Clinical Immunology 245 (2022) 109184

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Clinical Immunology
journal homepage: www.elsevier.com/locate/yclim

Single-cell mapping reveals dysregulation of immune cell populations and

VISTA+ monocytes in myasthenia gravis
Rui Fan a, 1, Wenjun Que a, b, 1, Zhuoting Liu a, Wei Zheng a, Xia Guo a, c, Linqi Liu a, Fei Xiao a, *
a
Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing 400016, China
b
Department of Blood Transfusion, the First Affiliated Hospital of Chongqing Medical University, No.1 Youyi Road, Yuzhong District, Chongqing 400016, China
c
Department of Neurology, the Second Affiliated Hospital of Zunyi Medical University, The Intersection of Xinlong Avenue and Xinpu Avenue in New Pu District, Zunyi
563000, China

A R T I C L E I N F O A B S T R A C T

Keywords: The pathogenesis and progression of myasthenia gravis (MG), an autoimmune disease, involve abnormal func­
Myasthenia gravis tion and composition of several immune cell populations. However, details of this dysregulation remain unclear.
Cohort We performed a cross-section analysis using cytometry time-of-flight on blood samples from 12 generalized MG
Immune
without glucocorticoid or other immunosuppressant treatment, and 10 sex- and age-matched healthy controls.
monocyte
NK cell
Combining data from an external validation cohort (MG n = 38, control n = 21), bulk-RNA sequencing and
Mass cytometry single-cell RNA sequencing, alterations in immune cell populations and differential expression of immune check
Immune check point point were revealed. Several switched memory B cell subsets (CD3- CD19+ CD27+ IgD- CD38+/− ) were
increased in MG patients. The number of HLA- DQ- CD38+ naïve B cells was higher in MG patients and
correlated with the quantitative MG score (QMG). Among NK cells, the number of CD56+ CD16+ NK cells and
CD56+ CD16+ CD8+ NK cells were decreased in MG patients and positively correlated with QMG. VISTA+
monocytes were increased in MG patients. Classical T cell subsets showed no significant change; however, the
expression of VISTA, LAG3, CTLA4, and CXCR5 was higher in T cells from MG patients. The expression of CD38
was higher in neutrophils from MG patients. The external validation cohort validated the dysregulation of NK cell
subtypes, and differences were also observed in subgroups of patients. Bulk-RNA sequencing also revealed
increased mRNA expression of VSIR in monocytes of MG patients compared to those from healthy controls, and
the antigen presentation and processing pathway was identified as enriched in the functional characterization of
VISTA+ monocytes via single-cell RNA sequencing. Our study revealed alterations in several immune cell subsets
and identified potential cellular biomarkers for MG diagnosis and disease severity assessment. In addition, the
abnormal expression of multiple immune checkpoints in MG provides further rationale for the investigation of
immune-checkpoint-related therapy.

1. Introduction
Myasthenia gravis (MG) is a typical T cell-dependent autoimmune
disease mediated by a series of autoantibodies, including anti-
acetylcholine receptor (AchR), anti-muscle-specific receptor tyrosine
kinase, anti-lipoprotein-receptor-related protein 4, anti-agrin, anti-rya­
nodine receptor, and anti-titin. [1] These antibodies are produced from
autoreactive B cells activated by CD4+ T cells and bind to functional
molecules in the postsynaptic membrane, causing impairment of signal
transmission in synapses and resulting in a series of clinical symptoms
that include ptosis, dysphagia, limb weakness, and even dyspnea. The
incidence rate of MG is 1.7 to 21.3 cases per million person-years, and
the prevalence rate is 15 to 179 cases per million persons [2]. The
Myasthenia Gravis Foundation of America's clinical classification is
based on disease severity; with class I corresponding to ocular MG
(OMG), and classes II to V to generalized MG (GMG) [3]. More than half
of OMG cases will progress to GMG within three years, and about 20% of
all MG cases progress to crisis within 1–3 years for unknown reasons.
Although anti-AchR is a pathogenic antibody in over 80% of MG
cases and is involved in the pathogenesis of MG via multiple

* Corresponding author at: Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, 1st Youyi Road, Yuzhong District, Chongqing
400016, China.
E-mail address: feixiao81@126.com (F. Xiao).
1
These authors contributed equally

https://doi.org/10.1016/j.clim.2022.109184
Received 4 September 2022; Received in revised form 21 October 2022; Accepted 5 November 2022
Available online 11 November 2022
1521-6616/© 2022 Elsevier Inc. All rights reserved.
R. Fan et al. Clinical Immunology 245 (2022) 109184

mechanisms, such as complement-mediated damage to the postsynaptic 2. Materials and methods

membrane, diminishing Ach-AchR binding, and increasing AchR
degradation [4], there is no significant correlation between antibody
titer and disease severity, and the underlying cause is still unclear.
Recently, several studies have indicated that specific immune cell sub­
sets, including follicular helper T cells (Tfh) and ThCD103, are different in
MG and healthy controls (HC), and their relative frequencies are
significantly associated with disease severity [5,6]. However, most
studies have focused on T and B cell subsets, whereas few have focused
on the dysregulation of natural killer (NK) or myeloid cell subsets via an
unbiased analysis method. The V-domain immunoglobulin suppressor of
T cell activation (VISTA), also known as PD-1H, is a novel immune
checkpoint (ICP) molecule mainly expressed on myeloid and T cells [7]
that is involved in multiple diseases such as cancer and systemic lupus
erythematosus (SLE) [8]. However, the role of VISTA in myasthenia
gravis remains largely unknown.
Mass cytometry/cytometry time-of-flight (CyTOF) is a rapidly
evolving technique that combines mass spectrometry and flow cytom­
etry, and provides significant advantages for the discovery of new cell
subsets, revealing abnormal immune statuses in autoimmune diseases
and oncologic disorders by simultaneous quantitative detection of more
than 40 proteins in a single cell. Recently, Ingelfinger et al. [6]
demonstrated via CyTOF that the main population of immune cells was
similar in MG patients and in healthy controls, and described an alter­
ation in the ThCD103 and ThGM cells from the blood and thymus in MG
patients. However, an in-depth investigation of other immune cell sub­
sets and novel ICP expression in MG is lacking at present. Moreover,
multiple studies have indicated that immune status, as well as the status
of the thymus and potential treatment strategies, are different in OMG
and in GMG, and we considered these factors to be potential con­
founders that may lead to biased results. We ruled out potential con­
founders in this study by recruiting 12 GMG patients and 10 age- and
sex-matched HCs. Phenotypic alterations of immune cell populations
were evaluated via CyTOF using 42 markers. The main altered cell
populations were validated by external validation cohort, main altered
ICP in mRNA level and functional characterization of novel cell popu­
lation revealed by bulk-RNA-sequencing and single-cell RNA-
sequencing.
2.1. Ethics statement
This project was approved by the Ethics Committee of the First
Affiliated Hospital of Chongqing Medical University (2020–382). Writ­
ten informed consent was obtained from all subjects.
2.2. Clinical features of enrolled participants
Patients with MG, and age- and sex-matched healthy controls (MG
group, n = 12; HC group, n = 10) (Table 1) were recruited from the
Department of Neurology of the First Affiliated Hospital of Chongqing
Medical University. Peripheral blood leukocytes were isolated within 12
h after blood collection, and antigen expression was detected using mass
cytometry. All enrolled patients satisfied the following conditions: 1)
Generalized myasthenia gravis; 2. Duration of the disease >3 months; 3)
Not receiving glucocorticoids or other immunosuppressant treatments,
or treatment having been discontinued for >3 months; 4) Not affected
by any other autoimmune, inflammatory, or infectious diseases, or
pregnancy; 5) Not affected by thymoma. Data from the external vali­
dation cohort (MG, n = 38; Control (CTRL), n = 21) were downloaded
from Mendeley Data (https://data.mendeley.com/datasets/nkcb8nc
7w8/1) [6].
2.3. Mass cytometry
Metal-tagged antibodies (Supplementary Table 1) were purchased
from Fluidigm Corporation (CA, USA). Cell labeling was performed as
previously described [9]. Briefly, after peripheral blood leukocytes were
isolated, single-cell suspensions were stained for 5 min with a 5 μM
solution of 194Pt cisplatin (Fluidigm) to distinguish between live and
dead cells, then blocked with phosphate buffered saline containing 5%
goat serum and 30% bovine serum albumin (BSA) for 30 min at 4 ◦ C.
Next, they were stained with different surface markers on ice for 30 min
at 4 ◦ C. The Foxp3 Fixation and Permeabilization kit (eBioscience) was
used according to the instructions of the manufacturer. After washing,
resuspension, and centrifugation, cells were incubated overnight. Next,
after being washed twice with Foxp3 permeabilization and fluorescence-
activated cell sorting (FACS) buffer, cells were incubated with

