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Ann. N.Y. Acad. Sci.

ISSN 0077-8923

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S
Issue: Myasthenia Gravis and Related Disorders
REVIEW

Mechanisms underlying B cell immune dysregulation


and autoantibody production in MuSK myasthenia gravis
Panos Stathopoulos,1 Aditya Kumar,1 Jason A. Vander Heiden, 1
Elba Pascual-Goñi,2
Richard J. Nowak,1 and Kevin C. O’Connor 1
1
Department of Neurology, Yale School of Medicine, New Haven, Connecticut. 2 Neurology Department, Hospital de la Santa
Creu i Sant Pau, Barcelona, Spain

Address for correspondence: Kevin C. O’Connor, Ph.D., Department of Neurology, Yale School of Medicine, Room 353J, 300
George Street, New Haven, CT 06511. kevin.oconnor@yale.edu

Pathogenic autoantibodies to muscle-specific tyrosine kinase (MuSK) can be found in patients with myasthenia
gravis (MG) who do not have detectable antibodies to the acetylcholine receptor. Although the autoantibody-
mediated pathology is well understood, much remains to be learned about the cellular immunology that contributes
to autoantibody production. To that end, our laboratory has investigated particular components associated with the
cellular immunopathology of MuSK MG. First, we found that B cell tolerance defects contribute to the abnormal
development of the naive repertoire, which indicates that dysregulation occurs before the production of autoanti-
bodies. Second, both the naive and antigen-experienced memory B cell repertoire, which we examined through the
application of high-throughput adaptive immune receptor repertoire sequencing, include abnormalities not found in
healthy controls. This highlights a broad immune dysregulation. Third, using complementary approaches, including
production of human monoclonal antibodies, we determined that circulating plasmablasts directly contribute to
the production of MuSK-specific autoantibodies in patients experiencing relapse following B cell depletion therapy.
These collective findings contribute to defining a mechanistic model that describes MuSK MG immunopathogenesis.

Keywords: myasthenia gravis; B cells; autoimmunity; immunopathology; autoantibodies; MuSK

Introduction shown that MuSK autoantibodies are pathogenic.6–8


In the AChR disease subtype, the IgG1 and IgG3
Myasthenia gravis (MG) can be considered a proto-
autoantibodies mediate immunopathology partly
type for peripheral autoantibody-mediated autoim-
through complement-dependent mechanisms. In
mune disorders.1,2 It is now well understood
contrast, MuSK autoantibodies are predominantly
that the molecular immunopathology of MG is
IgG4; this subclass does not effectively acti-
attributed to the presence of circulating autoanti-
vate complement.9 Thus, the immunopathology
bodies specifically targeting acetylcholine receptor
is mediated through autoantibody-dependent but
(AChR), muscle-specific tyrosine kinase (MuSK),
complement-independent mechanical disruption
or low-density lipoprotein receptor–related pro-
of the interaction between MuSK and the postsy-
tein 4 (LRP4).3–5 The MuSK MG disease subtype,
naptic protein LRP4 and collagen Q.10,11 Moreover,
which is the focus of this report, is characterized by
isolated Ag-specific Fabs are sufficient to induce
immunological and clinical features that are gen-
pathology in MuSK MG, which highlights the
erally distinct from AChR MG. These distinguish-
Fc-independent pathogenic mechanism of MuSK
ing features of MuSK MG include IgG4 subclass
autoantibodies that is not observed with AChR
involvement, favorable response to B cell deple-
autoantibodies.11–13
tion therapy, and a clinical course that frequently
The production of autoantibodies clearly impli-
involves bulbar symptoms (Table 1). Passive trans-
cates a principal role for B cells in the manifestation
fer and active immunization studies in animals have
of MG. Yet studies directed toward understanding

doi: 10.1111/nyas.13535
154 Ann. N.Y. Acad. Sci. 1412 (2018) 154–165 
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Stathopoulos et al. B cell abnormalities in MuSK myasthenia gravis

Table 1. Clinical and serological differences between AChR and MuSK MG2,16,59,73

Feature AChR MG MuSK MG

Bulbar symptoms Not predominant Predominant


Pure ocular form Frequent Very rare
Thymus involvement Frequent Very rare
Autoantibody titer association with symptoms Poor Present
Response to pyridostigmine Good Medium/poor
Autoantibody titer decrease post-rituximab Mild/none Pronounced
Post-rituximab relapse Frequent Rare

