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American Society of Hematology
2021 L Street NW, Suite 900,
Washington, DC 20036
Phone: 202-776-0544 | Fax 202-776-0545
bloodadvances@hematology.org
Alterations in B- and circulating T-follicular helper cell subsets in immune
thrombotic thrombocytopenic purpura
Tracking no: ADV-2022-007025R1
Jin-Sup Shin (University College London Hospitals, United Kingdom) Maryam Subhan (University
College London, United Kingdom) Geraldine Cambridge (UCL, United Kingdom) Yanping Guo (Cancer
Institute, UCL, United Kingdom) Rens de Groot (University College London, United Kingdom) Marie
Scully (5National Institute for Health Research Cardiometabolic Programme, UCLH/UCL Cardiovascular
BRC, London, UK, United Kingdom) Mari Thomas (5National Institute for Health Research
Cardiometabolic Programme, UCLH/UCL Cardiovascular BRC, London, UK, United Kingdom)
Abstract:
T follicular helper (Tfh) cells regulate development of antigen-specific B-cell immunity. We
prospectively investigated B-cell and cTfh subsets in 45 immune TTP patients at presentation and
longitudinally after rituximab (RTX). B-cell phenotype was altered at acute iTTP presentation with
decreased transitional cells and postgerminal centre (post-GC) memory B cells and increased
plasmablasts compared to healthy controls. A higher percentage of plasmablasts was associated with
higher anti-ADAMTS13 IgG and lower ADAMTS13 antigen levels. In asymptomatic patients with ADAMTS13
relapse, there were increased naïve B cells and a global decrease in memory subsets, with a trend
to increased plasmablasts. Total circulating Tfh (CD4+CXCR5+) and PD1+ Tfh cells were decreased at
iTTP presentation. CD80 expression was decreased on IgD+ memory cells and double negative memory
cells in acute iTTP. Longitudinal analysis: at repopulation after B cell depletion in de novo iTTP,
post-GC and double negative memory B cells were reduced compared to pre-RTX. RTX did not cause
alteration in cTfh frequency. The subsequent kinetics of naïve, transitional, memory B cells and
plasmablasts did not differ significantly between patients who went on to relapse vs those who
remained in remission. In summary, acute iTTP is characterised by dysregulation of B- and cTfh-cell
homeostasis with depletion of post-GC memory cells and cTfh cells and increased plasmablasts.
Changes in CD80 expression on B cells further suggest altered interactions with T cells.
Conflict of interest: COI declared - see note
COI notes: M.S. has received speaker's fees and honoraria from Alexion, Sanofi, Novartis, and
Takeda and has received research funding from Takeda. M.T. has received speaker's fees and
honoraria from Sanofi and Bayer. The remaining authors declare no competing financial interests.
Preprint server: No;
Author contributions and disclosures: J.S. designed research, recruited patients, performed
laboratory testing, collected data, analysed data and wrote the manuscript. M.O.S. designed
research, recruited patients, performed laboratory testing, collected data, analysed data and wrote
the manuscript. G.C. designed research, analysed data and wrote the manuscript. Y.G. performed
laboratory testing, collected data and reviewed the manuscript. R.dG. designed research, analysed
data and wrote the manuscript. M.S. designed research, recruited patients, analysed data and wrote
the manuscript. M.T. designed research, recruited patients, analysed data and wrote the manuscript.
Non-author contributions and disclosures: No;
Agreement to Share Publication-Related Data and Data Sharing Statement: For original data, please
contact jin-sup.shin@nhs.net.
Clinical trial registration information (if any):
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Alterations in B- and circulating T-follicular helper cell subsets in immune
thrombotic thrombocytopenic purpura
Authors
Jin-Sup Shin,1 Maryam Owais Subhan,2 Geraldine Cambridge,3 Yanping Guo,4 Rens de
Groot,2 Marie Scully,1,5 and Mari Thomas1,5
1Department of Haematology, University College London Hospital (UCLH), London, United Kingdom;
2Institute of Cardiovascular Science, University College London; 3Centre for Rheumatology Research,
University College London (UCL), London, United Kingdom; 4Cancer Research UK Flow Cytometry
Translational Technology Platform, Cancer Institute, UCL, London, United Kingdom; 5National
Institute for Health Research Cardiometabolic Programme, UCLH/UCL Cardiovascular BRC, London,
UK
Corresponding author contact information:
mari.thomas@nhs.net
Department of Haematology
University College London Hospitals
250 Euston Road, London
United Kingdom
NW1 2PG
Short title for the running head: B and cTfh cell changes in immune TTP
Word counts
- Text 3964
- Abstract 229
Figure/table count 6 figures 2 tables
Reference count 59
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Key Points
• Abnormal B-cell phenotype in acute iTTP with decreased transitional and
postgerminal centre memory cells and increased plasmablasts
• Decreased total and PD1+ circulating T-follicular helper cells and changes in B-cell
CD80 expression suggest altered B-T–cell interactions
ABSTRACT
T follicular helper (Tfh) cells regulate development of antigen-specific B-cell immunity. We
prospectively investigated B-cell and cTfh subsets in 45 immune TTP patients at
presentation and longitudinally after rituximab (RTX). B-cell phenotype was altered at acute
iTTP presentation with decreased transitional cells and postgerminal centre (post-GC)
memory B cells and increased plasmablasts compared to healthy controls. A higher
percentage of plasmablasts was associated with higher anti-ADAMTS13 IgG and lower
ADAMTS13 antigen levels. In asymptomatic patients with ADAMTS13 relapse, there were
increased naïve B cells and a global decrease in memory subsets, with a trend to increased
plasmablasts. Total circulating Tfh (CD4+CXCR5+) and PD1+ Tfh cells were decreased at
iTTP presentation. CD80 expression was decreased on IgD+ memory cells and double
negative memory cells in acute iTTP. Longitudinal analysis: at repopulation after B cell
depletion in de novo iTTP, post-GC and double negative memory B cells were reduced
compared to pre-RTX. RTX did not cause alteration in cTfh frequency. The subsequent
kinetics of naïve, transitional, memory B cells and plasmablasts did not differ significantly
between patients who went on to relapse vs those who remained in remission. In summary,
acute iTTP is characterised by dysregulation of B- and cTfh-cell homeostasis with depletion
of post-GC memory cells and cTfh cells and increased plasmablasts. Changes in CD80
expression on B cells further suggest altered interactions with T cells.