Table 1
Information of enrolled participants.
Participants Gender Age MGFA Thymoma Anti-AchR Anti-Musk Anti-LRP4 Course (Month)
(M/F) (Y) classification

MG1 F 72 IIIB None Positive Negative Negative 41

MG2 F 47 IIIA None Positive Negative Negative 19
MG3 M 40 IIIA None Positive Negative Negative 60
MG4 F 50 IIA None Positive Negative Negative 163
MG5 F 49 IIB None Positive Negative Negative 223
MG6 F 53 IIB None Positive Negative Negative 27
MG7 F 21 IIIA None Positive Negative Negative 10
MG8 F 48 IIA None Positive Negative Negative 225
MG9 M 57 IIIB None Positive Negative Negative 34
MG10 M 24 IIIB None Positive Negative Negative 21
MG11 M 44 IIA None Positive Negative Negative 18
MG12 M 27 IIIA None Positive Negative Negative 24
HC1 F 66 NA None ND ND ND NA
HC2 F 47 NA None ND ND ND NA
HC3 F 52 NA None ND ND ND NA
HC4 F 25 NA None ND ND ND NA
HC5 M 34 NA None ND ND ND NA
HC6 F 58 NA None ND ND ND NA
HC7 M 24 NA None ND ND ND NA
HC8 F 58 NA None ND ND ND NA
HC9 F 72 NA None ND ND ND NA
HC10 F 48 NA None ND ND ND NA

AchR: acetylcholine receptors; F, female; HC: healthy controls; LRP4: lipoprotein-receptor-related protein 4; M, male; MG: myasthenia gravis; MGFA: Myasthenia
Gravis Foundation of America; MuSK: muscle-specific kinase; NA, not applicable; ND, not done; Y, years.

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R. Fan et al. Clinical Immunology 245 (2022) 109184

intracellular antibodies in permeabilization buffer for 30 min at 4 ◦ C.
Finally, after washing and resuspension, cells were analyzed within 12 h
on a Helios CyTOF System (Fluidigm Corporation) at an event rate of
<500 events/s.
2.4. Data analysis
After data acquisition, .fcs and normalized files were obtained using
a Helio3 CyTOF mass cytometer (Fluidigm Corporation) and bead-based
normalization software [10]. CD45+ cells were gated after the elimi­
nation of calibration beads and cell fragments, and multi-omics analyses
were conducted to explore the potential biological function of novel
significantly different cell subsets (Fig. 1A). CD45+ live cells and other
immune cell populations were defined by loading data into FlowJo (http
s://www.flowjo.com/). The .fcs file was then loaded into R software
(version 4.3.1, The R Foundation, Vienna, Austria)) using the FlowCore
R package (http://bioconductor.org/packages/flowCore/). The unsu­
pervised clustering algorithm (PhenoGraph) and data transformation
were performed using the cytofkit R package [11], the tSNE algorithm
was performed using the Rtsne package (https://github.com/jkrijth
e/Rtsne/), and the visualization was performed using the ggplot2
(https://CRAN.R-project.org/package=ggplot2/) and the ggpubr pack­
age (https://CRAN.R-project.org/package=ggpubr).

Fig. 1. The composition of the population of the main immune cells differed in MG patients and HCs. A. Flowchart of experimental design. B. Heatmap showing
lineage markers among the main immune cell populations. C. tSNE plot of immune cell populations in MG patients and HCs. D. Differences in the population of the
main immune cells between MG patients and HCs. Statistical analysis was performed using unpaired t-tests (significance level: NS >0.05; *0.01 < p < 0.05; **0.001
< p < 0.01; *** < 0.001). DC: dendritic cells; HC: healthy controls; MG: myasthenia gravis; NK: natural killer cells; NKT: natural killer T cells. tSNE: t-distributed
Stochastic Neighbor Embedding.

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R. Fan et al. Clinical Immunology 245 (2022) 109184

2.5. Bulk-RNA-sequencing and single-cell RNA-sequencing data
The gene expression profiles of bulk-RNA-sequencing and single-cell
RNA-sequencing were obtained from the Gene Expression Omnibus
database (http://www.ncbi.nlm.nih.gov/geo) (GSE85452 and
GSE103544). In the GSE85452 dataset, CD14 + monocytes were iso­
lated from 13 patients with MG and 12 HC. In the GSE103544 dataset,
we obtained 856 RNA-sequencing data of CD14 + monocytes from two
healthy donors.
2.6. Semi-supervised clustering algorithm, differential expression gene
analysis and functional enrichment
Semi-supervised Category Identification and Assignment (SCINA)
[12] was conducted using the SCINA package to identify VISTA+
monocytes using single-cell RNA sequencing. The Seurat package was
used to construct a single-cell analysis file and identify differentially-
expressed genes (DEGs) using standard MAST and DESeq2 methods.
Four pseudobulk-based algorithms were applied using the Libra package

Fig. 2. Phenotypic alterations exhibited by B cells and T cells in MG. A. tSNE plot of B cell clusters corresponding to MG patients and HCs. B. Heatmap showing the
markers among B cell clusters identified by the PhenoGraph algorithm. C. Differences in the two main classical B cell subsets between MG patients and HCs. D.
Annotation of B cell clusters. E. F. Five significantly different subsets of B cells. G. Correlation between the frequency of HLA- DQ- CD38+ naïve B cells and QMG.
Statistical analysis performed using unpaired t-tests (significance level: NS >0.05; *0.01 < p < 0.05; **0.001 < p < 0.01; *** < 0.001) and Pearson correlation
analysis. HC: healthy controls; MG: myasthenia gravis. tSNE: t-distributed Stochastic Neighbor Embedding.