peripheral B cell functional abnormalities in MuSK (BCRs). This process is fundamental for gener-
MG remain very few in number. However, the suc- ating the wide diversity of the immunoglobulin
cessful introduction of biological therapeutics in repertoire, but it also creates autoreactive B cells
MG has renewed investigative interests in elucidat- alongside those that make up the non-self-reactive
ing the details of the contributions made by B cells to naive repertoire. To evade the development of
the production of MuSK MG autoantibodies.14,15 In an immune response against self, two separate
particular, several recent studies16–20 have demon- tolerance mechanisms counterselect B cells during
strated the benefits of B cell depletion ther- their development.21 The first is a central tolerance
apy through the use of rituximab. In addition checkpoint in the bone marrow between the
to diminished autoantibody titers and significant early immature and immature B cell development
clinical improvement, rituximab also allowed for stages, which removes a large population of B
tapering and subsequent discontinuation of other cells that express self-reactive BCRs.22 The sec-
immunotherapies in subsets of MG patients.16 This ond checkpoint selects against self-reactive new
treatment modality has thus offered a unique oppor- emigrant/transitional B cells before they enter the
tunity to gain further insight into which specific B mature naive B cell compartment. Defects in the
cell subsets produce pathogenic MG autoantibodies. integrity of these tolerance mechanisms can be
In this report, we focus on examining the con- demonstrated through quantifying the frequency
tributions of B cells to the immunopathology of of both polyspecific and autoreactive B cells
MuSK MG. A speculative model of autoantibody downstream of each checkpoint. A considerable
production in MuSK MG serves to navigate the number of autoimmune diseases include central
spectrum of cellular immunopathology that covers and peripheral B cell checkpoints that fail to enforce
early B cell development and repertoire formation tolerance,23–25 suggesting that the defect may be a
through MuSK autoantibody production by specific fundamental requirement of autoimmunity.
B cell subsets. Our recent findings show that abnor- Despite the detailed understanding of the
mally high frequencies of self-reactive naive B cells autoantibody-mediated pathophysiology of MuSK
accumulate in MuSK MG, indicating a breach in MG, little is known regarding how defects in
immune tolerance. Consistent with these tolerance autoimmune regulation, namely tolerance, con-
defects, we provide evidence of a distorted naive tribute to MuSK MG. In recent work, we asked
B cell repertoire that influences the memory B cell whether the naive B cell repertoire in MG shows evi-
compartment. Finally, through the study of patients dence of compromised tolerance due to checkpoint
receiving rituximab, we describe features of circu- defects, leading to the accumulation of autoreac-
lating memory B cells and plasmablasts that directly tive B cells in the naive compartment.26 Our study
contribute to MuSK autoantibody-production. included MuSK MG subjects and was designed
to determine whether B cell tolerance checkpoint
B cell tolerance defects in MuSK MG integrity was preserved in this form of MG. A well-
During early B cell development, immunoglobulin described approach22,27–29 to assess the two toler-
variable-region gene segments are stochastically ance checkpoints guiding B cell development was
recombined to generate functional antibodies that used. This approach was applied to measure the
are expressed on the cell surface as B cell receptors frequency of polyreactive and autoreactive BCRs in

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B cell abnormalities in MuSK myasthenia gravis Stathopoulos et al.

Figure 1. The fidelity of the B cell tolerance checkpoints is compromised in patients with MuSK MG. A representative example of
data analysis used to determine central B cell tolerance checkpoint fidelity is shown. The integrity of the tolerance checkpoint was
examined using a well-established assay, which operates by quantifying the fraction of naive B cell subsets that are polyreactive.
Recombinant antibodies, representing the BCRs, were generated from single new emigrant/transitional (CD19+ CD21lo CD10+
IgMhi CD27– ) B cells (which are downstream of the central B cell tolerance checkpoint) derived from two MuSK MG patients
(MG-MuSK-1, MG-MuSK-2) and two healthy controls (HD-1 and HD-2). The antibodies were then tested for reactivity with a
set of three structurally distinct antigens (dsDNA, lipopolysaccharide (LPS), and insulin) by enzyme-linked immunosorbent assay
(ELISA). The consolidated three-dimensional plots summarize the reactivity of each antibody from the subjects toward each antigen
in the ELISA. The absorbance values for LPS (y-axis), dsDNA (x-axis), and insulin (data point size) are plotted together. Green
symbols indicate antibodies that were positive for binding all three antigens (defined as polyreactive), and gray symbols indicate
those that were not positive for all three antigens. The values and pie charts shown in the bottom right corner of each graph indicate
the fraction of antibodies that were polyreactive (in green). Adapted from Ref. 26.

