Clinical and Experimental Immunology, 2022, 210, 201–216

https://doi.org/10.1093/cei/uxac104
Advance access publication 7 January 2023
Review

Review
Human B-cell subset identification and changes in
inflammatory diseases
Rebekah L. Velounias1 and Thomas J. Tull2,*,
1
Department of Immunobiology, King’s College London, Guy’s Hospital Campus, London, UK

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2
St John’s Institute of Dermatology, King’s College London, Guy’s Hospital Campus, London, UK
Correspondence: Thomas Tull, St John’s Institute of Dermatology, King’s college London, London, UK. Email: thomas.tull@kcl.ac.uk
*

Summary
Our understanding of the B-cell subsets found in human blood and their functional significance has advanced greatly in the past decade. This has
been aided by the evolution of high dimensional phenotypic tools such as mass cytometry and single-cell RNA sequencing which have revealed
heterogeneity in populations that were previously considered homogenous. Despite this, there is still uncertainty and variation between studies
as to how B-cell subsets are identified and named. This review will focus on the most commonly encountered subsets of B cells in human blood
and will describe gating strategies for their identification by flow and mass cytometry. Important changes to population frequencies and function
in common inflammatory and autoimmune diseases will also be described.

Keywords: B cell, systemic lupus erythematosus, autoimmunity, immune checkpoints

Abbreviations: ABC: age associated B cell; aNAV: activated naïve; ASC: antibody secreting cell; BAFF: B cell activating factor; BCR: B cell receptor; Breg: B
regulatory cell; CSM: class switched memory; DN: double negative; IgV: immunoglobulin variable; HCD: healthy control donor; GALT: gut associated lymphoid
tissue; IFN: interferon; MFI: median fluorescence intensity; MS: multiple sclerosis; MTG: mitotracker; MZB: marginal zone B cell; MZP: marginal zone precursor;
RA: rheumatoid arthritis; RH: rhodamine; SLE: systemic lupus erythematosus; SPADE: spanning tree progression of density normalized events; SS: Sjogren’s
syndrome; SSc: systemic sclerosis; TLR: toll like receptor; TNF: tumour necrosis factor.

Introduction cytokines as well as antigen presentation [13–20]. Their

A unified and consistent approach to the identification and
description of B-cell subsets is essential to ensure reproduci-
bility of scientific studies and that analogous conclusions can
be drawn from them. Although many recent advances have
been made in B-cell biology, significant areas of uncertainty
remain regarding the nature of certain B-cell subsets and their
role in homeostasis and inflammatory disease. This is likely to
have arisen due to a number of reasons. Firstly, whilst murine
models are a valuable tool to understand B-cell biology, there
are significant differences between murine and human B-cell
subsets which mean that it can be difficult to draw analogous
conclusions from them [1–4]. Secondly, the evolution of high
dimensional phenotypic techniques has identified many novel
subsets but further work is needed to expand on their func-
tional and pathogenic roles [5–8]. Thirdly, B cells in blood
often represent a developmental continuum and overlap
phenotypically without sharply demarcated gates to differen-
tiate them [5, 9–11]. And finally, there remain significant gaps
in our understanding of human B-cell maturation and there-
fore the developmental trajectories that link human B-cell
subsets to one another [12].
B cells are known to play important roles in a variety of
inflammatory diseases via the production of antibodies and
pathogenic role is supported by the response of a variety of
inflammatory conditions to B-cell directed therapies such
as rituximab and B-cell activating factor (BAFF) antagon-
ists [21–25]. Immune mediated inflammatory diseases such
as systemic lupus erythematosus (SLE), Sjogren’s syndrome
(SS), rheumatoid arthritis (RA), and COVID-19 are associ-
ated with significant changes in B-cell subset frequency which
is likely driven by inflammatory cytokines and pro-survival
factors as well as changes in B-cell differentiation and homing
[26–28].
In this review we will describe the main subsets of B cells in
peripheral blood and how these can be identified using flow
and mass cytometry, as well as important changes in B-cell
subsets in inflammatory immune mediated conditions. B1
cells will not be discussed in this review due to uncertainties
regarding their status and because they represent a rare subset
in adult human blood [29–31].
Identification of B cells in peripheral blood
The surface expression of both CD19 and CD20 are induced
early in B-cell development in the bone marrow [11, 32].
They are both expressed on B-cell subsets in blood but CD20