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R. Fan et al.
[13]. Transcription factor enrichment analysis was performed using
CHEA3 (https://maayanlab.cloud/chea3/). Gene ontology (GO) and
Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrich­
ment were performed using ClusterProfiler (https://guangchuangyu.
github.io/software/clusterProfiler), and visualization was performed
using the GOplot R package (https://cran.r-project.org/web/packa
ges/GOplot/citation.html).
2.7. Statistical analysis
Statistical analysis was performed using R software, and differences
between the two groups were evaluated using either an unpaired Stu­
dent's t-test or a Mann–Whitney U test. Correlations were assessed using
Spearman's analysis.
2.8. Role of the funders
The funders had no role in the study design, data collection, data
analyses, interpretation, or writing of this report.
3. Results
3.1. Composition of main immune cell populations differed in HC and MG
B cells, T cells, monocytes, NK, natural killer T cells (NKT cells),
neutrophils, and dendritic cells (DC) were identified by PhenoGraph
based on the expression of lineage markers, including CD3, CD19, CD14,
CD56, CD66b, and HLA-DR (Fig. 1B), and the dimensionality reduction
of these cell populations was assessed by t-distributed stochastic
neighbor embedding (tSNE) (Fig. 1C). Statistical analysis of the fre­
quencies of these main immune cell populations indicated that the fre­
quencies of B cells and monocytes were significantly higher in MG
patients compared to those those in HCs, but were not significantly
associated with quantitative MG score (QMG). No significant differences
were observed in T cells and other immune cell populations between the
two groups (Fig. 1D). However, tSNE plots revealed heterogeneity in NK
cells, monocytes, and neutrophils between the MG and HC groups.
3.2. Frequency of switched-memory B cells increased in MG
To further explore the heterogeneity of B cells in HCs and MG pa­
tients, and to identify the main B cell subsets that were increased in MG
patients, B cells were further divided into 18 clusters by unsupervised
clustering algorithms using PhenoGraph and reduced dimensionality by
tSNE (Fig. 2A and B). These clusters could then be classified into four
classical subsets according to the expression of IgD and CD27: switched
memory B cells (CD27 + IgD-), non-switched B cells (CD27 + IgD+),
naïve B cells (CD27-IgD+), and double negative B cells (CD27-IgD-).
Switched memory B cells and non-switched B cells were significantly
increased in MG patients (Fig. 2C). These four classical subsets of B cells
can be subdivided into 12 subsets in combination with other cell
markers (Fig. 2D), and we found that five B cell subsets were signifi­
cantly different between the HC and the MG groups (Fig. 2E, F). Cluster9
(HLA-DQ-CD38+ naïve B cells) was increased in MG patients and
significantly associated with QMG (Fig. 2G). Using the same approach, T
cells were subdivided in 30 clusters (Fig. S1A), but the frequencies of
classical subpopulations, including naïve T cells, effector memory T cells
(TEM), central memory T cells (TCM), and effector T cells (Teff), were
not significantly different between the HC and the MG group (Fig. S1B,
S1C). Interestingly, the expression of immune checkpoints in T cells,
including VISTA, CTLA4, and LAG3 (but not PD-1), was significantly
higher in MG patients, (Fig. S1D). Previous studies suggested that the
number of Tfh was increased in MG patients and correlated with QMG
[14], and our result showed that the expression of CXCR5 (a charac­
teristic marker for Tfh), was significantly increased in MG patients
(Fig. S1D).
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Clinical Immunology 245 (2022) 109184
3.3. Phenotypic alteration of NK cells in MG
In addition to T and B cells, other immune cells contribute to the
regulation of immune responses in direct or indirect ways. Approxi­
mately 33 clusters were identified by PhenoGraph in CD3- CD19- cells,
and CD14+ monocytes, neutrophils, CD56+ NK cells, and dendritic cells
(DCs) were identified using classical lineage markers (Fig. 3A and B,
Supplementary Table 2). Although the total frequency of neutrophils,
CD56+ NK cells, and DCs was not significantly different between the HC
and the MG groups, the tSNE map showed heterogeneity of these cell
populations between the groups (Fig. 3A). To explore the cell pop­
ulations that showed significant differences and were associated with
disease severity, a series of statistical analyses was conducted. Inter­
estingly, the main significantly different cell populations between the
HC and MG were NK cell subsets and it correlated with QMG. The fre­
quencies of cluster27 and cluster29 were increased in the MG group,
those of cluster25 and cluster26 were decreased in the MG group. The
frequency of cluster29 was negatively correlated with QMG; whereas
those of cluster7, 22, and 26 were positively correlated with QMG.
Notably, cluster27 and cluster29 corresponded to CD56+ CD16- NK
cells, and cluster25 and clutser26 to CD56+ CD16+ NK cells (Fig. 3B).
CD56+ NK cells were clustered by PhenoGraph and visualized by tSNE,
and the results showed that the position of CD56+ CD16+ NK cells was
almost empty in the MG group; conversely, the position of CD56+ CD16-
NK cells was almost empty in the HC group (Fig. 3C-E). The frequency of
CD56+ CD16+ NK cells was positively correlated with QMG. In addi­
tion, the frequency of CD56+ CD16+ CD8+ NK cells was positively
correlated to QMG and was significantly lower in in the MG group
compared to that in HCs (Fig. 3E and F). Five thousand cells were
randomly extracted from each sample in the external validation cohort,
and 25 clusters were identified by PhenoGraph based on the expression
of CD3, CD19, CD14, CD56, CD16, and CD8. Cluster8 and cluster19
were identified as CD56+ CD16+ NK cells, and cluster19 as CD56+
CD16+ CD8+ NK cells (Fig. S2A and B). We found that the frequency of
CD56+ CD16+ NK cells was significantly lower in the MG group
compared to that in the CTRL group, which validated the reliability of
our results. In addition, the results of the subgroup analysis showed that
the frequency of CD56+ CD16+ NK cells was lower in early-onset MG
(EOMG) than in late-onset MG (LOMG) cases, lower in patients with
azathioprine therapy than in patients without immunosuppressive
therapy, lower in patients with thymectomy than in those without thy­
mectomy, and lower in GMG patients than in OMG patients (Fig. S2C).
CD56+ CD16+ CD8+ NK cells showed a similar trend (Fig. S2D). The
frequencies of CD56+ CD16+ and CD56+ CD16+ CD8+ NK cells also
showed a high area under the curve for the diagnosis of MG (73% and
72%, respectively) (Fig. S2F). However, there was no significant corre­
lation between the frequencies of NK cell subsets and the titer of the anti-
AchR antibody (Fig. S2E).
3.4. VISTA+ monocytes were the mainly increased monocyte subset in
MG
Nine clusters were identified as monocytes (Supplementary Table 2):
cluster6, cluster8, and cluster9 comprised approximately 94% of the
total monocyte population. Cluster8 and cluster9 were significantly
decreased and increased, respectively, in the MG group (Fig. 4A).
Cluster9 was identified as CD14+ VISTA+ monocytes using the tSNE
plot (Fig. 4B). High-throughput microarray data from the GSE database
(GSE:85452, isolated CD14+ monocytes from 13 MG patients and 12
HCs) were analyzed for VISTA mRNA expression. The results showed
that VISR/C10orf54 mRNA was significantly increased in monocytes
from MG patients (Fig. 4C). Although VISTA is thought to serve pri­
marily as an inhibitory protein that limits immune responses and is most
highly expressed in monocytes and other potential antigen-presening
cells (APCs) [15], a recent study indicated that VISTA antibodies can
activate CD14+ monocytes and promote inflammation responses [16].
R. Fan et al. Clinical Immunology 245 (2022) 109184