the naive repertoire, which are inversely correlated healthy controls was compared to the same fraction
with the extent of negative selection, thereby pro- found in the MuSK MG study subjects (Fig. 1).
viding an assessment of checkpoint integrity. The median fraction of polyreactive B cells in the
As a means to display the assay output, we show healthy controls was 8.9%, while in MuSK MG the
an example of data we collected in evaluating the fraction was 51.7%. Overall, this study26 revealed
central tolerance checkpoint in MuSK MG. The that the MuSK MG subtype harbors defects in both
checkpoint integrity was measured by determining central and peripheral B cell tolerance checkpoints.
the frequency of new emigrant/transitional B cells Defective B cell tolerance may be a fundamental
that were characterized as polyreactive by displaying contributor to autoimmunity in MuSK MG. It is
reactivity toward three structurally distinct antigens: interesting to speculate that dysfunctional tolerance
double-stranded DNA (dsDNA), lipopolysaccha- is among the requirements needed for the disease
ride (LPS), and insulin. The fraction of B cells that to develop. The population of self-reactive naive B
were positive for binding all three antigens from the cells that accumulate because of defective tolerance

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Stathopoulos et al. B cell abnormalities in MuSK myasthenia gravis

checkpoints in MuSK MG may indirectly contribute circulating B cell repertoire. We focused on deter-
to the production of autoantibodies by serving mining whether the MuSK MG repertoires included
as precursors for autoantibody-producing B cells. abnormal clonal expansions, skewed immunoglob-
Conceivable approaches for testing such a hypothe- ulin gene usage (including the H, ␬, and ␭ V(D)J
sis could include extensively testing the reactivity of loci), and distinctive antigen-binding region prop-
the naive repertoire to MuSK and applying the toler- erties. We found that large, dominant clonal lineages
ance assay, described above, to germ-line precursors were not readily apparent in the memory compart-
of antigen-experienced, somatically hypermutated ment of MuSK MG repertoires. However, several
human MuSK autoantibody–producing B cells. repertoire-scale characteristics consistent with dys-
Given the pronounced clinical improvement regulation of B cell development and selection were
commonly observed with rituximab-mediated B cell evident.
depletion in MuSK MG, it is reasonable to ques- The most prominent repertoire abnormality
tion the ability of rituximab to restore checkpoint involved the biased use of variable (V) region gene
fidelity in MuSK MG, although this remains to be segments. We observed significant shifts in the rela-
tested. It has been shown that B cell depletion in tive abundance and variability of each V gene family
type 1 diabetes, where B cell tolerance is defec- between MuSK MG and HDs.36 In the naive com-
tive, does not result in B cell tolerance checkpoint partment of MuSK MG subjects, V-family biases
fidelity restoration.30 Consequently, in MuSK MG, were observed in the form of decreased usage of
it is anticipated that B cell depletion with rituximab IGHV3 as well as an increase in IGHV1 and IGHV4
would fail to correct elevated frequencies of polyre- usage. The same V-family biases observed in naive
active/autoreactive naive B cells. If these polyreac- MG repertoires were apparent in the class-switched
tive/autoreactive naive B cells are indeed precursors memory populations, particularly in the case of the
of MuSK autoantibody producers, one could assume IgG isotype. The IGHV usage biases in the naive
that the durability of MG treatment strategies that compartment of MuSK MG subjects were also asso-
eliminate B cells may not be long-lasting. The failure ciated with an increase in the variability of IGHV
to reset tolerance checkpoint defects and eliminate usage across different MuSK MG patients. To quan-
the self-reactive pool may contribute to relapses, tify this effect, we compared V-family variability
thus underlining the need for biomarkers serving as using the repertoire dissimilarity index (RDI).37 The
relapse predictors. RDI scores the aggregate difference in gene usage
between any pair of subjects (within the HD or
The circulating B cell repertoire in MuSK
MuSK MG cohort), providing a measure of how dis-
MG is distorted
similar two gene usage distributions are from each
High-throughput adaptive immune receptor reper- other. MuSK MG repertoires display considerably
toire sequencing (AIRR-seq) allows for the com- higher RDIs and more individual RDI variability
prehensive evaluation of BCR repertoire properties within both the naive and memory compartments
in health and disease. It provides the depth neces- compared with HD repertoires, suggesting that B
sary to adequately depict large populations, such as cell developmental abnormalities in MG may be
the circulating peripheral repertoire, which includes partially patient specific (Fig. 2). Furthermore, the
up to 1 × 1011 B cells in humans.31 An increas- most pronounced difference was observed within
ing compendium of AIRR-seq studies have shown the naive compartment, where the naive HD reper-
altered repertoires in a number of diseases, includ- toires show remarkable consistency in IGHV usage
ing celiac disease,32 IgG4-related disease (IgG4- in contrast to MuSK MG repertoires. Overall, these
RD),33 systemic lupus erythematosus,34 and mul- results show that the naive MuSK MG repertoire is
tiple sclerosis.35 abnormally formed and appears to propagate defor-
We recently applied this approach to compare mations into the postgerminal center memory com-
the broad repertoire features of both the naive partment for which it is a precursor.
and memory B cell compartments in MuSK MG These findings fit well with the defects we
patients with those of healthy donors (HDs).36 We observed in the fidelity of MuSK MG B cell tol-
reasoned that a disease driven by autoantibodies erance mechanisms. Given that properly function-
may have conspicuous abnormalities present in the ing tolerance checkpoints affect counterselection of