Received 4 August 2022; Revised 19 October 2022; Accepted for publication 15 November 2022
© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please
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202
expression is lost during terminal B-cell differentiation into
antibody secreting cells (ASCs) [33, 34]. CD19 therefore al-
lows identification of both B cells and ASCs. A gating strategy
using CD19 for the identification of both B cells and ASCs
in blood by flow cytometry is illustrated in Fig. 1A. An anti-
body panel of 9 ‘key’ markers can be used to identify the main
B-cell subsets in blood and these are summarized in Table 1.
Additional markers are also given in this table which can aid
gating and the identification of further subsets.
Mass cytometry utilizes the ionic signal from antibodies
tagged to rare earth metals and therefore is not limited by the
spectral overlap from fluorochrome-conjugated antibodies
that are used for flow cytometry [80]. Mass cytometry there-
fore allows single cells to be labelled with ~40 metal tagged
antibodies that can be directed to cell surface or intracel-
lular targets. A gating strategy to identify live single B cells
by mass cytometry is displayed in Fig. 1B. Further identi-
fication of B-cell subsets can then be performed by biaxial
gating or dimensional reduction and clustering methods.
Biaxial gating of mass cytometry data can be challenging as
the expression of B-cell subset markers often lack distinct
pinch point gates and metal minus one control samples that
guide the accurate placement of gates are impractical due to
cost. Dimensionality reduction and clustering techniques that
identify subsets based on the expression of multiple markers
can therefore be advantageous over biaxial gating. ViSNE, a
visualization tool for high-dimensional single-cell data based
on the t-Distributed Stochastic Neighbour Embedding algo-
rithm and uniform manifold approximation and projection
(UMAP) are common dimensionality reduction tools used to
visualize mass cytometry data [81, 82]. These algorithms can
be run using all panel markers or select B-cell subset markers
and allow the two-dimensional projection of B cells arranged
in multidimensional space. Representative viSNE plots of
CD19+ B cells from a healthy control donor (HCD) gener-
ated by running the algorithm on lineage markers are dis-
played in Fig. 1C. In these viSNE plots, memory B cells appear
as distinct islands but significant continuity exists between
CD10+CD24hiCD38hi transitional and CD10−CD24intCD38int
naïve B-cell subsets and manually gating such plots can
therefore be challenging (Fig. 1C). For this reason, span-
ning tree progression of density normalized events (SPADE)
run on ViSNE coordinates can be a useful tool to separate
subpopulations existing in phenotypic continuity (Fig. 1D)
[9, 10]. With SPADE, B-cell populations that are phenotyp-
ically similar to one another are displayed as nodes which
are interlinked by branches to form trees. Nodes representing
B-cell subsets can then be manually grouped together to form
bubbles representing that population (Fig. 1D). Whilst this
approach is in part directed it has the advantage over biaxial
gating in that multiple markers can be used simultaneously to
define B-cell subsets.
Transitional B cells
Transitional B cells are defined as recent emigrant B cells from
the bone marrow and represent the most immature B-cell
subset in peripheral blood [12]. Studies of B-cell repopulation
following bone marrow stem cell transplantation and B-cell
depletion with rituximab have identified sequential matur-
ation of transitional B cells through T1, T2, and T3 phases
[35, 36, 45, 83, 84]. This maturational sequence is associated
with the acquisition and loss of various markers. Notably,
Velounias and Tull
CD24, CD38, and CD10 are downregulated and these are
commonly utilized markers to distinguish transitional B-cell
subsets. T1 and T2 B cells share expression of CD10 with im-
mature B cells in the bone marrow, but this is not expressed
by T3 cells [37, 45]. T1 and T2 cells are often distinguished
from one another based on high and intermediate expression
of CD24 and CD38 (Fig. 2A). This approach is subjective as
the definition of high and intermediate expression can differ
between individuals and experiments given technical factors