Fig. 3. Phenotypic alterations of NK cells in MG. A. tSNE plot of CD3-CD19- cells corresponding to MG patients and HCs. B. Heatmap showing the markers among
clusters divided by the PhenoGraph algorithm. C. tSNE plot of CD56+ NK cells corresponding to MG patients and HCs. D. tSNE plot of CD16 expression in CD56+ NK
cells. E. Differences in NK subsets between MG patients and HCs. F. Correlation between frequencies of CD56+ CD16+ or CD56+ CD16+ CD8+ NK cells and QMG.
Statistical analysis performed using unpaired t-tests (significance level: ns >0⋅05; *0⋅01 < p < 0⋅05; **0⋅001 < p < 0⋅01; *** < 0⋅001) and Pearson correlation
analysis (significant level: ns >0.05; #0.01 < p < 0.05; ##0.001 < p < 0.01; ### < 0.001). HC: healthy controls; MG: myasthenia gravis. tSNE: t-distributed
Stochastic Neighbor Embedding.

To further explore the biological features of VISTA, identifying the gene
signature and performing the functional characterization of VISTA+
CD14+ monocytes was crucial. To achieve this, we used CD14+
monocyte single-cell RNA sequencing datasets from GSE103544 (856
CD14+ monocytes RNA-seq data), VSIR+ monocytes (VSIR+ Mo), and
VSIR- monocytes (VSIR-Mo), which were clearly identified by a semi-
supervised clustering algorithm known as SCINA (Fig. 4D and E). Four
pseudobulk-based algorithms (DESeq2-Wald, DESeq2-LRT, edgeR-LRT,
and edgeR-QLF) and two classical algorithms (MAST and DESeq2) were
used to identify significantly differentially expressed genes (DEGs).
VSIR+ Mo gene signatures were identified as intersections of the results
of the six algorithms. In total, 37 genes were upregulated and identified
as gene signatures, but no common downregulated genes were observed
(Fig. 4F-G and Fig. 5A, Supplementary File 1). Next, The 37 gene sig­
natures were subjected to KEGG pathway enrichment analysis and GO
functional enrichment analysis. Antigen processing and presentation,
and related functional pathways were the most significantly enriched
pathways (Fig. 5B and C). Eight genes, including HLA gene family CD74
and PSME, were included in antigen processing and presentation
pathway (Fig. 5D). From our mass cytometry data, we found that HLA-
DR expression was indeed increased in CD14+ VISTA+ monocytes
(Fig. 5E). Transcription factor enrichment analysis was performed on the
37 gene signatures, and the results showed that IRF8 and IRF5 were the
most enriched transcription factors (TFs), and that VSIR may be regu­
lated by TFEB (Fig. S3, Supplementary File2). In addition, although the
frequencies of neutrophil subsets were not significantly different, the
tSNE plot revealed heterogeneity in neutrophils from the MG and the HC
group. We found that CD38+ neutrophils were enriched in MG patients
(Fig. 5F), and the expression of CD38 in neutrophils from these patients
was significantly increased (Fig. 5G).
4. Discussion
Our study provides an expression landscape of multiple immune
markers in peripheral immune cells using CyTOF. We observed that the
frequencies of CD56+ CD16+ and CD56+ CD16+ CD8+ NK cells

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R. Fan et al. Clinical Immunology 245 (2022) 109184

Fig. 4. Alterations in CD14+ VISTA+ monocytes and identified gene signatures via single-cell RNA sequencing. A. Frequencies of cluster8 and cluster9 were
decreased and increased in MG, respectively. B. tSNE plot showing the alteration of cluster8 and cluster9; cluster9 was identified as VISTA+ CD14+ monocytes. C.
mRNA expression of VSIR in monocytes from MG patients (n = 13) and HCs (n = 12). D. Density plot showing the expression of VSIR in VSIR+ and VSIR- Mo. E.
Violin plot showing the expression of VSIR in VSIR+ and VSIR- Mo. F. Interaction of up-regulated genes identified by six algorithms. G. Interaction of down-regulated
genes identified by six algorithms. Statistical analysis performed using unpaired t-tests (significance level: NS >0.05; *0.01 < p < 0.05; **0.001 < p < 0.01; *** <
0.001). HC: healthy controls; MG: myasthenia gravis; p_DGEseq2_LRT: pseudobulk DESeq2 LRT; p_DGEseq2_wald: pseudobulk DESeq2 Wald; p_edgeR_LRT: pseu­
dobulk edgeR LRT; p_edgeR_QLF: pseudobulk edgeR QLF; VSIRneg_Mo: VSIR-Monocytes; VSIRpos_Mo: VSIR+Monocytes.

decreased and were significantly associated with the QMG score. In the
external validation cohort, we validated the differences exhibited by
these two NK cell subsets in the MG and the CTRL groups, and showed
significant differences between GMG and OMG patients, between pa­
tients treated with and without thymectomy, between patients treated
with and without immunosuppressive therapy, and between early onset
and late-onset patients. From a clinical perspective, our results show the
potential of CD56+ CD16+ and CD56+ CD16+ CD8+ NK cells as
diagnostic cellular markers and indicators of disease severity.
NK cells are innate lymphoid cells that are generally identified as
CD56+ CD3- in humans. Human NK cells can be classified into two
subsets (CD56bright and CD56dim) based on the density of the cell
surface marker CD56. Most human NK cells (approximately 90%) ex­
press CD56 at a relatively low-density (CD56dim), and this is associated

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R. Fan et al. Clinical Immunology 245 (2022) 109184

Fig. 5. Functional characterization of VSIR+ monocytes. A. Heatmap of gene signatures in VSIR+Mo and VSIR-Mo. B. Network graph of top10 GO enriched terms. C.
Network graph of top 10 KEGG enriched terms. D. Gene signatures enriched in the antigen processing and presentation pathway. E. Mass cytometry data showing the
expression of HLA-DR in VSIR+/− monocytes. F. tSNE analysis revealed heterogeneity of neutrophils in MG patients and HCs, and distinguished clusters of CD38+
cells. G. Expression of CD38 in neutrophils from MG patients and HCs. Statistical analysis performed using unpaired t-tests (significance level: ns >0.05; *0.01 < p <
0.05; **0.001 < p < 0.01; *** < 0.001). HC: healthy controls; MG: myasthenia gravis; VSIRneg_Mo: VSIR-Monocytes; VSIRpos_Mo: VSIR+Monocytes.