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B cell abnormalities in MuSK myasthenia gravis Stathopoulos et al.

leading up to relapse may reflect mechanisms tran-


spiring in the early stages of the disease.
It is recognized that B cell populations that do not
express CD20, such as long-lived plasma cells sit-
uated in the bone marrow, produce a considerable
portion of circulating immunoglobulin.40 Given the
absence of CD20 on their surface, they are neither
directly (by cell lysis) nor indirectly (by depletion
of their CD20+ progenitors) affected by rituximab.
This is supported by unchanged immunization-
induced and plasma cell–dependent antibody titers
to microbial antigens, such as tetanus, varicella, and
pneumococcus, following B cell depletion.16,41,42
Figure 2. Immunoglobulin variable-region gene segment Accordingly, the markedly diminished MuSK
usage is skewed in MuSK MG repertoires. Heavy-chain V gene
family usage variability for the naive and memory B cell com-
autoantibody titer, as early as 3 months after
partments. Usage variability is represented by the repertoire rituximab-mediated CD20+ B cell depletion,16 sug-
dissimilarity index (RDI) for naive (Naive-IgM) and memory gests that long-lived plasma cells are unlikely candi-
(Memory-IgG) V families (IGHV1 thru IGHV5) among healthy dates for MuSK autoantibody production. Rather,
donors (HDs) and MuSK MG patients. Each point indicates a short-lived antibody-secreting cells, such as plas-
pairwise RDI score (y-axis) for two subjects, with the mean score
for each set of subjects marked by a horizontal bar and a density
mablasts, are more viable candidates. As only a small
estimate indicated by the shaded region. RDI scores are grouped fraction of these cells expresses CD20, the effective-
by status comparisons for HD in blue and MuSK MG (MuSK) ness of rituximab in MuSK MG may depend upon
in green. Adapted from Ref. 36. depleting a pool of plasmablast-progenitor CD20+
memory B cells.43,44 One alternative version of this
model takes into consideration that a fraction of
a considerable fraction of developing B cells,21 it plasmablasts43 may be CD20+ ; consequently, the
is reasonable to conclude that unique BCR reper- autoantibody-expressing plasmablast fraction may
toire characteristics would be conspicuous in a naive be directly depleted by rituximab.
B cell compartment that is shaped without proper We were able to support this hypothesis through
tolerance regulation. Consequently, we suggest that our recent observations of MuSK MG patients
these repertoire abnormalities are consistent with, who experienced a post-rituximab relapse.38 To
and may be reflective of, inadequate counterselec- investigate the details of autoantibody production
tion during B cell development in MuSK MG. and thereby refine the mechanisms of cellular
immunopathology in MuSK MG, we tested
MuSK autoantibody production: lessons
whether antigen-specific memory B cells and
from B cell depletion therapy
autoantibody-expressing plasmablasts emerge dur-
The specific B cell subsets that produce MuSK MG ing disease relapse. We first isolated CD27+ B cells
autoantibodies have been inadequately defined. We from post-rituximab relapse MuSK MG patients
studied such subsets in MuSK MG patients treated and controls and stimulated them with CpG, IL-21,
with rituximab who relapsed after having achieved IL-6, and CD40L to induce differentiation into
sustained remission.38 Because of the pronounced antibody-producing cells. We showed that the
clinical improvement commonly observed with CD27+ B cell population derived from MuSK MG
CD20 depletion in MuSK MG, immunosuppres- relapse patients, but not those of the controls, were
sive agents, such as steroids, mycophenolate mofetil, capable of MuSK autoantibody production upon
azathioprine, and other broad-acting agents, can stimulation. Since CD27+ B cells can include both
be tapered and often completely withdrawn.16,19,39 memory and short-lived antibody-secreting cells
Once the confounding effects of such therapeutics (plasmablasts), we next focused exclusively on the
are absent, the reemergence and evolution of the B plasmablasts in order to determine whether they
cell compartment can be investigated and associ- contributed to autoantibody production. Employ-
ated with clinical status. The postdepletion period ing multichromatic flow cytometry, we found

