Download IgG4+ clones are dominantly present in the B

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
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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
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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
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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.
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