with high cytotoxicity. In contrast, the other human NK cell type
(approximately 10%) expresses CD56 at a relatively high density
(CD56bright) in the peripheral blood. This cell type is less cytotoxic but
more efficient in cytokine and chemokine production compared to the
CD56dim subset [17]. Notably, recent studies have shown that classi­
fying the cytotoxicity and cytokine-producing ability of NK cells by
expression of CD56 is not a strict method; for example, cytokine-
producing CD56bright NK cells may acquire cytotoxicity equal to
CD56dim upon specific stimulation by cytokines or receptor activation,
and CD56dim NK cells may produce IFN-γ or other functional cytokines
under specific conditions [17]. Crinier et al. [18] isolated CD45+ CD3-
CD19- CD14- CD56+ cells from human blood and subjected them to
single-cell RNA sequencing. The results showed that NK cells were
divided into two clusters by an unsupervised clustering algorithm. One
was characterized by FCGR3A (encoding CD16), with gene signatures of
a CD56+ FCGR3A+ NK cell cluster enriched in cytolysis function, while
the gene signature of the other NK cell cluster was enriched in response
to cytokines and regulation of cytokine production functions. CD16
(FCGR3A) is a low-affinity FcyRIII expressed on a small portion of
CD56bright (50–70%) and in the majority of CD56dim cells (95%) [19],
and mediates antibody-dependent cellular cytotoxicity (ADCC) by
binding to IgG-coated targets [21,22]. In our study, CD56+ CD16+ NK