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Stathopoulos et al. B cell abnormalities in MuSK myasthenia gravis

plasmablast response was polyclonal/oligoclonal in


that patient. Given that these mAbs were derived
from several different clones, a continuum of
binding strengths is not unexpected. Indeed, the
plasmablast-derived mAbs displayed a range of
MuSK-binding capacities. With some behaving as
strong binders, others appeared to be relatively
weaker while remaining positive compared with the
AChR-derived controls, all of which were negative.
This range of binding strengths is in agreement
with other studies of human monoclonal recom-
binant immunoglobulins derived from patients
with autoantibody-mediated diseases. This includes
pemphigus,45,46 neuromyelitis optica,47 and AChR
Figure 3. Summary of MuSK cell-based assay data performed MG,48 all of which include mAbs reported to exhibit
with plasmablast-derived monoclonal antibodies (mAbs).
Results are presented as Δ% positive (for MuSK binding) cells
a range of binding capacities. What is important
on the y-axis. Δ% positive cells = (% frequency of positive to consider in addition to the binding strength of
MuSK–green fluorescent protein (GFP)–transfected cells/% fre- these MuSK-specific mAbs, which was measured
quency of MuSK–GFP–transfected cells) – (% frequency of with an in vitro assay, is understanding the rela-
positive GFP–transfected cells/% frequency of GFP–transfected tionship between their binding strength and their
cells). Bars represent means, and dots represent individual
mAb values. Testing of all samples was performed in dupli-
pathogenic capacity. It would be presumptuous to
cate. Plasmablast-derived mAb from MuSK MG post-rituximab assume that those with lower binding capacity may
relapse patients MuSK 1 (n = 4), MuSK 2b (n = 33), MuSK 3 not be pathogenic, given that human-derived MuSK
(n = 45), and AChR control patients AChR 7 (n = 15) and AChR mAbs have not yet been studied. This gap in our
8 (n = 11) are shown. The 4A3 mAb is a humanized, murine knowledge highlights the value of these mAbs as
hybridoma–derived MuSK-specific mAb that was used as a pos-
itive control. The dotted line represents the Δ% positive cells
tools for improving our understanding of MuSK
cutoff (control mean + 4SD = 21.9). Adapted from Ref. 38. MG pathology. We anticipate that their role in
pathology will be evaluated using in vitro models
of AChR-mediated signaling activity and broader
mildly elevated plasmablast populations (defined effects through in vivo studies with rodent mod-
as CD19+ CD27hi CD38hi cells34 ) in MuSK MG els. In addition, they will be useful in furthering
patients experiencing disease exacerbation/relapse. our understanding of how the structural character-
From these populations, single plasmablasts were istics of MuSK mAbs of the IgG4 subclass play a
sorted into individual wells in 96-well plates. role in their antigen recognition and pathogenic-
Antibody heavy- and light-chain variable regions ity. In particular, an interesting feature of secreted
were then amplified and cloned so that monoclonal IgG4 antibodies is their ability to engage in “Fab-
recombinant antibodies (mAb), each representing arm exchange,” where a monospecific IgG4 anti-
a single plasmablast, could be expressed. The body may swap a heavy- and light-chain pair with
evaluation of binding to MuSK-, clustered AChR-, another IgG4 to become bispecific.49 The biologi-
and GFP-transfected human embryonic kidney cal consequences of Fab-arm exchange are not yet
cells showed that a considerable number (seven) of fully understood. It has recently been shown that
the 82 MuSK plasmablast-derived mAbs, but none Fab-arm-exchanged autoantibodies are present in
of the 26 control (AChR MG) plasmablast-derived MuSK MG.12 Their presence in patients further
mAbs, bound specifically to MuSK (Fig. 3). highlights that our MuSK-specific mAbs, which dis-
Of the seven MuSK-specific mAbs we identified, play a range of binding capacities, may play impor-
six were derived from the same patient’s plasmablast tant roles in MuSK MG pathology. This is because
compartment. Of these six, three were individual one arm of the autoantibody may be swapped out
members of a clone, while the remaining three for another with a different specificity (perhaps also
were unique clones. With these early-stage find- targeting the neuromuscular junction), producing a
ings in mind, we reasoned that the MuSK-specific bivalent autoantibody with an augmented binding

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B cell abnormalities in MuSK myasthenia gravis Stathopoulos et al.