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INTRODUCTION
Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening thrombotic
microangiopathy mediated by an immunoglobulin G (IgG) antibody against the
metalloprotease ADAMTS13 that enhances its clearance or inhibits its VWF processing
activity.1 In iTTP, there is an incompletely understood loss of tolerance resulting in a shift
from immune homeostasis to autoimmunity. This involves dendritic cells which acquire
antigens derived from ADAMTS13 that activate cross-reactive naïve CD4+ T cells which
differentiate into autoreactive effector CD4+ T cells.2,3 Mature autoreactive B cells recirculate
into the germinal centre of secondary lymph nodes where they are stimulated by compatible
antigens in the presence of the autoreactive T helper cells and differentiate into
autoantibody-producing plasma cells or long-lived memory B cells.4
B cell depletion therapy with rituximab (RTX; a chimeric monoclonal antibody against the
pan-B cell marker, CD20) has been demonstrated to be effective in reducing relapse rates in
iTTP and prolonging disease-free survival in acute episodes, compared to PEX and steroid
alone.5-7 Giving rituximab pre-emptively in patients at high risk of a clinical relapse (based on
a fall in ADAMTS13 activity levels to<10-20%, i.e. ‘ADAMTS13 relapse’) reduces clinical
relapse rates compared to historical controls.8
A recent Genome Wide Association Study (GWAS) of iTTP confirmed associations with
SNPs at the HLA locus and identified a novel association on chromosome 3.9 The locus on
chromosome 3 contains five genes, one of which is the CD80 gene. CD80 is a co-stimulator
for T lymphocyte activation. After activation through the B-cell receptor (BCR) or IL4, B cells
express CD80, which interacts with CD28 on T cells to provide co-stimulation signals.10 The
HLA-DRB1*11 and DRB1*03 genes are known risk factors for iTTP, suggesting the MHC
class II protein variants they encode have optimal affinity for certain ADAMTS13 peptides
recognised by CD4+ T-cell receptors in iTTP patients.11,12 Two peptides derived from the
CUB-2 domain of ADAMTS13 are presented on HLA-DRB1*11 and HLA-DRB1*03
respectively and recognised by iTTP patient-derived CD4+ T cells.13,14
CD4+ T cells are pivotal in development of iTTP as CD4+ T cell help is required in the
production and affinity maturation of ADAMTS13-directed antibodies.3 Within the CD4+T cell
population, the T follicular helper (Tfh) subset are vital for supporting antibody-mediated
immune responses by providing co-stimulation signals through CD40L and IL-21 production,
which promote the growth, differentiation and class-switching of antigen-activated naïve B
cells.15-17 Tfh cells constitutively express the chemokine receptor CXCR5 which facilitates
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migration into germinal centres (GC).18 Tfh cells express co-stimulatory molecules such as
inducible co-stimulatory molecule (ICOS) and immune-regulatory molecules such as
programmed death-1 (PD-1) as their transcription factors, which can be used to further
define Tfh cells.19 More specifically, PD-1+ICOS- Tfh cells seem to represent quiescent and
PD-1+ICOS+ Tfh cells recently activated memory Tfh cells20,21
Circulating Tfh (cTfh) cells also express the GC homing receptor CCR7.22 Expansion of cTfh
cells has been associated with the development of several autoimmune diseases.23-29
The aim of this study was to investigate the B cell and cTfh subset distribution in iTTP
patients, both at presentation and longitudinally after anti-CD20 therapy in relation to clinical
and laboratory parameters and B cell kinetics. The role of cTfh has not previously been
investigated in iTTP. The temporal relationships between B and T cell subpopulation at
critical stages throughout the course of disease will improve the understanding of the
pathogenesis of iTTP, potentially provide biomarkers to predict relapse and may identify new
avenues for therapeutic intervention.
METHODS
Patients and controls:
We prospectively enrolled iTTP patients from September 2018 - June 2021 through the
United Kingdom TTP Registry (database and Biobank of UK TTP - Multicentre Research
Ethics Committee [MREC]:08/H0810/54 and MREC:08/H0716/72). The study was conducted
according to the Declaration of Helsinki. Blood samples were obtained from age and sex-
matched healthy controls (HC), with no history of autoimmune disease or
immunosuppressant medication. Definitions of iTTP diagnosis, remission and relapse were
based on previous studies.30-32
Patients were divided into two groups: de novo acute iTTP episodes and asymptomatic
ADAMTS13 relapses (treated with pre-emptive RTX). Acute presentations of iTTP were
treated with PEX, corticosteroids and RTX (4-8 doses 375 mg/m2 to normalize ADAMTS13
activity). Time after RTX was defined as time since first RTX infusion. Caplacizumab was
given to 18/22 of patients.
Patients with previously diagnosed iTTP who were in clinical remission (normal platelet
count) but developed a new severe ADAMTS13 deficiency were referred to as ADAMTS13
relapse episodes.32 These patients received pre-emptive RTX (four doses of
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200/500/375mg/m2 one week apart). Dosing varied at clinician discretion or as part of a
separate randomised control trial (Elective Rituximab in TTP trial;[REC]17/LO/1055).
Blood samples and PBMC isolation:
Heparinised blood samples collected from patients prior to initiation of RTX or other
immunosuppression and HC were analysed within 24 hours or frozen at -80˚C and stored for
later analysis. Samples were also collected one month and three months post RTX and then
three monthly thereafter until clinical or ADAMTS13 relapse, or end of study (whichever
came first). Peripheral blood mononuclear cells (PBMC) were isolated by diluting blood (1:1)
with phosphate-buffered saline(PBS) 1X and layered over Ficoll-Paque Premium 1084R
(Sigma-Aldrich).33
Flow cytometry
PBMC (approximately 1 ×106/sample) were incubated with conjugated antibodies for 20
minutes in the dark at room temperature. Cells were washed twice and resuspended in PBS
and samples were analysed on a Cytoflex S cytometer (Beckman Coulter) (Supplementary
Materials). Each sample was divided into two antibody panels: for B cell subsets and Tfh
cells (Supplementary Materials: Table 1a & 1b). Viability was determined using
LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (InvitrogenTM). Analysis was performed on
FlowJo software version 10 (FlowJo LLC).