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R. Fan et al.
cell populations were decreased in MG patients, which may imply that
cytotoxicity and ADCC are decreased in NK cells from these patients. Liu
et al. [22] evaluated the killing activity of NK cells from MG patients and
reported the killing ability of these cells on CD4+ T cells was impaired.
Chien et al. [23] evaluated NK cell cytotoxicity in MG patients treated
with or without plasmapheresis, and with or without immunosuppres­
sants, and reported that it was decreased after plasmapheresis and in the
group treated with immunosuppressants. Our study further reveals that
the lower cytotoxicity of NK cells was secondary to the reduction in the
number of CD56+ CD16+ NK cells. Reportedly, cytokines are closely
related to the phenotype and function of NK cells, and cytokine dysre­
gulation has been observed in MG patients [25,5]. Previous research
demonstrated concentration-dependent downregulation of CD16
expression at membrane surface of NK cells induced by IL-18 [26] and
IL-18 levels were reported to be higher in MG patients than in HCs [27].
Tfh cells were significantly increased in MG patients, and IL-21 is the
characteristic cytokine produced by these cells. Kasaian et al. [28]
indicated the production of IL-21 by activated T cells may limit the NK
cell response. We suggest that the lower number of CD56+ CD16+ NK
cells in MG patients could be due to regulation of cytokines related to NK
function, and the frequency of CD56+ CD16+ NK cells may reflect
regulation ability, with a higher number of CD56+ CD16+ NK cells
indicating stronger regulation ability and relatively mild disease
severity, and vice versa. In addition, an abnormal percentage of NK cells
was also observed in other diseases, such as SLE and rheumatoid
arthritis (RA) (both the frequency of CD3- CD56+ NK cells and their
cytotoxicity were lower than in the control group) [29], type 1 diabetes
(the frequency of CD3- CD56+ NK cells was lower in recent-onset T1D
cases compared to those of long-standing T1D and to controls) [30], and
neuromyelitis optica syndrome disease (the frequency of CD3- CD56+
CD16+ NK cells was lower than in the control group) [31]. CD56+
CD16+ CD8+ NK cells were also identified in our study, and showed
trend that was similar to that of CD56 + CD16+ cells. McKinney et al.
[32] suggested that CD8+ NK cells are associated with the outcome of
relapsing/remitting multiple sclerosis as well as with the regulation of
CD4+ T cell activation and proliferation. In HIV, the frequency of highly
functional CD8+ NK cells is negatively correlated with HIV-related
disease markers [33]. Recent research on CD8+ NK cells mainly
focused on infectious diseases, and few studies have focused on auto­
immune diseases. Therefore, the role of CD8+ NK cells in autoimmune
diseases is still poorly understood and needs to be further explored.
We also found that the frequency of VISTA+CD14 + monocytes in
the MG group was markedly increased, and the mRNA level of VSIR was
also significantly increased in monocytes from MG patients, suggesting
that increased VISTA protein levels may be due to increased levels of
mRNA. VISTA is a novel immune checkpoint that is highly expressed on
myeloid cells and T cells [34] and is considered to have therapeutic
potential because of its suppressive ability on the immune system, as
immune response improved after anti-VISTA therapy or in cases of
VISTA deficiency. Han et al. [35] suggested that the expression of VISTA
is increased in SLE and discoid lupus erythematosus; VISTA knockout
mice with a BALB/c background spontaneously develop autoimmune
diseases resembling human lupus. Furthermore, the immune response
was reduced in MRL/lpr mice after treatment with monoclonal antibody
against VISTA, which suggests a therapeutic potential of VISTA in
autoimmune diseases. However, no study has explored the relationship
between MG and VISTA. Although emerging data has revealed an
important role for VISTA in the regulation of innate immune cell func­
tion, there is still controversy on whether VISTA is an activating or an
inhibitory receptor in monocytes. In myeloid cells, overexpression of
VISTA reduced MHC-II expression, and anti-VISTA (a VISTA agonist)
downregulates MHC-II genes in monocytes [15]; however, another
monoclonal antibody against VISTA upregulates MHC-II genes [16].
These contradictory results have limited the development of anti-ICP
therapies based on VISTA. To further explore the biological function
of VISTA in monocytes, gene signatures for VISTA+ CD14+ monocytes
9
Clinical Immunology 245 (2022) 109184
were identified and a functional characterization was performed using
single-cell RNA sequencing. Our results showed that 37 genes, including
the HLA gene family, were upregulated in VISTA+ monocytes, and these
gene signatures were enriched in antigen presentation/processing in GO
and KEGG. Moreover, IRF8 and IRF5 were the most enriched factors
according to the transcription factor enrichment analysis. To the best of
our knowledge, this study is the first to identify VISTA+ monocytes and
reveal their gene signatures.
Our results suggest the potential value of cellular therapies related to
VISTA, such as downregulating VISTA+ monocytes to reduce their an­
tigen processing/presentation ability in autoimmune diseases, and
adoptive VISTA+ monocyte therapy to enhance the anti-tumor ability of
the immune system. In addition, expression of ICPs (including VISTA,
LAG3, and CTLA4) was also higher in T cells from MG patients, sug­
gesting that the expression of these ICPs is increased by some unknown
mechanism to regulate immune response, but the feedback is not suffi­
cient to elicit disease remission. This potential mechanism needs to be
explored in further research.
Activation of functional populations of T and B cells is the typical
mechanism of MG pathogenesis, but several studies have indicated that
the percentage of classical T cell subsets, including Th1/2/9/17, TEM,
and TCM [5,6] are similar to those observed in HCs, while the Tfh subset
is significantly altered [14]. In our study, a marked alteration in T cell
subsets was not observed; however, CXCR5 expression was higher in MG
patients than in HCs. Upregulation of the expression of multiple ICPs in
the T cells of MG patients may imply functional dysregulation of T cells.
B cells play a fundamental role in multiple autoimmune diseases, and
B cell populations undergo a wide range of alterations (including
transformation into memory B cells and transitional B cells) before
giving rise to antibody-secreting cells (ASCs) at different stages of
autoimmune diseases. In SLE, IgD- CD27- memory B cells [35,36],
CD27- IgD- CD95+ memory B cells [38], and CD23- IgD+ CD27- acti­
vated naïve B cells [39] are increased and correlate with disease
severity. In MG, CD19- CD138- ASCs are also increased and correlate
with disease severity [40], and memory B cells (CD19 + CD27 + CD38-)
are increased in GMG and OMG compared with HCs [41]. Our results
demonstrate that switched memory B cells (CD19+ CD27+ IgD-),
including CD38+ and CD38-, are significantly increased, but this in­
crease did not correlate with disease severity. CD38 is a type II glyco­
protein widely expressed in almost all developmental stages of B cells,
and is highly expressed in early B cells [42], suggesting that it may be
involved in developmental pathways, including activation and prolif­
eration [43]. CD38 is also expressed in plasmablasts, and in short-lived
and long-lived plasma cells, implying that CD38 may be an interesting