Figure 4. Speculative mechanistic hypothesis for the production of MuSK MG autoantibodies. The proposed mechanistic path
to autoantibody production in MuSK MG begins with naive B cells (steps 1 and 2). These cells develop in the presence of defective
B cell tolerance checkpoints, which results in a naive population that includes an abnormally high population of self-reactive cells
and a skewed repertoire (indicated by light blue cells). Activation and differentiation of the naive compartment to effector B cell
populations (memory and antibody-secreting) may require antigen exposure and help from T cells (step 3), but direct evidence of
such in MuSK MG is required. The memory B cell compartment (step 4) includes a skewed repertoire closely resembling that of
the abnormal naive compartment (indicated by light blue cells). Circulating short-lived plasmablasts and bone marrow–inhabiting
plasma cells (steps 5 and 6) may contribute to MuSK MG autoantibody production. However, observations following B cell
depletion therapy with rituximab in MuSK MG suggest that plasma cells may not play a major role. B cell depletion therapy
eliminates CD20+ memory and naive B cells but does not directly eliminate plasmablasts or plasma cells, the majority of which
do not express appreciable levels of CD20. After CD20-targeted depletion, serum autoantibody titers markedly diminish in MuSK
MG, suggesting that long-lived plasma cells are unlikely candidates for autoantibody production. Rather, short-lived plasmablasts
are more viable candidates. As only a small fraction of these cells express CD20, the effectiveness of B cell depletion therapy may
depend upon depletion of a pool of plasmablast-progenitor CD20+ memory B cells. Conversely, autoantibody titers that remain
elevated following CD20 targeted depletion may be the product of longer lived plasma cells.

capacity compared with the parent MuSK monova- naive B cells owing to defective central and periph-
lent mAb. eral tolerance checkpoints.26 The memory B cell
In summary, these collective results suggest that compartment repertoire reflects abnormalities of
oligoclonal-circulating plasmablasts contribute to the deformed naive repertoire.36 In addition, we
MuSK autoantibody production. Furthermore, postulate that this deformed memory repertoire
these cells appear to emerge in the reconstituted B harbors MuSK-specific B cells. The memory B cell
cell repertoire of patients experiencing relapse after pool can then supply the antibody-secreting B cell
B cell depletion therapy. To the best of our knowl- pool, which comprises a spectrum of mainly CD20–
edge, these are the first reported human-derived short-lived (plasmablasts) and long-lived antibody
MuSK-specific mAbs,38 and the results provide a secretors (plasma cells). Our more recent study
more granular understanding of the mechanisms points toward the role of such peripheral blood
governing MuSK autoantibody production. plasmablasts in MuSK autoantibody production.38
Apart from our recent laboratory-based findings,
A speculative model of MuSK MG this model is supported by clinical observations in
immunopathology different diseases associated with IgG4 autoantibod-
Taken together, the findings detailed above sup- ies, including MuSK MG, pemphigus, thrombotic
port a speculative model describing the role of thrombocytopenic purpura (TTP), and membra-
B cell subsets in MuSK MG immunopathology nous nephropathy. Such observations include the
(Fig. 4). In brief, the model suggests that pathol- rather rapid decrease of disease-specific autoanti-
ogy begins with the accumulation of autoreactive body titers occurring after B cell depletion with

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rituximab.16,39,50–52 On the basis of our proposed autoantibody-mediated diseases, such as pemphi-