B cell immunophenotyping and gating strategy
We used the mature B cell ‘Bm1–Bm5’ classification to identify B cell subsets based on co-
expression of IgD and CD38. B cell subsets include transitional B cells, naive B cells,
memory populations including IgD+ memory cells, post germinal centre (post-GC) cells,
double negative memory cells and plasmablasts.34,35 Antibodies used for the B cell panel
were CD19 PECy7, CD27 APC, IgD BV421, CD38 PE. Following on from our GWAS finding
that the CD80/POGLUT1 locus is associated with iTTP,9 we also analysed the level of CD80
expression (median fluorescence intensity or MFI) on B-cells subsets in iTTP.36 Gating
strategies are shown in Supplemental Figure 1.
Circulating Tfh immunophenotyping and gating strategy
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The total cTfh population was defined by CD3+ T cells that expressed CD4+CXCR5+. The
different subsets within the total cTfh population were defined by expression of activation
markers: PD1+ cTfh, ICOS+ cTfh and PD1+ICOS+ cTfh. In the cTfh panel, PBMC were
stained with CD3 PECy7, CD4 PerCP, CXCR5 FITC, PD1 APC and ICOS BV421
antibodies. The gating strategy and representative plots are shown in Supplemental Figure
2.
ADAMTS13 assays
ADAMTS13 activity was analysed by fluorescence resonance energy transfer (FRETS
(normal range: 60%-123%).37 ADAMTS13 antigen levels were quantified using in-house
developed enzyme-linked immunosorbent assay, previously described (normal range: 74%
to 134%).38 ADAMTS-13 activity and antigen levels were expressed as a percentage relative
to pooled normal plasma (PNP). Anti-ADAMTS13 IgG levels were measured with in-house
ELISA (normal range <6.1%) with concentration of anti-ADAMTS13 IgG calculated as a
percentage relative to an index plasma from a patient with a high auto-antibody titre
(assigned a value of 100%).7
Statistical Analysis
Mann-Whitney U and Wilcoxon tests were used for paired and unpaired continuous
variables, respectively. The X2 and Fisher’s exact tests were performed for categorical
variables. Spearman correlation test was used to measure the possible relationship between
two variables of interest. Statistical analysis was performed using GraphPad Prism 8
(GraphPad Software Inc., La Jolla, CA, USA).
Data Sharing statement
For original data, please contact jin-sup.shin@nhs.net.
RESULTS
Patient demographics and ADAMTS13 biomarkers
Demographics of iTTP patients and HC and ADAMTS13 assay results are shown in
Supplemental Table 2a). There were 45 unique patients with iTTP involving 46 episodes: 22
de novo acute episodes and 24 asymptomatic ADAMTS13 relapses treated with pre-emptive
rituximab.
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In acute iTTP episodes, 82% (18/22) cases had cardiac involvement and 50% (11/22)
neurological. 14% (3/22) of acute episodes required organ support on the intensive care unit.
All acute episodes were treated with PEX, corticosteroids and RTX. 82% (18/22) patients
received anti-VWF nanobody, Caplacizumab. Additional immunosuppression included
mycophenolate mofetil (27%, 6/22) and bortezomib (9%, 2/22). All but one of ADAMTS13
relapse episodes in this study had previously received rituximab treatment, either during an
acute presentation or previous ADAMTS13 relapse episode. In this cohort, the median
number of previous TTP episodes was 3 (range 1-8 episodes), with a median duration since
the most recent TTP episode being 21 months (range 13-191 months), (Supplemental Table
2b).
Post germinal centre memory B cells are decreased and plasmablasts increased at
iTTP presentation. (Figure 1)
The total number of CD19+ B cells at presentation of acute iTTP or preceding pre-emptive
rituximab for ADAMTS13 relapse episodes was not significantly different to HC. At
presentation in acute iTTP episodes, post-GC memory cells were decreased compared to
healthy controls (HC 13.7% (range, 3.1-28.8%) vs acute iTTP 7.9% (range, 1.8-27.8%),
p=0.008), whereas plasmablasts were increased (HC 0.7% (range, 0.2-2.5%) vs acute iTTP
1.6% (range, 0.2-15.4%), p=0.007), (Figure 1).
B cell repopulation after RTX recapitulates ontogeny beginning with naïve B cell exit from the
bone marrow followed by gradual maturation of memory subsets over time. In ADAMTS13
relapse cases, there was an increased proportion of naïve B cells compared to healthy
controls and a trend to increased transitional cells and plasmablasts (Figure 1). There was a
marked reduction in all memory subsets.
Altered circulating T follicular helper cell subsets at iTTP presentation: decreased
total cTfh and PD1+ cTfh (Figure 2)
The relative proportions of CD4+ cTfh subsets in acute TTP and ADAMTS13 relapse were
determined and compared with HC (Figure 2). The frequency of total cTfh and PD1+ cTfh
were significantly reduced in acute iTTP compared to HC. This may be suggestive of
migration of circulating Tfh cells into germinal centers. There were no significant differences
seen in ICOS+ or PD1+ICOS+ cTfh cells. In ADAMTS13 relapses, no differences were seen
in total, PD1+ or PD1+ICOS+ cTfh cells. However, ICOS+ cTfh cells were increased in
patients compared to HC.
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Relationship between ADAMTS13 parameters, circulating Tfh cells and B cell subsets
at iTTP presentation
In the acute iTTP group, median time to achieve a normal platelet count from presentation
was 4 days (range, 2-11) and median time to normalization of ADAMTS13 activity was 33
days (range 3-382; interquartile range, 22-124). At presentation in acute iTTP episodes, a
higher percentage of plasmablasts appears to be associated with lower antigen levels (r= -
0.41, p=0.055) (Figure 3). Indeed, a plasmablast level of >3% was associated with IgG
antibody level of >50% (Table 1). This may be due to increased production of anti-
ADAMTS13 IgG antibody from a larger number of plasmablasts, which in turn results in
increased ADAMTS13 clearance. In both acute iTTP cases and ADAMTS13 relapses, there
was no correlation between total cTfh cells and any of the B cell subsets (data not shown).
Expression of CD80 on B cell subsets at acute presentation and post RTX
In acute iTTP episodes, CD80 MFI was decreased in IgD+ memory cells and double
negative memory cells compared to HC (Figure 4). However, in ADAMTS13 relapse cases,
CD80 MFI was significantly increased in post-GC and double negative memory cells
compared to HC). A possible explanation for this difference between acute iTTP and
asymptomatic ADAMTS13 relapse may be that in asymptomatic falls in ADAMTS13 activity
prior to an acute clinical relapse, it is possible to detect activated memory B cell populations
due to less ‘background noise’ or before they marginate.
A summary of the B and T follicular helper cell immunophenotyping results at acute iTTP
presentation and ADAMTS13 relapse is shown in Table 2.
