The part of the sequence encoding CDRH3 is indicated by a horizontal line below the gene sequences. repertoires. Analysis of immunoglobulin heavy chain gene rearrangements amplified from genomic DNA template of circulating B cells of the allergic subjects in this study, compared to healthy control subjects. The usage frequency of IGHV (a) and IGHJ (b) gene subgroups is shown. For each subgroup, the frequency is shown in the following order ANGPT2 (from left to right): normal repertoires of other studies, samples obtained from non-vaccinated donors at time 0 (S1) and 1 year (S3), and samples obtained from vaccinated donors at time 0 (S1), 2 months (S2) and 1 year (S3). Frequency of mutation in the IGHV gene (c), the mean CDRH3 length (d) and the mean calculated hydrophobicity (e) is illustrated. The top and bottom panels in each figure section report unmutated sequences (top) and mutated sequences (bottom), respectively. Supplementary Figure E4 Analysis of the CDR3 lengths of the immunoglobulin heavy chain gene rearrangements amplified from cDNA template. Each panel represents sequences derived from a different isotype, in the blood or nasal biopsy of allergic patients receiving or not receiving immunotherapy at Fumagillin Fumagillin different time points. Sequences are collapsed by unique sequences, defined as those sequences having the same V identity without allele, same J without allele and the same CDR3 sequence. Supplementary Figure E5 Analysis of the V mutation levels of the immunoglobulin heavy chain gene rearrangements amplified from cDNA template. Each panel represents sequences derived from a different isotype in the blood or nasal biopsy of allergic patients receiving or not receiving immunotherapy at different time points. Sequences are collapsed by unique sequences, defined as those sequences having the same V identity without allele, same J without allele and the same CDR3 sequence. Significance of p<0.001 (*) was determined by pairwise T-test using Bonferroni correction Supplementary Figure E6 Rearrangement of IT2-P11 as proposed by IMGT V-QUEST tool (10). The analysis suggests that the gene may represent a public rearrangement as it appears to have been established largely from individual IGHV and IGHJ genes without involvement of an IGHD gene and with the addition of only six N nucleotides (indicated by a horizontal line above the sequences). The part of the sequence encoding CDRH3 is indicated by a horizontal line below the gene sequences. Bases of the IGHV and IGHJ genes likely to have been trimmed of during the rearrangement process are not shown. Only those bases of IT2-P11 that were not encoded by PCR primers are shown. Bases showing identity between the IT2-P11 gene and its closest germline gene counterparts are highlighted with a grey background. Supplementary Figure E7 Analysis of isotype expression, tissue localization, and clonal persistence of B cells belonging to clonal lineages containing IgE-expressing members in SIT vs Non-SIT patients. (A) Isotype expression by allergen-specific B cell clones containing IgE members. Clones containing only IgE members are not shown on graph, but the difference between groups was not significant. Fumagillin (B) Tissue and blood distribution of allergen-specific B cell clones containing IgE-expressing members. Non-SIT patients had a higher proportion of IgE clones detected in their biopsy samples. Significance was determined by Fishers exact test (two-tailed p-values * = 0.01 to 0.05, ** = 0.001 to 0.01, *** = 0.0001 to 0.001, **** = < 0.0001). (C) Shows samples in more than one time point during the SIT course. Numbers above bars indicate the total number of clusters representing the given category. Sequences from the 2-month time point were excluded since this sample was only collected for SIT patients. Supplementary Figure E8 Trees describing putative evolutionary relationships between unique sequences belonging to different clone sets. Clone sets related to scFv IT2-B12 (A), IT2-B23 (B), IT2-P614 (C), IT4-B119 (D), IT4-B148 (E), IT2-B227 (F), IT4-P62 (G), IT6-B22 (H), IT2-P536 (I), IT2-B239 (J), IT6-nD12 (K) are shown. Phage sequences are represented by diamond nodes, while clones identified in the nasal biopsies are shown as rectangles, and clones derived from PBMC sequences are displayed as circles. The colors indicate the time points where clones were identified (red.
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