such as fluorescent signal intensity. T1 cells are known to have
low expression of CD21 and the use of this marker in con-
junction with CD24 and CD38 can be useful to guide the T1
gate (Fig. 2A) [35].
The use of additional markers can also help to accurately
identify T1, T2, and T3 subsets and are represented in Fig. 2B
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and C as SPADE trees. T1 cells have relatively high expression
of β1 integrin, which is likely to be important for interaction
with bone marrow stromal cells [85]. T1 cells also lack ex-
pression of CD62L and CCR7 (Fig. 2C), which is likely to
result in a lack of homing capability as seen in studies of both
murine and human transitional B cells [9, 86, 87]. IgM was
traditionally thought to be downregulated through T1–T3 de-
velopment but T2 and T3 populations are both heterogeneous
in their expression of this marker (Fig. 2D). IgMhi T2 cells
have high expression of β7 integrin and are enriched in gut
associated lymphoid tissue (Fig. 2E) [9]. Furthermore, IgMhi
T2 cells align with cells undergoing marginal zone B (MZB)
cell development in terms of surface and transcriptomic fea-
tures [9]. This suggests that a gut homing T2 MZP population
exists in humans as has been demonstrated in mice [88–90].
T3 cells are a late transitional B-cell subset and are thought
to represent an intermediate stage of development between
T2 and naïve B cells. In keeping with this, T3 cells appear in
the blood after T2 cells during B-cell repopulation post B-cell
ablation therapy and differentiate into naïve B cells in vitro
[44, 45]. Transitional B cells can be distinguished from naïve
B cells as they lack expression of the ABCB1 cotransporter,
which as a result prevents the extrusion of dyes such as rhoda-
mine (RH) or mitotracker (MTG) [43]. T3 cells can therefore
be gated as CD27−IgD+CD10−RHhi/MTGhi (Fig. 3A and B).
The distinction between RH low and high populations can
be guided by the signal obtained from CD27+ memory B cells
which also lack to ABCB1 cotransporter and therefore are
RHhi (Fig. 3C). The expression of CD45RB in T3 cell popu-
lations is heterogeneous (Fig. 3D) and CD45RBhi cells also
have higher expression of IgM and CD1c and align with cells
undergoing MZB cell differentiation (Fig. 3E) [9, 46]. The
use of dye extrusion is essential for the distinction of T3 cells
from naïve B cells as they otherwise share an overlapping sur-
face phenotype (Fig. 3F).
Multiple abnormalities in transitional B-cell subsets have
been reported in inflammatory diseases. Transitional B cells
harbour a high proportion of autoreactive cells which are re-
moved through sequential maturation into naïve B cells in
healthy individuals and this checkpoint is defective in SLE
[91]. The mechanisms that underlie this checkpoint failure
are not known but are may be secondary to aberrant tran-
sitional B-cell maturation or migration into lymphoid tissue.
Transitional B cells from patients with SLE have been reported
to have lower expression of the gut homing receptor β7 in-
tegrin suggestive of defective gut homing capacity and β7hi
T2 cells are depleted in lupus nephritis [9, 39]. Transitional
B cells have been shown to be increased in frequency in SLE
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Figure 1: identification of B cells in peripheral blood. (A) Flow cytometry plots showing a gating strategy used to identify CD19+ B cells and
CD19+CD27hiCD38hi ASCs. Data are from PBMCs from a representative healthy control donor (HCD). (B) A gating strategy of mass cytometry data
to identify live CD19+ B cells. Antibodies to CD3 and CD14 were conjugated to the same metal and therefore created a ‘dump’ channel which allows
exclusion of T cells and cells of myeloid origin. Data are from a female representative healthy control donor. (C) ViSNE plots generated using the
following markers; CD10, CD24, CD38, CD27, CD45RB IgD, IgM, IgA, and IgG. The plots are from a female HCD and depict expression of lineage
markers used to identify B-cell subsets. (D) SPADE on viSNE plots demonstrating expression of B-cell lineage markers used to classify CD19+ B cells
into transitional (TS), naïve, activated naive (aNAV), marginal zone B cell (MZB), and their precursors (MZP) and IgG+ and IgA+ memory subsets. Data
adapted from Tull TJ et al [9]
204 Velounias and Tull

