**2.4 Statistical analysis**

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

Immunoglobulin Sequences (http://www.imgt.org/). Sequences were analysed using JOINSOLVER (http://joinsolver.niaid.nih.gov/) and IMGTV-QUEST. Silent and replacement somatic mutations were identified by comparison with the germline gene sequence; in early experiments the ratio of replacement to silent mutations was considered to be evidence of affinity selection if significantly higher than 3:1. To correct for the inherent bias towards replacement mutations in CDRs, we have more recently applied the method of Hershberg to determine whether affinity selection has occurred in B-cell clones from ectopic germinal centres (Hershberg *et al.*, 2008). This method employs an algorithm that allows for the effects of microsequences in the complementarity determining regions (CDRs) and the bias towards transition mutations. Clonally related sets of rearranged V-genes were identified by their use of the same germline V, (D) and J exons and shared junctional sequences. Genealogical trees were constructed by analysis of shared and unshared mutations using the parsimony method of phylogenetic analysis (PAUP, (Swofford, 1993)), enabling the assignment of sequences from parent and daughter cells that have been produced during clonal proliferation, thus providing clear evidence of the presence of

clonally proliferating, somatically mutating B-cells within the germinal centre.

**display** 

**2.3 Cloning antigen-specific autoantibodies from germinal centre B-cells by 'phage** 

In order to confirm the antigen specificity of B-cells generated in ectopic germinal centres, and to analyse in detail the relationship between their mutations and antigen specificity, we reconstituted the rearranged Ig V-genes as single chain Fv (scFv) or Fab antibodies by 'phage display. Single chain Fv antibodies comprise the heavy and light chain variable region domains linked by a short peptide. Although linked together by a short additional peptide sequence, the VH and VL domains are able to fold into their natural 3-dimensional conformation and pair correctly, as the antibody produced by a B-cell or plasma cell. They contain the antigen binding site, and therefore mimic the antigen specificity of the original antibodies from which they were derived. A caveat that must be born in mind is that the original H and L chain pairings are unknown, except when both genes are cloned from a single cell. The detailed methodology has been described elsewhere (Stott & Sims, 2000; Matthews *et al.*, 2002). Rearranged VH and VL-genes amplified either from microdissected germinal centres or pooled V-genes from the same B-cell clone, were used to construct scFvs using a (Gly4Ser)3 linker DNA. The resulting scFv library, comprising a pool of randomly linked VH-VL genes, was then inserted into the phagemids pCANTAB6 or pHEN2 continuously with the gene encoding the bacteriophage coat protein P3, and grown in *E. coli* in the presence of helper phage. The resulting scFv-P3 fusion protein was expressed on the surface of bacteriophage or as soluble scFv by transfection into a non-permissive, or permissive, strain of *E. coli* respectively. Alternatively, Fab libraries were constructed using whole light chain cDNA and DNA encoding the VH region and the first constant region domain of the heavy chain by similar techniques (Matthews et al., 2002). An advantage of amplifying directly from genomic DNA is that the distribution of cloned V-genes reflects the usage of those genes by B-cells and plasma cells more accurately than amplification from cDNA, which is biased towards plasma cells. The library of scFvs or Fabs attached to bacteriophage by the P3 'phage coat protein was then panned on plastic plates coated with either a whole extract of the target tissue, or purified recombinant antigen, to identify selfreactive antibodies. Bound 'phage were washed, eluted, re-grown in *E. coli* and panning The method of Hershberg *et al*. (2008), described in section 2.2 above, was used to determine the significance of replacement mutations in rearranged Ig V-genes cloned from germinal centre B-cells, as evidence for affinity maturation of the antibodies expressed by them. The distribution of VH gene families and individual V, D and J exons was assessed using twotailed 2 analysis, corrected for multiple comparisons.
