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IgG4+ Clones Identified by Next-Generation Sequencing Dominate the B-Cell Receptor Repertoire in IgG4-Associated Cholangitis Lucas J Maillette de Buy Wenniger, Marieke E Doorenspleet, Paul L Klarenbeek, Joanne Verheij, Frank Baas, Ronald P Oude Elferink, Paul P Tak, Niek de Vries, Ulrich Beuers Supplementary Figure Legends HEP-12-1534-R Supplementary figure 1: IgG4+ clones are dominant in blood of IgG4-associated cholangitis (IAC), but not in healthy (HC) or disease (DC) controls. (A) The contribution of individual clones to the total BCR repertoire in all individuals (including IgA+, IgD+, IgG+ and IgM+ clones). Scatterplot showing clonal abundance as percentage of the IgG+ BCR repertoire (each dot represents an individual clone). IgG4+ clones are marked in red. (B) The number of dominant clones (clonal frequency >0.5% of the total BCR repertoire) found in IAC, HC and DC. Supplementary table 1: The CDR3 amino acid sequence of dominant IgG4+ BCR clones shows no consistent homology. Characteristics are summarized of all individual IgG4+ dominant clones, including IGHV and IGHJ usage, CDR3 amino acid sequence, the frequency of the clone as the percentage of the total IgG+ BCR repertoire, as well as the percentage of nucleotides mutated in the Vregion and the percentage of mutation marked as non-silent mutations. The relatively high percentage of mutations and the fact that the majority of these mutations result in amino acid changes support the notion that these clones have undergone affinity maturation. Supplementary figure 2: The IGHV and IGHJ usage of the full BCR repertoire is similar in IAC patients, disease controls and healthy controls. Bar chart showing the use of individual IGHV genes (A) and IGHJ genes (B) as percentage of all BCR clones. Supplementary figure 3: IgG4+ clones are enriched in inflamed papilla tissue. To investigate the possibility that the observed overlap of dominant IgG4+ clones was due to contamination from peripheral blood, we re-analyzed the repertoires in tissue and blood with this in mind. We reasoned that if contamination would explain the overlap between peripheral blood and tissue, the most frequent clones should be similar in both compartments. To this end, in both patients of which we had both tissue and peripheral blood (IAC4 and IAC5), we 2 HEP-12-1534-R took the 25 most dominant clones from peripheral blood and subsequently determined their frequency within the tissue sample and vice versa (top 25 clones listed in Figure 3A-B). We observed in both IAC patients that although some of the top 25 clones in blood can also be found in tissue, a substantial number of these were not among the most dominant clones in tissue or could not be retrieved at all. When dividing these clones in IgG4+ and IgG4-, we observed that IgG4+ clones are among the highest ranked clones, while clones with another isotype than IgG4 were only found as low-ranked clones or not found at all (Figure C, MannWhitney U test p<0.0001 for IAC4 and IAC5 combined, p=0.0003 in separate analyses; filled dots represent the clones found in IAC4, open dots were found in IAC5). Therefore, the IgG4+ clones seem to be enriched in the inflamed tissue rather than showing overlap based on contamination of blood in the tissue. We arbitrarily chose to include the top 25 clones but including the top 50 or top 200 clones gave similar results (but were predominantly enlarging the IgG4- group). Supplementary figure 4: Graphical representation of the experimental procedures workflow. Samples were collected from peripheral blood or tissue and mRNA was isolated and cDNA was synthetized for downstream application. A linear amplification was performed, using a primer set covering all functional Vheavy genes. This product was then used either for the determination of the total BCR repertoire (V-CDR3-J amplification) or for the subtyping of individual clones (V-CDR3-C amplification). For the former, a PCR using primerB as a forward primer and a generic primer specific for all functional Jheavy genes containing the primerA as reverse primer was performed. For the latter, the Ig isotypes were determined using primerB as a forward primer, and primers specific for the IgA, IgD, IgM and IgG isotype as reverse primers. Sequencing was performed on both pools of sequences (both V-CDR3-J and V-CDR3-C) according to the manual for 454 amplicon sequencing on a genome sequencer FLX (using primerA and primeB sequences). Using custom-made bioinformatics algorithms, the frequencies of individual clones were determined based on their unique VDJ 3 HEP-12-1534-R rearrangement and CDR3 sequence and matched with their isotype and subclass characteristics. Supplementary table 2: Summary of clinical, serological, radiological and histological data on the studied IAC patients and the controls with pancreaticobiliary disease. A color code was used to mark the observations that could be suggestive (light green) or are strongly suggestive of the presence of IgG4-related disease (bright green) and those that could be suggestive (light orange) or are strongly suggestive of either PSC or pancreaticobiliary malignancy (orange). For all patients, the ratio of serum IgG4 over serum IgG1 was calculated. An arbitrary ratio of serum IgG4/IgG1 > 1 was considered to be supportive of the diagnosis of IgG4-related disease as this reflected a genuine relative increase in serum IgG4 as opposed to a general overproduction of all IgG subclasses including IgG4. In order to investigate the presence of histological indications for the presence of IgG4related disease we searched the histological database for available tissue sections of all analyzed patients. In cases where this had not yet been done and if a tissue block was available an immunohistochemical staining for IgG4 was performed. All available tissue specimens from all patients were analyzed by an experienced hepatological pathologist who was blinded to the clinical diagnosis and scored for the published classical signs of IgG4related disease (2): the presence of a dense lymphoplasmacellular infiltrate, storiform fibrosis, background eosinophilia and obliterative phlebitis. The infiltration of IgG4-positive plasma cells was assessed by counting the maximal number of these cells per high power field (HPF). In one of the PSC patients low numbers of infiltrating IgG4+ cells were observed in stomach and duodenal papilla tissue, and up to 10 IgG4+ cells per high power field were found in a sigmoidal biopsy. One PSC patient (DC1) with elevated serum IgG4 underwent short-term prednisolon treatment without biochemical response. In two out of three patients with a malignancy, sections of the tumor showed infiltration with low numbers of IgG4+ plasma cells. N.d. not done. N.a. not applicable. 4