Table 1: Lineage markers to define B-cell subsets with expected frequencies in healthy peripheral blood and studies reporting altered frequency or
function in autoimmune rheumatic diseases and COVID-19

Subset Key markers Additional markers and subsets Frequency Disease associations
(% CD19+)

T1 CD27−IgD+IgM+CD10+ β1hiβ7loCCR7loCD62Llo [9, 37] 1−4 [37, 38] SLE [38, 39]
CD21loCD24+++CD38+++ [35–37] SSc [40]
COVID [41, 42]
T2 CD27−IgD+IgM+CD10+ β1hi/intCD62Lhi/int 2−10 [28, 38] SLE [9, 28, 38, 39]
CD21hiCD24++CD38++ [35–37] IgMhiT2: IgMhiCCR7hiβ7hi [9] SSc [40]
IgMloT2: IgMloCCR7loβ7lo [9] COVID [41, 42]
T3 CD27−IgD+IgM+CD10−CD24+ IgMhiT3: IgMhiCD1chiβ7hiCD45RBint/hi [9, 46] 9−13 [9, 45] SLE [9, 44]
CD38+RHhi/MTGhi [43–45] IgMloT3: IgMloCD1cloβ7loCD45RBlo/int [9]
Naive CD27−IgD+IgM+CD10−CD24+ 40−60 [9, 45] SLE [47, 48]

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CD38+RHlo/MTGlo [43, 44] SSc [49]
Naïve MZP CD27−IgD+IgMhiCD10 CD1chi [9, 46] 0.5−3 [9, 46] SLE [9]
−
CD24+CD38+RHlo/
MTGloCD45RBhi [46, 50]
Activated CD27−IgD+CD10−CD21lo FRCL5+, CXCR5+, T-bet+ [26] 0.1−3 [9] SLE [26, 27, 51]
naive CD24loCD38lo [26, 27] COVID [42]
MZB CD27+IgD+IgM+ [10] CD1chi [52] 2−30 [53, 54] SLE [54, 55]
MZB1: β7hiCCR7hi [6] SS [56, 57]
MZB2: β7loCCR7lo [6] SSc [58]
RA [59]
COVID [42, 60]
IgM only CD27+IgD−IgM+ [61, 62] CD45RB+ [5] 10 [63]
CSM CD27+IgD−IgM− [5, 61] CD45RB+, IgG, IgA or IgE+ [64] 5−40 [26, 53] SLE [54, 65]
SS [66]
RA [67, 68]
ANCA vasc [69]
DN CD27−IgD− [70, 71] DN1: CD21int/hi, CXCR5+ [26, 41] 1−10 [26, 71] SLE [26, 27, 74]
DN2: CD21lo, FRCL5+, CXCR5−, T-bet+, RA [75–77]
CD11chi [26, 41] SSc [49]
DN3: CD21−CD11c−CXCR5− [72] COVID [41, 42, 78, 79]
DN4: CD21+CD11c+ [73]
ANCA vasc = ANCA-associated vasculitis; B1 = B1 integrin; B7 = B7 integrin; CSM = class switched memory; DN = double negative; MZP = marginal zone
precursor; SLE = systemic lupus erythematosus; SS = Sjorgen’s syndrome; SSc = Systemic sclerosis;.