Longitudinal analysis: effect of RTX
Flow cytometric analysis of B cell subsets and cTfh cells was performed for patients pre-RTX
and longitudinally 1 month, 3 months, 6, months, 9 months, 12 months etc until end of
study/relapse. After RTX treatment, all 46 episodes achieved B cell depletion (defined by a
laboratory CD19+ count <0.005 ×109/L), except 1 patient in the ADAMTS13 relapse cohort
(Supplemental Figure 3 (a) – (f) for acute iTTP cases; data not shown for ADAMTS13
relapse episodes). In contrast, RTX did not cause any significant alterations in cTfh numbers
(Supplemental Figure 3 (g) – (k) for acute iTTP cases; data not shown for ADAMTS13
relapse episodes). B cell return occurred at median 10 months (range 6-14) months) in the
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acute iTTP group and 8 months (range 0.25-15 months) in ADAMTS13 relapse group. The
timepoint of B cell return was not associated with ADAMTS13 or clinical relapse.
Comparison of B cell subsets frequencies prior to RTX treatment and at B cell return
We then compared two specific timepoints: pre-RTX and B cell return. Repopulation
following B cell depletion in the acute iTTP group demonstrated relatively higher proportion
of plasmablasts but a reduction of in post-GC and double negative memory B cells (Figure
5a). In the ADAMTS13 relapse group where all except 1 patient had received historical RTX,
plasmablasts were increased in frequency at B cell return compared to pre-treatment levels
but the distribution of B cell subsets was otherwise similar before and after re-treatment
(Figure 5b).
Longitudinal follow up: relapses vs sustained remissions
We prospectively investigated changes in B-cell subsets after RTX in 20 iTTP patients (16
from our initial cohort and 4 additional patients) longitudinally until a subsequent clinical or
ADAMTS13 relapse, and compared to patients who remained in remission over an
equivalent period.
There were 10 patients in the relapse cohort (3 followed after an acute episode, 7 after
elective rituximab) and 10 in the remission cohort (4 acute and 6 elective episodes at t=0).
Median age was 51y (range 38-81) and 45.5y (21-68) respectively. Median follow up was15
months (range 6-24). One patient in the relapse group did not achieve B-cell depletion
(CD19+ count 0.005x10^9/L). Time to B-cell return was similar: 8 months (4-13 months) in
relapses vs 8months in remission (0.25-15.5 months). All subsequent relapses were
ADAMTS13 relapses and occurred at a median of 14 months (9-25 months).
Longitudinal analysis showed that in both groups B-cell return after rituximab develops along
normal B-cell ontogeny. The kinetics of naïve, transitional, memory B-cells and plasmablasts
did not differ significantly between patients who subsequently went on to relapse vs those
who remained in sustained remission. (Figure 6) No alterations in B-cell subsets were
identified prior to a relapse.
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DISCUSSION
Interactions between T and B cells which occur within germinal centres (GC) of secondary
lymphoid organs (SLO) are critical for the development of humoral immune responses. Tfh
cells, a distinct subset of T helper cells, have been recognised in recent years as a crucial
regulator of GC formation, B cell development and long‑term humoral memory generation,
and have a significant role in the pathogenesis of autoimmune diseases.17 The mechanism
involved in the loss of tolerance and subsequent development of anti-ADAMTS13 antibodies
in iTTP patients is still largely unknown and we currently lack the ability to predict relapse
accurately. The observed association between the MHC class II allele HLA DRB1*11 and
development of iTTP implies a role for helper CD4+ T cells in the initiation of autoimmune
reactivity against ADAMTS13.11,12,39
This is the first prospective study to perform a comprehensive analysis of B cell subsets and
cTfh cell changes in iTTP before and after immunosuppressive therapy. B cell phenotype is
altered at acute iTTP presentation with decreased post-GC memory B cells and an increase
in plasmablasts in iTTP compared to healthy controls. Potential mechanisms for the reduced
proportion of memory B cells among circulating B cells at presentation of iTTP include 1)
some B cells dying in the early memory B cell stages and never becoming memory B cells,
2) hyperactivation of T and B cells in iTTP resulting in an elevated differentiation of memory
B cells into antibody-producing plasma cells or 3) migration of memory B cells into lymphoid
tissue.40-42 Similar reductions in the memory B cell compartments have been described in
sarcoidosis, systemic sclerosis and Sjogren’s syndrome.40,43-48
Comparing immunophenotype results between different studies is complex due to the variety
of staining and gating protocols used, and clinical and demographic characteristics of the
patient cohort, but some general conclusions can be made regarding iTTP in comparison to
other autoimmune diseases. We found an increased ratio of naïve to switched memory B
cells and higher plasmablasts in the acute patients. In contrast, in patients with active SLE
there is an apparent expansion of memory vs naïve B cell subsets correlating in part with
disease activity, but this is due to a naïve B cell lymphopaenia.49 Expansion of plasmablasts
also occurs in SLE but is relatively higher than we found in iTTP. In Sjogrens syndrome,
transitional and naïve B cells seem expanded compared to memory subsets, thus closer to
the iTTP result, but in RA patients naïve and memory populations are generally similar to
healthy age matched controls.50 More recently, attention has focussed on the double
negative (DN) (IgD-CD27-) B cell subset. In systemic sclerosis, SLE and RA, the relative
importance of this subset, especially in relation to therapy resistance, has been recognised.
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The DN B cell population contains both activated and antibody secreting post germinal
centre B cells and very early switched B cells (with fewer mutated Ig transcripts).51,52 This
population was not different from HC in the iTTP patients in relation to relapse, but additional
studies would be needed to assess functional properties of B cells within this population in
iTTP.
The relative contribution of different naive and antigen-experienced B cell subsets to
pathogenesis and response to therapies will depend on the position and circumstances
underlying specific breaches of tolerance and thus ultimately depend on the specificity of the
autoreactive B cell receptor(s). The different patterns we have described in iTTP compared
to other systemic autoimmune diseases indicate a lack of apparent expansion of memory
populations in the peripheral blood and thus the probability of rapid sequestration or
expansion of autoantibody producing clones within tissues or lymphoid organs.
At acute iTTP presentation, we also demonstrate a novel association between higher
plasmablast frequency and higher ADAMTS13 IgG antibody level and a trend towards
reduced ADAMTS13 antigen levels. This suggests a potential underlying mechanism for
iTTP development, where rapid differentiation to auto-antibody producing plasmablasts
leads to increased production anti-ADAMTS13 IgG which in turn results in increased
ADAMTS13 clearance.