target for targeted therapy aimed at depleting ASCs. In addition, the
percentage of HLA- DQ- CD38+ naïve B cells was higher in MG patients
and was correlated with disease severity. The expression of CD38 was
significantly increased in MG patients; however, the expression of CD38
in total T cells and B cells was not significantly different, and functional
characterization of HLA- DQ- CD38+ naïve B cells through future
research is needed. CD38 is required for migration of neutrophils to the
site of infection or inflammation [44], and CD38 expression is closely
correlated with chemotactic migration to formylmethionine leucyl-
phenylalanine in localized aggressive periodontitis patients [45].
Neutrophil numbers are significantly higher in the spleens and livers of
wild type BALB/c mice upon Listeria infection, but lower in the spleen of
CD38 knockdown mice [46]. Neutrophil migration can mediate joint
damage and persistent inflammation by migrating into synovial joints
and triggering Rheumatoid arthritis [47]. We suggest that MG neutro­
phils may preferentially migrate to the neuromuscular junction and
enhance the inflammatory response due to their high levels of expres­
sion of CD38; however, the regulatory mechanism needs to be further
explored. The primary limitation of our research is the limited sample
size due to the strict inclusion criteria adopted. To address this short­
coming, we validated our results with an external validation cohort. In
addition, this study was a cross-sectional study, which could not
R. Fan et al.
dynamically assess the immune changes, especially the crosstalk be­
tween different immune cells. Therefore, future longitudinal follow-up
studies are necessary.
5. Conclusion
In summary, we revealed the dysregulation of multiple immune cell
populations, identified several cellular biomarkers related to MG disease
severity, and highlighted large alterations in NK cell subsets. Further­
more, we discovered alterations in VISTA in MG patients and identified
the characteristics of VISTA+ monocytes.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.clim.2022.109184.
Funding
This work was supported by Future Medical Youth Innovation Team
Program of Chongqing Medical University (No. W0043), the Fifth Senior
Medical Talents Program of Chongqing for Young and Middle-aged,
Middle-aged Medical Excellence Team Program of Chongqing, and
Chongqing chief expert studio project, China.
Data statement
Data are available on request to the authors.
Contributors
The study was conceived, designed by F.X., R.F. and W.J.Q.; Z.T.L.,
W.Z., X.G., L.Q.L. contributed to sample collection and clinical assess­
ments; R.F. performed mass cytometry data analysis and bioinformatic
analysis; R.F. and W.J.Q. drafted the manuscript and all authors dis­
cussed the results and contributed to the writing of the paper. R.F and W.
J.Q. contributed equally to this work as co-first authors.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Data availability
Data will be made available on request.
Acknowledgments
We greatly appreciate the all participating patients and thank the
mass cytometry facility (PLT Tech, China) for mass cytometry experi­
ment and single-cell sample preparation.
References
[1] N.E. Gilhus, J.J. Verschuuren, Myasthenia gravis: subgroup classification and
therapeutic strategies, Lancet Neurol. 14 (2015) 1023–1036, https://doi.org/
10.1016/S1474-4422(15)00145-3.
[2] A.S. Carr, C.R. Cardwell, P.O. McCarron, J. McConville, A systematic review of
population based epidemiological studies in Myasthenia Gravis, BMC Neurol. 10
(2010) 46, https://doi.org/10.1186/1471-2377-10-46.
[3] J.M. Ponseti, E. Espín, M. Armengol, Diagnóstico y tratamiento de la miastenia
grave, Med. Clin. 115 (2000) 264–270, https://doi.org/10.1016/S0025-7753(00)
71529-6.
[4] M.I. Leite, S. Jacob, S. Viegas, J. Cossins, L. Clover, B.P. Morgan, D. Beeson,
N. Willcox, A. Vincent, IgG1 antibodies to acetylcholine receptors in ‘seronegative’
myasthenia gravis†, Brain. 131 (2008) 1940–1952, https://doi.org/10.1093/
brain/awn092.
[5] X. Huan, S. Luo, H. Zhong, X. Zheng, J. Song, L. Zhou, J. Lu, Y. Wang, Y. Xu, J. Xi,
Z. Zou, S. Chen, C. Zhao, In-depth peripheral CD4+ T profile correlates with
myasthenic crisis, Ann Clin Transl Neurol. 8 (2021) 749–762, https://doi.org/
10.1002/acn3.51312.
[6] F. Ingelfinger, S. Krishnarajah, M. Kramer, S.G. Utz, E. Galli, M. Lutz, P. Zwicky, A.
U. Akarca, N.P. Jurado, C. Ulutekin, D. Bamert, C.C. Widmer, L. Piccoli, F. Sallusto,
10
Clinical Immunology 245 (2022) 109184
N.G. Núñez, T. Marafioti, D. Schneiter, I. Opitz, A. Lanzavecchia, H.H. Jung, D. De
Feo, S. Mundt, B. Schreiner, B. Becher, Single-cell profiling of myasthenia gravis
identifies a pathogenic T cell signature, Acta Neuropathol. 141 (2021) 901–915,
https://doi.org/10.1007/s00401-021-02299-y.
[7] S. Qin, L. Xu, M. Yi, S. Yu, K. Wu, S. Luo, Novel immune checkpoint targets: moving
beyond PD-1 and CTLA-4, Mol. Cancer 18 (2019) 155, https://doi.org/10.1186/
s12943-019-1091-2.
[8] G. Wang, R. Tai, Y. Wu, S. Yang, J. Wang, X. Yu, L. Lei, Z. Shan, N. Li, The
expression and immunoregulation of immune checkpoint molecule VISTA in
autoimmune diseases and cancers, Cytokine Growth Factor Rev. 52 (2020) 1–14,
https://doi.org/10.1016/j.cytogfr.2020.02.002.
[9] Z. Wu, M. Wang, G. Liang, P. Jin, P. Wang, Y. Xu, Y. Qian, X. Jiang, J. Qian,
M. Dong, Pro-Inflammatory Signature in decidua of recurrent pregnancy loss
regardless of embryonic chromosomal abnormalities, Front. Immunol. 12 (2021),
772729, https://doi.org/10.3389/fimmu.2021.772729.
[10] R. Finck, E.F. Simonds, A. Jager, S. Krishnaswamy, K. Sachs, W. Fantl, D. Peer, G.
P. Nolan, S.C. Bendall, Normalization of mass cytometry data with bead standards,
Cytometry A. 83 (2013) 483–494, https://doi.org/10.1002/cyto.a.22271.
[11] H. Chen, M.C. Lau, M.T. Wong, E.W. Newell, M. Poidinger, J. Chen, Cytofkit: A
bioconductor package for an integrated mass cytometry data analysis pipeline,
PLoS Comput. Biol. 12 (2016), e1005112, https://doi.org/10.1371/journal.
pcbi.1005112.
[12] Z. Zhang, D. Luo, X. Zhong, J.H. Choi, Y. Ma, S. Wang, E. Mahrt, W. Guo, E.
W. Stawiski, Z. Modrusan, S. Seshagiri, P. Kapur, G.C. Hon, J. Brugarolas, T. Wang,
SCINA: a semi-supervised subtyping algorithm of single cells and bulk samples,
Genes (Basel) 10 (2019) E531, https://doi.org/10.3390/genes10070531.
[13] J.W. Squair, M. Gautier, C. Kathe, M.A. Anderson, N.D. James, T.H. Hutson,
R. Hudelle, T. Qaiser, K.J.E. Matson, Q. Barraud, A.J. Levine, G.L. Manno, M.
A. Skinnider, G. Courtine, Confronting false discoveries in single-cell differential
expression, Nat. Commun. 12 (2021), https://doi.org/10.1038/s41467-021-
25960-2.
[14] S. Ashida, H. Ochi, M. Hamatani, C. Fujii, K. Kimura, Y. Okada, Y. Hashi,
K. Kawamura, H. Ueno, R. Takahashi, T. Mizuno, T. Kondo, Immune skew of
circulating follicular helper T cells associates with myasthenia gravis severity,
Neurol Neuroimmunol Neuroinflamm. 8 (2021), e945, https://doi.org/10.1212/
NXI.0000000000000945.
[15] E. Ma, Z. Y, S. E, B. Cm, M. R, C. C, N. Rj, VISTA: A Target to Manage the Innate
Cytokine Storm, Front. Immunol. 11 (2021), https://doi.org/10.3389/
fimmu.2020.595950.
[16] B.M. Rogers, L. Smith, Z. Dezso, X. Shi, E. DiGiammarino, D. Nguyen,
S. Sethuraman, P. Zheng, D. Choi, D. Zhang, A. Nguyen, K. McGuire, W. Liu,
N. Chung, D.T. Chao, S. Ye, G.R. Starbeck-Miller, VISTA is an activating receptor in
human monocytes, J. Exp. Med. 218 (2021), https://doi.org/10.1084/
jem.20201601.
[17] B. Zitti, Y.T. Bryceson, Natural killer cells in inflammation and autoimmunity,
Cytokine Growth Factor Rev. 42 (2018) 37–46, https://doi.org/10.1016/j.
