46 CHAPTER 3 we took biopsies from these distinct two regions from 12 different inflamed knee joints in 7 individual RA patients. The ST biopsies were analyzed for BCR repertoires and compared with paired peripheral blood (PB) samples. The SP and IP regions showed comparable numbers of BCR-clones (mean ± SD: 1,337 ± 651 versus 1,420 ± 552 respectively) and highly expanded BCR-clones (HECs; mean ± SD: 29.7 ± 6.7 versus 32.2 ± 8.9), with a comparable impact of these HECs on the total repertoire (mean ± SD: 55.1% ± 18.0 versus 50.8% ± 17.9). In contrast, PB showed a significantly higher number of BCR-clones (p < 0.0001; 5,407 ± 2,533), a lower number of HECs (p < 0.0001; 5.3 ± 5.2), and these HECs accounted for significantly less impact on the total repertoire (p < 0.0001; 15.6% ± 24.1) (Figures 1A–C). In order to determine similarity between different BCR repertoires we compared the most expanded BCR-clones between the two biopsied regions, using PB as control. Of the 25 most expanded BCR-clones in the SP region on average 16.7% (mean, SD 12.5) were also present in the top-25 clones in the IP region (“Clonal retrieval”). However, significantly fewer of the expanded BCR clones in SP (p<0.001; 1% ± 2.5%) and IP (p<0.01; 2% ± 2.7%) could be retrieved in PB samples (Figures 1D–F). To analyze similarity for the complete BCR, rather than the most dominant clones only, we use the Chao-modified Sørensen index [11]. This index gives an estimate for the combined frequency of all shared clones while correcting for shared clones not observed due to incomplete sampling. The comparison of the SP and IP region showed a mean similarity index of 0.32 (SD 0.17; n=12), significantly higher than the score of 0.06 observed when comparing ST-SP or ST-IP with PB (mean, SD 0.08 and 0.07 respectively, n=12; p<0.001, Figure 1G). Thus, the BCR repertoire in inflamed ST of RA patients shows similarity at different locations within same joint, and shows sharing of some dominant BCR clones, whereas hardly any overlap was observed when comparing ST to PB.
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