model, this decrease could be attributed to the gus, TTP, and membranous nephropathy.
depletion of CD20+ memory B cells and the Clinical relapses after achieving remission with
consequent ablation of the continuous supply of rituximab and associated biomarkers are interesting
antibody-secreting cells. The terminated supply and relevant points of comparison between MuSK
would affect a decrease in the population of short- MG and pemphigus or membranous nephropathy.
lived antibody producers. Alternatively, a fraction Since pemphigus has been studied more extensively
of plasmablasts may be CD20+ ,43 the consequence than MuSK MG, and given that the two conditions
of which is their direct depletion, which con- are analogous, as described above, clinical and sero-
tributes to decreased titers and a favorable clinical logical observations in pemphigus could inform
response. anticipated responses to rituximab in MuSK MG.
The model mechanism we propose for describ- Our group has been following a rituximab-treated
ing the post-rituximab MuSK autoantibody titer MuSK MG cohort for almost 10 years.19,38,39 Our
decrease is well supported by observations in other experience is comparable with that of other cen-
IgG4 autoantibody–related conditions. These con- ters (Table 2). Some larger series16,39 did not report
ditions could be viewed as a disease group that relapses with a median follow-up that was approx-
shares common pathogenic characteristics. Among imately 2–4 years. In contrast to these series, iso-
those, pemphigus is a well-characterized and exten- lated patients that have relapsed have been reported
sively studied autoimmune mucocutaneous blister- with similar follow-up periods.19,65,66 Moreover, we
ing disease with an immunopathological profile that recently reported three such relapses.38 In pemphi-
is in many ways parallel to that of MuSK MG. First, gus, clinical relapses post-rituximab vary from a
pemphigus pathology is mediated by autoantibodies reported low of 0–20% (intensive rituximab dosing
against desmosome proteins desmoglein (DSG)-1 combined with IVIg and 10 years of follow-up)57 to
and DSG3.53 These autoantibodies are predomi- 50–80% (occurring on average after 1.5 years with
nately of the IgG4 subclass (and secondarily of the standard rituximab dosing).57,58 Relapsing pemphi-
IgG1 subclass as in MuSK MG) and correlate with gus patients had either increased or persistently high
disease activity.50,54–59 Second, pemphigus responds levels of pemphigus autoantibodies.56–58 Further-
well to B cell depletion with rituximab and does so in more, in membranous nephropathy, an increase or
a manner that is quite similar to MuSK MG.50,56–58,60 reemergence of PLA2R antibodies predicted clini-
Importantly, rituximab induces a significant decline cal relapse.64 In our recent report,38 all MuSK MG
in serum anti-DSG1 and anti-DSG3 antibodies patients experiencing relapse had measurable MuSK
along with clinical improvement.50,56,60,61 In addi- autoantibodies. Given that MuSK MG autoantibod-
tion to pemphigus, analogous clinical and serolog- ies show correlations with clinical fluctuations,9,59
ical responses to rituximab have been reported in they could provide a useful post-rituximab relapse
TTP and membranous nephropathy.51,52 In TTP, biomarker as in the case of pemphigus and mem-
rituximab induces a significant decrease of the pre- branous nephropathy.67
dominantly IgG4, pathogenic disintegrin and met- Our proposed model postulates that autoreac-
alloproteinase with a thrombospondin type 1 motif, tive naive B cells differentiate into memory B cells
member 13- (ADAMTS13-) autoantibodies, accom- with the help of MuSK-specific T cells. Further,
panied by recovery in ADAMTS13 activity and clin- the distorted memory B cell compartment likely
ical improvement.51,62 Similarly, in membranous includes MuSK-specific memory B cells that dif-
nephropathy, the PLA2R antibodies, which are pri- ferentiate to MuSK autoantibody–secreting cells.
marily of the IgG4 subclass,63 decrease with ritux- Given the analogies of MuSK MG and pemphi-
imab therapy.52,64 Collectively, these data provide gus, our model is supported by studies that have
evidence that rituximab affects a decrease of IgG4 explored antigen-specific B and T cell repertories
autoantibody titers in a variety of similar autoim- in pemphigus. Specifically, circulating DSG1- and
mune disorders. It could therefore be inferred DSG3-specific IgG+ B cells were examined before
that our proposed model, which features short- and after rituximab. B cells were stained with fluo-
lived plasmablasts in MuSK MG cellular pathogen- rescently labeled recombinant DSG1 or DSG3, and
esis, is similar to those that describe other IgG4 their frequency was determined by flow cytometry.

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B cell abnormalities in MuSK myasthenia gravis Stathopoulos et al.

Table 2. Summary of the available literature describing the use of rituximab in MuSK MG

Follow-up MGFA-PIS at the Relapse


Study N Protocol (months) end of follow-up (n)