In asymptomatic patients with ADAMTS13 relapse, alterations in B cell subsets prior to pre-
emptive RTX therapy were pronounced with significantly increased naïve B cell population,
global decrease in all memory subsets and trend towards increased plasmablasts. These
subset alterations most likely represent changes related to historic RTX therapy with long-
term suppressive effects of RTX on memory cell subsets. This also explains the largely
similar distribution of B cell subsets before and after pre-emptive RTX treatment. Following B
cell depletion therapy, B cells repopulate primarily from bone marrow-derived naive B cells,
with delayed regeneration of memory B cells.53,54 Poor memory B cell reconstitution may
reflect long-term effects of RTX on B cells, described both after organ transplantation and in
other autoimmune diseases.55-57
Total cTfh (CD4+CXCR5+) and PD1+ Tfh cells were decreased in acute iTTP patients at
presentation compared to HC. A similar decrease in cTfh has been observed in sarcoidosis
with infiltration of Tfh into skin lesions, suggesting that cTfh are recruited into affected sites.47
In acute iTTP, a potential explanation for the concomitant decrease in post-GC memory cells
and cTfh cells may be that they are localising in germinal centres within lymphoid tissue. In
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contrast, in asymptomatic patients with ADAMTS13 relapse, there were no significant
differences in total cTfh cells and PD1+ cTfh. This may be because this is a different stage
of the disease process or possibly that the effect of previous Rituximab alters the interplay of
B and cTfh cells. Tfh helper T cells utilise CXCR5 positivity to access germinal centres (GC).
In GC, they produce IL21 which plays a key role in class switch and affinity maturation
leading the generation of memory B cells and plasma cells from antigen-activated naïve B
cells. Other peripheral blood helper T cells (CXCR5-; CXCR2 and PD1+) can also produce
IL21 and have been identified as a source of extrafollicular T cell help to B cells in patients
with other autoimmune diseases.58 Although there are no frank inflammatory sites or
lymphoid clusters in iTTP, it is possible that aberrant interactions between ADAMTS13-
specific memory B cells and other T helper cells may occur in extrafollicular sites in spleen
for example.
We also compared B and cTfh frequencies at 2 different timepoints following RTX infusion:
prior to RTX and at B cell return. B cell depletion was achieved in all patients by 1 month.
Subsequent B cell reconstitution occurred at 10 months in the acute iTTP cohort and 8
months in the asymptomatic ADAMTS13 relapse cohort. At B cell return after a de novo
acute iTTP episode, plasmablast levels were significantly raised with a trend towards
increased transitional and naïve cells. Importantly, no patients relapsed at the time of B cell
return. The time interval between B cell return and relapse suggests that additional
differentiation and selection of specific autoreactive B cell clones from naive populations may
play a key role, although expansion of ADAMTS13 specific memory B cell populations in
lymphoid tissues to critical levels cannot be ruled out. RTX did not cause any significant
alterations in cTfh frequency.
Our data also reveals altered expression of CD80 on B cells in iTTP, with decreased
expression in IgD+ memory cells and double negative memory cells in acute iTTP episodes
and increased expression in post-GC memory and double negative memory cells in
ADAMTS13 relapses, prior to RTX therapy. CD80 and CD86 are expressed on antigen
presenting cells such as dendritic cells and activated B cells, and have the capacity to
stimulate or inhibit T cell responses through their receptors CD28 and cytotoxic T
lymphocyte-associated antigen 4 (CTLA-4) respectively.10 Dendritic cells exposed to
ADAMTS13 have previously been shown to present ADAMTS13 peptides, with preferential
HLA-DRB1* 11–dependent presentation of CUB2-derived peptides, which the authors
hypothesised may contribute to the onset of acquired TTP by stimulating low-affinity self-
reactive CD4+ T cells.2,3
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Manipulation of the CD80/CD86 pathway has shown efficacy in the clinical setting, with
Abatacept (a CTLA-4 immunoglobulin which targets autoimmune B cells by reducing
CD80/CD86 expression) approved for the treatment of rheumatoid arthritis.59 Of interest, the
CD80 locus was identified in the GWAS for iTTP and contained a SNP in the 3’ UTR that
was in high linkage with the lead SNP.9 Further investigation into the link between
possession of this SNP with translation and expression of CD80 on different cell types is
ongoing. Additional studies are required to understand how these differences in expression
could translate into functional properties of CD80/86 in iTTP patients.
In the longitudinal analysis, no alterations in B-cell subsets were identified prior to a relapse,
suggesting temporal relationships between B sub-populations cannot be used as a
biomarker to better predict relapses.
Taken together, our findings give a novel insight into the role of B and cTfh cells in the
development of iTTP. We propose that de novo acute iTTP is characterised by dysregulation
of B and cTfh cell homeostasis with decreased circulating subsets of GC memory cells and
cTfh cells and an increased frequency of plasmablasts in the circulation, perhaps reflecting
the increase in cognate interaction between antigen-specific T and B cell populations in
secondary lymphoid tissue. Changes in the frequency of CD80 on B cells suggests altered
interactions with T cells. The finding of alteration in CD80 expression further supports the
recent novel association between iTTP and five alleles within a haploblock on chromosome
3 – one of which encodes CD80.9 Although our studies did not address the questions of
antigen selectivity and exquisite specificity of the autoimmune response to ADAMTS13 in
iTTP, the novel findings will direct further functional analysis, and provide potentially
interesting targets for further research and therapeutics in iTTP.
AUTHORSHIP CONTRIBUTIONS
Contributions: J.S. designed research, recruited patients, performed laboratory testing,
collected data, analysed data and wrote the manuscript. M.O.S. designed research,
recruited patients, performed laboratory testing, collected data, analysed data and wrote the
manuscript. G.C. designed research, analysed data and wrote the manuscript. Y.G.
performed laboratory testing, collected data and reviewed the manuscript. R.dG. designed
research, analysed data and wrote the manuscript. M.S. designed research, recruited
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patients, analysed data and wrote the manuscript. M.T. designed research, recruited
patients, analysed data and wrote the manuscript.
DISCLOSURE OF CONFLICTS OF INTEREST
M.S. has received speaker’s fees and honoraria from Alexion, Sanofi, Novartis, and Takeda
and has received research funding from Takeda. M.T. has received speaker’s fees
and honoraria from Sanofi and Bayer. The remaining authors declare no competing financial
interests.