cytogfr.2018.08.001.
[18] C. A, M. P, E. B, P. C, G. J, B. A, S. L, C.-M. I, E. M, G.-M. M, J. S, B. E, H. S, H. J, U.
S, V. F, N.-M. E, V. E, High-dimensional single-cell analysis identifies organ-specific
signatures and conserved NK Cell subsets in humans and mice, Immunity 49
(2018), https://doi.org/10.1016/j.immuni.2018.09.009.
[19] M.A. Cooper, T.A. Fehniger, M.A. Caligiuri, The biology of human natural killer-
cell subsets, Trends Immunol. 22 (2001) 633–640, https://doi.org/10.1016/s1471-
4906(01)02060-9.
[21] K. Kitaya, Accumulation of uterine CD16(− ) natural killer (NK) cells: friends, foes,
or Jekyll-and-Hyde relationship for the conceptus? Immunol. Investig. 37 (2008)
467–481, https://doi.org/10.1080/08820130802191292.
[22] R.-T. Liu, W. Li, D. Guo, C.-L. Yang, J. Ding, J.-X. Xu, R.-S. Duan, Natural killer cells
promote the differentiation of follicular helper T cells instead of inducing apoptosis
in myasthenia gravis, Int. Immunopharmacol. 98 (2021), 107880, https://doi.org/
10.1016/j.intimp.2021.107880.
[23] P.J. Chien, J.H. Yeh, H.C. Chiu, Y.M. Hsueh, C.T. Chen, M.C. Chen, C.M. Shih,
Inhibition of peripheral blood natural killer cell cytotoxicity in patients with
myasthenia gravis treated with plasmapheresis, Eur. J. Neurol. 18 (2011)
1350–1357, https://doi.org/10.1111/j.1468-1331.2011.03424.x.
[25] X. Huan, S. Luo, H. Zhong, X. Zheng, J. Song, L. Zhou, J. Lu, Y. Wang, Y. Xu, J. Xi,
Z. Zou, S. Chen, C. Zhao, In-depth peripheral CD4+ T profile correlates with
myasthenic crisis, Ann Clin Transl Neurol. 8 (2021) 749–762, https://doi.org/
10.1002/acn3.51312.
[26] C.M. Nielsen, A.-S. Wolf, M.R. Goodier, E.M. Riley, Synergy between Common γ
Chain Family Cytokines and IL-18 Potentiates Innate and Adaptive Pathways of NK
Cell Activation, Front. Immunol. 7 (2016) 101, https://doi.org/10.3389/
fimmu.2016.00101.
[27] S. Jander, G. Stoll, Increased serum levels of the interferon-gamma-inducing
cytokine interleukin-18 in myasthenia gravis, Neurology. 59 (2002) 287–289,
https://doi.org/10.1212/wnl.59.2.287.
[28] M.T. Kasaian, M.J. Whitters, L.L. Carter, L.D. Lowe, J.M. Jussif, B. Deng, K.
A. Johnson, J.S. Witek, M. Senices, R.F. Konz, A.L. Wurster, D.D. Donaldson,
M. Collins, D.A. Young, M.J. Grusby, IL-21 limits NK cell responses and promotes
antigen-specific T cell activation: a mediator of the transition from innate to
adaptive immunity, Immunity. 16 (2002) 559–569, https://doi.org/10.1016/
s1074-7613(02)00295-9.
[29] Y.-W. Park, S.-J. Kee, Y.-N. Cho, E.-H. Lee, H.-Y. Lee, E.-M. Kim, M.-H. Shin, J.-
J. Park, T.-J. Kim, S.-S. Lee, D.-H. Yoo, H.-S. Kang, Impaired differentiation and
cytotoxicity of natural killer cells in systemic lupus erythematosus, Arthritis
Rheum. 60 (2009) 1753–1763, https://doi.org/10.1002/art.24556.
R. Fan et al.
[30] M. Rodacki, B. Svoren, V. Butty, W. Besse, L. Laffel, C. Benoist, D. Mathis, Altered
natural killer cells in type 1 diabetic patients, Diabetes. 56 (2007) 177–185,
https://doi.org/10.2337/db06-0493.
[31] L. Khani, M.H. Jazayeri, R. Nedaeinia, M. Bozorgmehr, S.M. Nabavi, G.A. Ferns,
The frequencies of peripheral blood CD5+CD19+ B cells, CD3-CD16+CD56+ NK,
and CD3+CD56+ NKT cells and serum interleukin-10 in patients with multiple
sclerosis and neuromyelitis optica spectrum disorder, Allergy, Asthma Clin.
Immunol. 18 (2022) 5, https://doi.org/10.1186/s13223-021-00596-5.
[32] M. Ef, C. I, H. Km, S. De, C. C, M. G, C. Ej, Z. Ss, S. Kgc, A CD8 + NK cell
transcriptomic signature associated with clinical outcome in relapsing remitting
multiple sclerosis, Nature, Communications. 12 (2021), https://doi.org/10.1038/
s41467-020-20594-2.
[33] F. Ahmad, H.S. Hong, M. Jäckel, A. Jablonka, I.-N. Lu, N. Bhatnagar, J.
M. Eberhard, B.A. Bollmann, M. Ballmaier, M. Zielinska-Skowronek, R.E. Schmidt,
D. Meyer-Olson, High frequencies of polyfunctional CD8+ NK cells in chronic HIV-
1 infection are associated with slower disease progression, J. Virol. 88 (2014)
12397–12408, https://doi.org/10.1128/JVI.01420-14.
[34] I. Le Mercier, W. Chen, J.L. Lines, M. Day, J. Li, P. Sergent, R.J. Noelle, L. Wang,
VISTA regulates the development of protective antitumor immunity, Cancer Res.
74 (2014) 1933–1944, https://doi.org/10.1158/0008-5472.CAN-13-1506.
[35] X. Han, M.D. Vesely, W. Yang, M.F. Sanmamed, T. Badri, J. Alawa, F. López-
Giráldez, P. Gaule, S.W. Lee, J.-P. Zhang, X. Nie, A. Nassar, A. Boto, D.B. Flies,
L. Zheng, T.K. Kim, G.W. Moeckel, J.M. McNiff, L. Chen, PD-1H (VISTA)-mediated
suppression of autoimmunity in systemic and cutaneous lupus erythematosus, Sci.
Transl. Med. 11 (2019) eaax1159, https://doi.org/10.1126/scitranslmed.aax1159.
[36] C. Wei, J. Anolik, A. Cappione, B. Zheng, A. Pugh-Bernard, J. Brooks, E.-H. Lee, E.
C.B. Milner, I. Sanz, 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. 178 (2007) 6624–6633, https://doi.org/
10.4049/jimmunol.178.10.6624.
[38] A.M. Jacobi, K. Reiter, M. Mackay, C. Aranow, F. Hiepe, A. Radbruch, A. Hansen,
G.-R. Burmester, B. Diamond, P.E. Lipsky, T. Dörner, Activated memory B cell
subsets correlate with disease activity in systemic lupus erythematosus: delineation
11
Clinical Immunology 245 (2022) 109184
by expression of CD27, IgD, and CD95, Arthritis Rheum. 58 (2008) 1762–1773,
https://doi.org/10.1002/art.23498.
[39] C.M. Tipton, C.F. Fucile, J. Darce, A. Chida, T. Ichikawa, I. Gregoretti, S. Schieferl,
J. Hom, S. Jenks, R.J. Feldman, R. Mehr, C. Wei, F.E.-H. Lee, W.C. Cheung, A.
F. Rosenberg, I. Sanz, Diversity, cellular origin and autoreactivity of antibody-
secreting cell population expansions in acute systemic lupus erythematosus, Nat.
Immunol. 16 (2015) 755–765, https://doi.org/10.1038/ni.3175.
[40] C.-J. Zhang, Y. Gong, W. Zhu, Y. Qi, C.-S. Yang, Y. Fu, G. Chang, Y. Li, S. Shi,
K. Wood, S. Ladha, F.-D. Shi, Q. Liu, Y. Yan, Augmentation of Circulating Follicular
Helper T Cells and Their Impact on Autoreactive B Cells in Myasthenia Gravis,
J. Immunol. 197 (2016) 2610–2617, https://doi.org/10.4049/jimmunol.1500725.
[41] Y. Hu, J. Wang, J. Rao, X. Xu, Y. Cheng, L. Yan, Y. Wu, N. Wu, X. Wu, Comparison
of peripheral blood B cell subset ratios and B cell-related cytokine levels between
ocular and generalized myasthenia gravis, Int. Immunopharmacol. 80 (2020),
106130, https://doi.org/10.1016/j.intimp.2019.106130.
[42] R.-R. H, M.-G. Mt, P. R, L.-S. R, S.-A. L, CD38 expression in early B-cell precursors
contributes to extracellular signal-regulated kinase-mediated apoptosis,
Immunology. 144 (2015), https://doi.org/10.1111/imm.12370.
[43] M.-O. N, M.-G. Me, F. K, S.-A. L, CD38 cross-linking enhances TLR-induced B cell
proliferation but decreases IgM plasma cell differentiation, Eur. J. Immunol. 37
(2007), https://doi.org/10.1002/eji.200636453.
[44] F.E. Lund, D.A. Cockayne, T.D. Randall, N. Solvason, F. Schuber, M.C. Howard,
CD38: a new paradigm in lymphocyte activation and signal transduction, Immunol.
Rev. 161 (1998) 79–93, https://doi.org/10.1111/j.1600-065x.1998.tb01573.x.
[45] T. Fujita, A. Kantarci, M.L. Warbington, K.H. Zawawi, H. Hasturk, H. Kurihara, T.
E. Van Dyke, CD38 expression in neutrophils from patients with localized
aggressive periodontitis, J. Periodontol. 76 (2005) 1960–1965, https://doi.org/
10.1902/jop.2005.76.11.1960.
[46] T. Lischke, K. Heesch, V. Schumacher, M. Schneider, F. Haag, F. Koch-Nolte, H.-
W. Mittrücker, CD38 controls the innate immune response against Listeria
monocytogenes, Infect. Immun. 81 (2013) 4091–4099, https://doi.org/10.1128/
IAI.00340-13.
[47] W. Hl, M. Rj, E. Sw, The multifactorial role of neutrophils in rheumatoid arthritis,
Nat. Rev. Rheumatol. 10 (2014), https://doi.org/10.1038/nrrheum.2014.80.