Hain, et al.74 1 375 mg/m2 weeks 1, 2, 3, and 4 and months 2, 3, 4, 12 I 0


and 5
Lin, et al.75 1 375 mg/m2 weeks 1, 2, 3, and 4 18 U 0
Thakre, et al.76 1 375 mg/m2 weeks 1, 2, 3, and 4 (cycle); cycle Q6M 13 CSR 0
Baek, et al.77 1 375 mg/m2 weeks 1, 2, 3, and 4 (cycle); cycle Q6M 1 18 CSR 0
×, 375 mg/m2 Q10W for 6 months
Illa, et al.18 3 375 mg/m2 weeks 1, 2, 3, and 4; Q2M 9 MM 0
Lebrun, et al.17 3 375 mg/m2 weeks 1, 2, 3, and 4; Q2M for 6 months 12–25 CSR, MM 0
Zebardast, et al.20 4 375 mg/m2 weeks 1, 2, 3, 4, 5, and 6 (one cycle); 13–27 CSR, PR, MM 0
repeat after 1 year (weeks 1, 2, 3, 4, and 5)
Maddison, et al.78 3 375 mg/m2 weeks 1, 2, 3, and 4; Q1M for 3 months 4–18 PR, I 0
Blum, et al.65 3 500 mg weeks 1 and 3 or 500 mg weeks 1, 2, 3, and 4 12–47 CSR, PR 2
or 500 mg/m2 week 1 (last two patients relapsed)
Stein, et al.79 2 1000 mg months 1 and 2 or 375/m2 weeks 1, 2, 3, 14–18 PR 0
and 4
Burusnukul, et al.80 2 375/m2 weeks 1, 2, 3, and 4 21–24 CSR 0
Nowak, et al.19 8 375 mg/m2 weeks 1, 2, 3, and 4 (cycle); cycle Q6M 12–30 CSR, PR, MM, I 2
Guptill, et al.66 6 375 mg/m2 weeks 1, 2, 3, and 4, then Q1M for 1–2 2–40 MM, PR, I 1
months. Repeat in the case of relapse
Diaz–Manera, et al.16 6 375 mg/m2 weeks 1, 2, 3, and 4, Q2M for 2 months, 6–60 CSR, PR, MM 0
retreat if symptomatic
Catzola, et al.81 1 375 mg/m2 weeks 1, 2, 3, and 4 and 3 months 20 MM 0
Keung, et al.39 9 375 mg/m2 weeks 1, 2, 3, and 4 (cycle), cycle Q6M 26–72 CSR, MM 0
stop at 2–5 cycles
Yi, et al.82 1 1000 mg × 6 in 8 weeks 34 W 0
Guptill, et al.14 3 Not available 13–36 MM, I 0
Govindarajan, et al.83 1 375 mg/m2 weeks 1, 3, 13, and 23 15 CSR 0
Stathopoulos, et al.38 4 375 mg/m2 weeks 1, 2, 3, and 4, repeat in 6 months 25–96 CSR, E 3
(cycle). Then repeat in the case of relapse.
Abbreviations: CSR, complete stable remission; I, improved; MGFA, MG Foundation of America clinical classification; MM, minimal
manifestations; PIS, postintervention status classification; PR, pharmacologic remission; Q1M, every month; Q2M, every 2 months;
Q6M, every 6 months; Q10W, every 10 weeks; U, unchanged; W, worse.

Clinical and serological response correlated with is the removal of CD20+ B cells that function as
disappearance of circulating DSG-specific IgG+ B antigen presenting cells. Moreover, the decrease of
cells, whereas relapsing patients showed high lev- DSG3-specific CD4+ T cells preceded the decline
els of the same.58,60 Moreover, peripheral blood in DSG3 serum autoantibodies. The presence of
immunophenotyping in these responding patients antigen-specific cells (T and B) that parallel dis-
showed an increase of CD19+ CD27– naive B cells ease activity in pemphigus supports the existence of
and a decrease in CD19+ CD27+ memory B cells. pathogenic, MuSK-specific memory B cells and T
In patients with long-lasting remissions, circulating helper cells, given the similarities between the two
DSG-specific B cells could not be detected. In addi- disorders.
tion to B cell studies, the T cell compartment has also Additional data supporting our MuSK MG
been explored in pemphigus.61 Analysis of DSG3- model, in the context of B cell depletion, are found
reactive T cell subsets showed that, while rituximab in the field of IgG4-related disease. IgG4-RD is
did not alter the overall pool of peripheral T cells, a multiorgan immune-mediated disorder charac-
DSG3-specific CD4+ T cells declined significantly. terized by IgG4 lymphoplasmacytic infiltration of
One potential explanation for this effect on T cells affected tissues and fibrosis.68,69 Many patients show

162 Ann. N.Y. Acad. Sci. 1412 (2018) 154–165 


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Stathopoulos et al. B cell abnormalities in MuSK myasthenia gravis

elevated serum IgG4,69 but its role is unknown. In part, by the National Institute of Neurological Dis-
contrast to MuSK MG, there is no known anti- orders and Stroke (NINDS) of the National Insti-
genic target. Nevertheless, IgG4-RD activity cor- tutes of Health under award number U01NS084495.
relates with the presence of peripheral, clonally J.A.V.H. is supported, in part, by National Institutes
expanded IgG4 plasmablasts.70 Moreover, IgG4-RD of Health award number R01AI104739. The authors
responds to B cell depletion with rituximab.71 Rit- thank Karen Boss for editorial assistance.
uximab decreases total serum IgG4 antibodies, but
Competing interests
also reduces IgG4-expressing plasma cells in tissue.71
Interestingly, peripheral blood plasmablasts decline K.C.O. has received personal compensation in the
during remission and rise before clinical relapse70 past year from Genentech for educational activ-
and are thus a potentially useful biomarker for ities and from Proclara Biosciences and Editas
disease monitoring during rituximab therapy.72 Medicine for consulting services. R.J.N. reports sup-
The post-rituximab disappearance of CD20– plas- port through an investigator-initiated trial agree-
mablasts and plasma cells suggests that these popu- ment from Genentech for placebo/drug for the
lations are short-lived and continuously supplied currently underway clinical trial (www.clincial tri-
by a CD20+ memory pool that is depleted by als.gov, NCT02110706).
rituximab.
In conclusion, the mechanistic model that we
have developed to describe the contributions of B References
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