REFERENCES
1. Kremer Hovinga JA, Coppo P, Lämmle B, Moake JL, Miyata T, Vanhoorelbeke K. Thrombotic
thrombocytopenic purpura. Nature reviews Disease primers. 2017;3:17020-17020.
2. Sorvillo N, van Haren SD, Kaijen PH, et al. Preferential HLA-DRB1*11-dependent presentation
of CUB2-derived peptides by ADAMTS13-pulsed dendritic cells. Blood. 2013;121(17):3502-3510.
3. Verbij FC, Turksma AW, de Heij F, et al. CD4+ T cells from patients with acquired thrombotic
thrombocytopenic purpura recognize CUB2 domain-derived peptides. Blood. 2016;127(12):1606-
1609.
4. Carsetti R, Rosado MM, Wardmann H. Peripheral development of B cells in mouse and man.
Immunol Rev. 2004;197:179-191.
5. Westwood JP, Webster H, McGuckin S, McDonald V, Machin SJ, Scully M. Rituximab for
thrombotic thrombocytopenic purpura: benefit of early administration during acute episodes and
use of prophylaxis to prevent relapse. J Thromb Haemost. 2013;11(3):481-490.
6. Froissart A, Buffet M, Veyradier A, et al. Efficacy and safety of first-line rituximab in severe,
acquired thrombotic thrombocytopenic purpura with a suboptimal response to plasma exchange.
Experience of the French Thrombotic Microangiopathies Reference Center. Crit Care Med.
2012;40(1):104-111.
7. Scully M, Cohen H, Cavenagh J, et al. Remission in acute refractory and relapsing thrombotic
thrombocytopenic purpura following rituximab is associated with a reduction in IgG antibodies to
ADAMTS-13. Br J Haematol. 2007;136(3):451-461.
8. Westwood JP, Thomas M, Alwan F, et al. Rituximab prophylaxis to prevent thrombotic
thrombocytopenic purpura relapse: outcome and evaluation of dosing regimens. Blood Adv.
2017;1(15):1159-1166.
9. Stubbs M, Coppo P, Cheshire C, et al. Identification of a novel genetic locus associated with
immune mediated thrombotic thrombocytopenic purpura. Haematologica. 2021.
10. Sansom DM, Manzotti CN, Zheng Y. What's the difference between CD80 and CD86? Trends
Immunol. 2003;24(6):314-319.
11. SCULLY M, BROWN J, PATEL R, MCDONALD V, BROWN CJ, MACHIN S. Human leukocyte
antigen association in idiopathic thrombotic thrombocytopenic purpura: evidence for an
immunogenetic link. Journal of Thrombosis and Haemostasis. 2010;8(2):257-262.
12. COPPO P, BUSSON M, VEYRADIER A, et al. HLA-DRB1*11: a strong risk factor for acquired
severe ADAMTS13 deficiency-related idiopathic thrombotic thrombocytopenic purpura in
Caucasians. Journal of Thrombosis and Haemostasis. 2010;8(4):856-859.
Downloaded from http://ashpublications.org/bloodadvances/article-pdf/doi/10.1182/bloodadvances.2022007025/1895989/bloodadvances.2022007025.pdf by guest on 11 May 2022
15
13. Laurent G, Sandrine D, Ivan P, et al. The ADAMTS131239–1253 peptide is a dominant HLA-
DR1-restricted CD4+ T-cell epitope. Haematologica. 2017;102(11):1833-1841.
14. Johana H, Silvia DA, Nuno AGG, et al. Dissecting the pathophysiology of immune thrombotic
thrombocytopenic purpura: interplay between genes and environmental triggers. Haematologica.
2018;103(7):1099-1109.
15. Breitfeld D, Ohl L, Kremmer E, et al. Follicular B helper T cells express CXC chemokine
receptor 5, localize to B cell follicles, and support immunoglobulin production. J Exp Med.
2000;192(11):1545-1552.
16. Kim CH, Rott LS, Clark-Lewis I, Campbell DJ, Wu L, Butcher EC. Subspecialization of CXCR5+ T
cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells. J Exp Med.
2001;193(12):1373-1381.
17. Crotty S. Follicular helper CD4 T cells (TFH). Annu Rev Immunol. 2011;29:621-663.
18. Rasheed AU, Rahn HP, Sallusto F, Lipp M, Müller G. Follicular B helper T cell activity is
confined to CXCR5(hi)ICOS(hi) CD4 T cells and is independent of CD57 expression. Eur J Immunol.
2006;36(7):1892-1903.
19. Webb LMC, Linterman MA. Signals that drive T follicular helper cell formation. Immunology.
2017;152(2):185-194.
20. He J, Tsai LM, Leong YA, et al. Circulating precursor CCR7(lo)PD-1(hi) CXCR5(+) CD4(+) T cells
indicate Tfh cell activity and promote antibody responses upon antigen reexposure. Immunity.
2013;39(4):770-781.
21. Locci M, Havenar-Daughton C, Landais E, et al. Human circulating PD-1+CXCR3-CXCR5+
memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody
responses. Immunity. 2013;39(4):758-769.
22. Morita R, Schmitt N, Bentebibel SE, et al. Human blood CXCR5(+)CD4(+) T cells are
counterparts of T follicular cells and contain specific subsets that differentially support antibody
secretion. Immunity. 2011;34(1):108-121.
23. Ma J, Zhu C, Ma B, et al. Increased frequency of circulating follicular helper T cells in patients
with rheumatoid arthritis. Clin Dev Immunol. 2012;2012:827480.
24. Zhu C, Ma J, Liu Y, et al. Increased frequency of follicular helper T cells in patients with
autoimmune thyroid disease. J Clin Endocrinol Metab. 2012;97(3):943-950.
25. Simpson N, Gatenby PA, Wilson A, et al. Expansion of circulating T cells resembling follicular
helper T cells is a fixed phenotype that identifies a subset of severe systemic lupus erythematosus.
Arthritis Rheum. 2010;62(1):234-244.
26. Zhang X, Liu S, Chang T, et al. Intrathymic Tfh/B Cells Interaction Leads to Ectopic GCs
Formation and Anti-AChR Antibody Production: Central Role in Triggering MG Occurrence. Mol
Neurobiol. 2016;53(1):120-131.
27. Gensous N, Charrier M, Duluc D, et al. T Follicular Helper Cells in Autoimmune Disorders.
Front Immunol. 2018;9:1637.
