Single cell analysis of B lymphocytes from Wegener's granulomatosis: B cell receptors display affinity maturation within the granulomatous lesions.

Increased amounts of anti-neutrophil cytoplasm antibody (ANCA) directed against proteinase 3 (PR3) are a diagnostic and pathogenic hallmark of full-blown Wegener's granulomatosis (WG). Aggregates of B lymphocytes proximal to PR3+ cells as well as plasma cells have been described as substantial components of Wegener's granuloma and could participate in forming tertiary lymphoid structures, which might promote autoantibody formation. Our aim was a molecular analysis of single B cells in order to develop a methodological approach that allows examination of potential ANCA formation in the tissue. Single B cells from cryo-conserved endonasal biopsies of three WG patients were isolated, using laser-assisted microdissection. Subsequently, their immunoglobulin variable heavy (VH) and light (Vkappa, Vlambda) chain genes were analysed by single cell polymerase chain reaction and direct sequencing. Sixteen immunoglobulin VH-Vkappa or VH-Vlambda chain gene couples were characterized. Twelve of these immunoglobulin gene couples resembled memory B cells. Two offsprings of one B cell were detected, indicating clonal expansion. VH genes representing 39 single B cells of WG tissues displayed significantly more mutations when compared with VH genes from peripheral blood of a healthy donor. The findings confirm and extend our previous results, arguing for an initial selection and affinity maturation of B cells within Wegener's granuloma. Further, the methodology provides the initial basis for the recombinant generation of antibodies derived from tissue cells.

[B lymphocyte differentiation in granulomatous tissues of the lung and the nasal mucosa in Wegener's granulomatosis: origin of anti-neutrophil cytoplasmic antibody formation?]. / B-Lymphozyten-Differenzierung in granulomatösen Geweben der Lunge und der Nasenschleimhäute beim Morbus Wegener: Ursprungsort der ANCA-Bildung?

Wegener's granulomatosis (WG) starts with granulomatous inflammation of the respiratory tract before it converts into a potentially organ and life threatening systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA). The site of formation of the highly specific ANCA directed against "Wegener's autoantigen" proteinase 3 (PR3) is still unknown. Previously, we have shown that follicle-like B lymphocytic infiltrates in the vicinity to PR3 expressing cells in WG-granulomata. We characterized the immunoglobulin-VH repertoire in lung and nasal granulomata (paraffin embedded) from four WG patients. A total of 115 individual VH genes were characterized and compared to 84 VH genes from the peripheral blood of a healthy donor. We found an increased frequency of mutations with a bias to amino acid exchanges within the antigen binding sites (CDR) 1 and 2 in WG tissue. A large number of mutations led to negatively charged amino acids and may increase affinity to the positively charged PR3. Furthermore, the occurrence of differently mutated members of one B cell clone indicates clonal expansion and intraclonal diversification by an antigen, e.g. PR3. Several WG tissue derived genes displayed similarities to published sequences from peripheral PR3 ANCA producing B cells. Thus, granulomata of the lower and upper respiratory tract contain follicle-like B cell clusters with a selected VH repertoire infiltrate in WG. WG granulomata could be the place of autoantigen presentation and formation of high-affinity ANCA within neoformed ectopic or tertiary lymphoid-like tissue areas.

B lymphocyte maturation in Wegener's granulomatosis: a comparative analysis of VH genes from endonasal lesions.

BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) are highly specific for Wegener's granulomatosis (WG). Evidence for a pivotal role of PR3-ANCA in the induction of vasculitis has been demonstrated. B cell clusters have been observed within endonasal biopsy specimens. OBJECTIVES: To determine whether B cell selection and maturation take place in granulomatous lesions of WG. METHODS: Granulomatous lesions and the immunoglobulin (VH) gene repertoire from nasal tissue of six WG patients-two active and two smouldering localised WG (ANCA negative, restricted to respiratory tract), plus one active and one smouldering PR3-ANCA positive generalised WG-were characterised by immunohistochemistry, polymerase chain reaction, cloning, DNA sequencing and database comparison. RESULTS: B lymphocyte-rich, follicle-like areas were observed proximal to PR3 positive cells and plasma cells in granulomatous lesions; 184 VH genes from these granulomatous lesions were compared with 84 VH genes from peripheral blood of a healthy donor. The mutational pattern of VH genes from active WG resembled memory B cells. Structural homologies of VH genes from granulomatous lesions to PR3-ANCA encoding genes were detected. Significantly more genes (55%, 45%, and 53%, respectively) from active WG compared with the healthy repertoire carried mutations to negatively charged amino acids within the binding site coding regions, favouring affinity to the positively charged PR3. CONCLUSIONS: Selection and affinity maturation of potentially PR3-ANCA producing autoreactive B cells may start in granulomatous lesions, thereby contributing to disease progression from ANCA negative localised to PR3-ANCA positive generalised WG.