogical trees were constructed and mutations numbered as described in the legend to Figure 5. Bracketed figures representing additional silent mutations. Dashed circles represent hypothetical

antigen-specific B-cell immune responses within the muscle.

Fig. 7. Immunohistochemical staining of antigen-specific muscle-infiltrating B-cells and plasma cells.

A – D: Infiltrating B-cells and plasma cells (red) within inflamed muscle; E & F: Proliferating B-cells (CD20+ B-cells – Fluorescein-Avidin D (green), Ki67+ - Texas Red-Avidin D (red)). Slides A, B, C & E are from 2 DM patients; D & F are from a PM patient. Original magnification for images A - D: 400x; E & F: 630x. Scale Bar (E & F) represents 15 µm. Arrows indicate double positive staining.

#### **4.2.2 The Ig V-gene repertoire and clonally proliferating, muscle-infiltrating B-cells**

Analysis of the repertoire of rearranged Ig V-genes expressed by infiltrating B-cells and plasma cells revealed significant biases for and against individual gene segments and families relative to the normal peripheral blood B-cell and the germline gene repertoires. Vgene usage varied between patients and myositis subsets and, in a few instances, differed significantly between the DM and PM subsets. Interestingly, naïve or unmutated B-cells (0-2 mutations per VH gene) constituted almost 50% of the B-cells in DM, but <10% in PM, where a large proportion of sequences was highly mutated (c.30% >20 mutations). As expected, mutations were prevalent within CDRs 1 & 2. A total of nine clonally related sequences was found in five of the IM patients studied; 2 DM and 3 PM patients, each with up to four different clones comprising between two and ten clonal variants (Figure 8). These clonally related sequences provide evidence for specific, antigen-driven B-cell immune responses within the inflamed muscle. However, using the method of Hershberg *et al.* (2008), we found no evidence of positive selection in the CDRs of clonally related sequences, nor in any sequences isolated from the DM patients, and only in a small percentage from the PM patients. Finally, using biotinylated recombinant antigens, we identified antigen-specific B and plasma cells in infiltrates from the five out of twelve patients whose autoantibody specificities were known, including Jo-1 (Figure 9).

Parallel studies (Bradshaw *et al.*, 2007) also demonstrated B-cell responses in muscle of 3 DM, 2 PM and 7 IBM patients but very few CD19+ or CD20+ cells were observed, the

Fig. 7. Immunohistochemical staining of antigen-specific muscle-infiltrating B-cells and

**4.2.2 The Ig V-gene repertoire and clonally proliferating, muscle-infiltrating B-cells**  Analysis of the repertoire of rearranged Ig V-genes expressed by infiltrating B-cells and plasma cells revealed significant biases for and against individual gene segments and families relative to the normal peripheral blood B-cell and the germline gene repertoires. Vgene usage varied between patients and myositis subsets and, in a few instances, differed significantly between the DM and PM subsets. Interestingly, naïve or unmutated B-cells (0-2 mutations per VH gene) constituted almost 50% of the B-cells in DM, but <10% in PM, where a large proportion of sequences was highly mutated (c.30% >20 mutations). As expected, mutations were prevalent within CDRs 1 & 2. A total of nine clonally related sequences was found in five of the IM patients studied; 2 DM and 3 PM patients, each with up to four different clones comprising between two and ten clonal variants (Figure 8). These clonally related sequences provide evidence for specific, antigen-driven B-cell immune responses within the inflamed muscle. However, using the method of Hershberg *et al.* (2008), we found no evidence of positive selection in the CDRs of clonally related sequences, nor in any sequences isolated from the DM patients, and only in a small percentage from the PM patients. Finally, using biotinylated recombinant antigens, we identified antigen-specific B and plasma cells in infiltrates from the five out of twelve patients whose autoantibody

Parallel studies (Bradshaw *et al.*, 2007) also demonstrated B-cell responses in muscle of 3 DM, 2 PM and 7 IBM patients but very few CD19+ or CD20+ cells were observed, the

630x. Scale Bar (E & F) represents 15 µm. Arrows indicate double positive staining.

specificities were known, including Jo-1 (Figure 9).

A – D: Infiltrating B-cells and plasma cells (red) within inflamed muscle; E & F: Proliferating B-cells (CD20+ B-cells – Fluorescein-Avidin D (green), Ki67+ - Texas Red-Avidin D (red)). Slides A, B, C & E are from 2 DM patients; D & F are from a PM patient. Original magnification for images A - D: 400x; E & F:

plasma cells.

majority of B-lineage cells being CD138+ plasma cells that had class switched to either IgG or IgA. Clonally related sequences were isolated from whole muscle sections from ten of the twelve myositis patients, with up to four different clonal sets isolated from each muscle sample. Further studies also support the absence of classical ectopic germinal centre structures and the clonal expansion and maturation of B-cells within inflamed muscle (Salajegheh *et al.*, 2010). Collectively this and our work strongly suggest the participation of antigen-specific B-cell immune responses within the muscle.

Fig. 8. Oligoclonal expansion of B-cells and plasma cells in inflammatory myopathies

Representative examples of clonal genealogical trees constructed from sequences isolated from muscleinfiltrating B-cells and plasma cells in individual patients, representing the minimum number of cell divisions required to generate each daughter cell. Clone A from a DM patient; clone B from a PM patient. The letters in the circles refer to individual sequences isolated from each B-cell clone. Genealogical trees were constructed and mutations numbered as described in the legend to Figure 5. Bracketed figures representing additional silent mutations. Dashed circles represent hypothetical intermediates whose sequences were not isolated from the muscle-infiltrating population.

Fig. 9. Infiltrating antigen-specific B-cells and plasma cells in inflammatory myopathies

CD20+ B-cells and antigen-specific cells were visualised by Fluorescein-Avidin D (green) and Texas Red-Avidin D (red) respectively. Original magnification was 630X. Arrows identify antigen-specific cells within the muscle-infiltrating population of polymyositis patients.

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer 417

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.

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

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

**5.2 The immune response to breast cancer** 

infiltration.

who do not overexpress HER-2.