28. Park HJ, Kim DH, Lim SH, et al. Insights into the role of follicular helper T cells in
autoimmunity. Immune Netw. 2014;14(1):21-29.
29. Ueno H. T follicular helper cells in human autoimmunity. Curr Opin Immunol. 2016;43:24-31.
30. Scully M, Cataland S, Coppo P, et al. Consensus on the standardization of terminology in
thrombotic thrombocytopenic purpura and related thrombotic microangiopathies. J Thromb
Haemost. 2017;15(2):312-322.
31. Zheng XL, Vesely SK, Cataland SR, et al. ISTH guidelines for the diagnosis of thrombotic
thrombocytopenic purpura. Journal of Thrombosis and Haemostasis. 2020;18(10):2486-2495.
32. Cuker A, Cataland SR, Coppo P, et al. Redefining outcomes in immune TTP: an international
working group consensus report. Blood. 2021;137(14):1855-1861.
33. Turner RJ, Geraghty NJ, Williams JG, et al. Comparison of peripheral blood mononuclear cell
isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39
and CD73. Purinergic Signal. 2020;16(3):389-401.
Downloaded from http://ashpublications.org/bloodadvances/article-pdf/doi/10.1182/bloodadvances.2022007025/1895989/bloodadvances.2022007025.pdf by guest on 11 May 2022
16
34. Bohnhorst J, Bjørgan MB, Thoen JE, Natvig JB, Thompson KM. Bm1-Bm5 classification of
peripheral blood B cells reveals circulating germinal center founder cells in healthy individuals and
disturbance in the B cell subpopulations in patients with primary Sjögren's syndrome. J Immunol.
2001;167(7):3610-3618.
35. Shi Y, Agematsu K, Ochs HD, Sugane K. Functional analysis of human memory B-cell
subpopulations: IgD+CD27+ B cells are crucial in secondary immune response by producing high
affinity IgM. Clin Immunol. 2003;108(2):128-137.
36. Agematsu K, Hokibara S, Nagumo H, Komiyama A. CD27: a memory B-cell marker. Immunol
Today. 2000;21(5):204-206.
37. Kokame K, Nobe Y, Kokubo Y, Okayama A, Miyata T. FRETS-VWF73, a first fluorogenic
substrate for ADAMTS13 assay. Br J Haematol. 2005;129(1):93-100.
38. Alwan F, Vendramin C, Vanhoorelbeke K, et al. Presenting ADAMTS13 antibody and antigen
levels predict prognosis in immune-mediated thrombotic thrombocytopenic purpura. Blood.
2017;130(4):466-471.
39. John ML, Hitzler W, Scharrer I. The role of human leukocyte antigens as predisposing and/or
protective factors in patients with idiopathic thrombotic thrombocytopenic purpura. Ann Hematol.
2012;91(4):507-510.
40. Bohnhorst JO, Thoen JE, Natvig JB, Thompson KM. Significantly depressed percentage of
CD27+ (memory) B cells among peripheral blood B cells in patients with primary Sjögren's syndrome.
Scand J Immunol. 2001;54(4):421-427.
41. Hansen A, Reiter K, Ziprian T, et al. Dysregulation of chemokine receptor expression and
function by B cells of patients with primary Sjögren's syndrome. Arthritis & Rheumatism.
2005;52(7):2109-2119.
42. Bohnhorst JØ, Bjørgan MB, Thoen JE, Jonsson R, Natvig JB, Thompson KM. Abnormal B Cell
Differentiation in Primary Sjögren's Syndrome Results in a Depressed Percentage of Circulating
Memory B Cells and Elevated Levels of Soluble CD27 That Correlate with Serum IgG Concentration.
Clinical Immunology. 2002;103(1):79-88.
43. Lee N-S, Barber L, Akula SM, Sigounas G, Kataria YP, Arce S. Disturbed Homeostasis and
Multiple Signaling Defects in the Peripheral Blood B-Cell Compartment of Patients with Severe
Chronic Sarcoidosis. Clinical and Vaccine Immunology. 2011;18(8):1306-1316.
44. Kudryavtsev I, Serebriakova M, Starshinova A, et al. Imbalance in B cell and T Follicular
Helper Cell Subsets in Pulmonary Sarcoidosis. Scientific Reports. 2020;10(1):1059.
45. Binard A, Le Pottier L, Devauchelle-Pensec V, Saraux A, Youinou P, Pers J-O. Is the blood B-
cell subset profile diagnostic for Sjögren syndrome? Annals of the Rheumatic Diseases.
2009;68(9):1447-1452.
46. Ibrahem HM. B cell dysregulation in primary Sjögren's syndrome: A review. Jpn Dent Sci Rev.
2019;55(1):139-144.
47. Ly NTM, Ueda-Hayakawa I, Nguyen CTH, Okamoto H. Exploring the imbalance of circulating
follicular helper CD4+ T cells in sarcoidosis patients. Journal of Dermatological Science.
2020;97(3):216-224.
48. Szabó K, Jámbor I, Szántó A, et al. The Imbalance of Circulating Follicular T Helper Cell
Subsets in Primary Sjögren’s Syndrome Associates With Serological Alterations and Abnormal B-Cell
Distribution. Frontiers in Immunology. 2021;12:789.
49. Odendahl M, Jacobi A, Hansen A, et al. Disturbed peripheral B lymphocyte homeostasis in
systemic lupus erythematosus. J Immunol. 2000;165(10):5970-5979.
50. Hansen A, Odendahl M, Reiter K, et al. Diminished peripheral blood memory B cells and
accumulation of memory B cells in the salivary glands of patients with Sjögren's syndrome. Arthritis
Rheum. 2002;46(8):2160-2171.
51. Wei C, Anolik J, Cappione A, et al. A new population of cells lacking expression of CD27
represents a notable component of the B cell memory compartment in systemic lupus
erythematosus. J Immunol. 2007;178(10):6624-6633.
Downloaded from http://ashpublications.org/bloodadvances/article-pdf/doi/10.1182/bloodadvances.2022007025/1895989/bloodadvances.2022007025.pdf by guest on 11 May 2022
17
52. Soto L, Ferrier A, Aravena O, et al. Systemic sclerosis patients present alterations in the
expression of molecules involved in B cell regulation. Frontiers in Immunology. 2015;6.
53. Leandro MJ, Cambridge G, Ehrenstein MR, Edwards JC. Reconstitution of peripheral blood B
cells after depletion with rituximab in patients with rheumatoid arthritis. Arthritis Rheum.
2006;54(2):613-620.
54. Cambridge G, Stohl W, Leandro MJ, Migone TS, Hilbert DM, Edwards JC. Circulating levels of
B lymphocyte stimulator in patients with rheumatoid arthritis following rituximab treatment:
relationships with B cell depletion, circulating antibodies, and clinical relapse. Arthritis Rheum.
2006;54(3):723-732.
55. Bemark M, Holmqvist J, Abrahamsson J, Mellgren K. Translational Mini-Review Series on B
cell subsets in disease. Reconstitution after haematopoietic stem cell transplantation - revelation of
B cell developmental pathways and lineage phenotypes. Clin Exp Immunol. 2012;167(1):15-25.
56. Roll P, Palanichamy A, Kneitz C, Dorner T, Tony HP. Regeneration of B cell subsets after
transient B cell depletion using anti-CD20 antibodies in rheumatoid arthritis. Arthritis Rheum.
2006;54(8):2377-2386.
57. Anolik JH, Barnard J, Owen T, et al. Delayed memory B cell recovery in peripheral blood and
lymphoid tissue in systemic lupus erythematosus after B cell depletion therapy. Arthritis Rheum.
2007;56(9):3044-3056.
58. Makiyama A, Chiba A, Noto D, et al. Expanded circulating peripheral helper T cells in
systemic lupus erythematosus: association with disease activity and B cell differentiation.
Rheumatology (Oxford). 2019;58(10):1861-1869.
59. Lorenzetti R, Janowska I, Smulski CR, et al. Abatacept modulates CD80 and CD86 expression
and memory formation in human B-cells. J Autoimmun. 2019;101:145-152.
Downloaded from http://ashpublications.org/bloodadvances/article-pdf/doi/10.1182/bloodadvances.2022007025/1895989/bloodadvances.2022007025.pdf by guest on 11 May 2022
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TABLES
Table 1: A high plasmablast frequency is associated with a higher ADAMTS13 IgG
antibody level at acute iTTP presentation.
Plasmablasts <3% (n=5) Plasmablasts >3%
(n=17)
pvalue *
ADAMTS13 antigen %
(median (range))
5 (0.4 - 86) 1.9 (0.6 - 2.2) 0.4
ADAMTS13 IgG
antibody % (median
(range))
32 (2-113) 64 (52 - 127) 0.02
*Mann Whitney U test
Table 2: Summary of B and cTfh cell subsets at acute iTTP presentation and
ADAMTS13 relapse compared to healthy controls
B and cTfh cell subsets at iTTP presentation
compared to healthy controls (HC)
Acute iTTP
presentation
(n=22)
ADAMTS13
relapse (n=24)
B cell subsets (% CD19+ B cells):
Transitional cells (IgD+CD38++)
Naive cells (IgD+CD38+)
IgD+ memory cells (IgD+CD38-)
Post GC memory cells (IgD–CD38+)
Double-negative memory cells (IgD–CD38–)
Plasmablasts (IgD–CD38++)
↓
→
→
↓
→
↑
→
↑
↓
↓
↓
→
CD80 expression on B cell subsets:
IgD+ memory cells (IgD+CD38-)
Post GC memory cells (IgD–CD38+)
Double-negative memory cells (IgD–CD38–)
↓
→
↓
→
↑
↑
Circulating T follicular helper cells (% CD3+CD4+ cells):
CD4+ CXCR5+ (Total cTfh cells)
CD4+ CXCR5+ PD1+
CD4+ CXCR5+ ICOS+
↓
↓
→
→
→
↑
↓ decreased compared to HC; → no difference compared to HC; ↑ increased compared to
HC
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19
FIGURE LEGENDS
Figure 1: Percentages of (a) transitional cells (IgD+CD38++), (b) naïve cells (IgD+CD38+)
(c) IgD+ memory B cells (IgD+ CD38-), (d) post germinal centre memory B cells (IgD-
CD38+) and (e) double negative memory B cells (IgD-CD38-) and (f) plasmablasts (igD-
CD38++) in patients with acute iTTP episodes (n=22), ADAMTS13 relapse (n=24) and HC
(n=27).
A13 relapse, ADAMTS13 relapse; Post-GC memory, post germinal centre memory cells; DN
memory cells, double negative memory cells
Figure 2: Percentages of (a) total cTfh (CD4+CXCR5+), (b) PD1+ cTfh
(CD4+CXCR5+PD1+), (c) ICOS+ cTfh (CD4+CXCR5+ICOS+) and (d) PD1+ICOS+ cTfh
(CD4+CXCR5+PD1+ICOS+) in patients with acute iTTP episodes (n=34), ADAMTS13
relapse (n=27) and HC (n=27). A13 relapse, ADAMTS13 relapse.
Figure 3: Relationship between ADAMTS13 antigen levels and plasmablast frequency.
Spearman correlation analysis was performed and p<0.05 indicates that the difference is
statistically significant.
Figure 4: Analysis of CD80 expression (median fluorescence intensity) on B cell subsets
defined by IgD/CD38. (a) Acute iTTP cases and (b) ADAMTS13 relapse cases.
Figure 5: Pairwise comparison of B cell subset frequencies before and after rituximab by
Wilcoxon signed–rank test. a) Acute iTTP episodes, pairwise comparison of 10 patients, b)
ADAMTS13 relapse episodes, pairwise comparison of 13 patients.
Figure 6: Longitudinal changes in B-cell subsets after RTX therapy for an acute iTTP
episode or ADAMTS13 relapse (ADAMTS13 activity 15%) until subsequent ADAMTS13
relapse, and compared to patients who remained in remission over an equivalent period.
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(a) (b) (c) (d) (e) (f)
Figu re 1
Figure 1
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(a) (b) (c) (d)
(
a
)
)
Figu re 2
Figure 2
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0 20 40 60 80 100
0
1
2
3
4
5
9
10
15
16
Correlation: ADAMTS13 Antigen
& Plasmablasts
ADAMTS13 anti
g
en
(
%
)
Plasmablasts (%)
r= -0.41
p= 0.055
F
i
gur e 3
Figure 3
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(a) Acute iTTP episodes (b) ADAMTS13 relapse
Figure 4
Figure 4
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(a) (b) (c) (d) (e) (f)
(
c
)
(
(
e
)
(
f
)
(a) (b) (c) (d) (e) (f)
F
i
gur e 5 a
)
Figu re 5 b)
Figure 5
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Figure 6
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