and this has been correlated with serum BAFF levels [28,
38, 92]. The important role of BAFF as a survival factor is
also supported by the increased frequency of transitional B
cells in patients with multiple sclerosis that are treated with
fingolimod and interferon-β which are both known to in-
crease serum BAFF levels [93]. Transitional B cells in SLE
have also been shown to be hyperresponsive to B-cell receptor
(BCR) signalling which is likely secondary to type I interferon
[94, 95]. Increased frequencies of transitional B cells have also
been reported in a number of other autoimmune diseases such
as juvenile dermatomyositis, primary SS (pSS), and systemic
sclerosis (SSc) [40, 96, 97]. Altered T1:T2 cell ratios have also
been identified in patients with renal transplant rejection and
are postulated to be an important predictor of graft loss [98,
99]. Finally, a reduction in transitional B cells has been seen in
COVID-19 infection which correlates with infection severity
[41, 42].
Transitional B cells are enriched in B regulatory cells
(Bregs) that are best described as B cells that can suppress
T-cell responses mainly via the production of IL-10 [100,
101]. Breg dysfunction has been identified in a number of
autoimmune diseases. CD24hiCD38hi B cells in SLE have less
capacity to inhibit CD4 T-cell cytokine production [100].
Induction of Bregs by plasmacytoid dendritic cells has also
been reported to be defective in SLE which is thought to be
mediated by type I interferon [102]. Breg dysfunction in SLE
may also be mediated by defective B cell homing or matur-
ation in GALT, which is known to be important for Breg
induction [9, 39, 103]. Reduced numbers of CD24hiCD38hi
B cells have been identified in patients with RA and they
failed to repress Th17 responses but maintained their ability
to suppress Th1 responses [104]. Furthermore, treatment of
RA with tumour necrosis factor alpha (TNF-α) antagonists
has been demonstrated to increase the number of CD27+
Bregs [105]. Primary SS has been associated with increased
frequency of CD24hiCD38hi B cells but these cells fail to sup-
press T cell TNF-α or IFN-γ secretion [97]. Furthermore,
adoptive transfer of CD24hiCD38hi B cells from patients with
pSS into mice with experimental SS demonstrated failure of
suppression of T follicular cell expansion [106]. Reduced
Breg frequency or defective Breg function has also been re-
ported in MS, SSc, pemphigus, and myasthenia gravis [107–
111]. Breg dysfunction has therefore been identified in a
number of autoimmune diseases but further work is required
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Figure 2: transitional B-cell subsets and phenotype. (A) Flow cytometry plots showing a gating strategy used for identification of T1 cells (CD27−IgD+
CD10+CD24+++CD38+++) and T2 cells (CD27−IgD+CD10+CD24++CD38++). Data are from PBMCs from a representative HCD. (B) Schema highlighting the
transitional B-cell population from the SPADE on viSNE plot in Fig. 1D. (C) SPADE on viSNE plots of TS B cells exported from TS SPADE bubble from
Fig 1D demonstrating marker expression and classification of transitional B-cell populations. T1 cells were defined as CD10+CD24+++CD38+++CD21lo, T2
as CD10+CD24++CD38++CD21hi, and T3 as CD10 − CD24+CD38+CD21hi. Data are from a representative female HCD. (D) SPADE on viSNE plot displaying
IgM expression. IgMhi T2 and T3 populations are shown by dotted red and light blue lines, respectively. IgMlo T2 and T2 populations are shown by dotted
green and purple lines, respectively. (E) Flow cytometry plot showing gating strategy of T2 B cells into T2 IgMhi and T2 IgMlo subpopulations and β7
integrin median fluorescence intensity (MFI) in these populations. Data are from six HCD
206 Velounias and Tull

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Figure 3: T3 and naïve B-cell subsets. (A) Flow cytometry plots showing a gating strategy used to identify CD27−IgD+CD10− B cells. (B) Flow cytometry
plots demonstrating gating strategies to identify naïve (CD27−IgD+CD10−RH123lo) and T3 (CD27−IgD+CD10−RH123hi) populations. (C) Flow cytometry plot
demonstrating the placement of the RH123hi gate from the signal from CD27+ memory B cells. Data are from a representative HCD. (D) Flow cytometry
plot depicting a gating strategy used to identify variants of T3 cells based on CD45RB. (E) Comparative histograms taken from flow cytometry data
displaying IgM and CD1c MFI in T3 CD45RBlo T3 CD45RBhi populations. (F) Comparative histograms taken from flow cytometry data displaying MFI of
CD24, CD21, CD38, CD5, IgM, and IgD between T3 and naïve B cells. (G) Comparative histograms taken from flow cytometry data displaying MFI of
IgM and CD1c between MZP and naive B cells. (H) Gating strategy of flow cytometry data to identify T3 and aNAV cells based on their expression of
CD21, CD24, and CD38

to identify the pathogenic significance and the mechanisms
that underlie this.
Naïve B cells
Naïve B cells represent B cells that have not encountered
antigen and have not undergone germinal centre matur-
ation. They therefore lack CD27 expression and have a low
level of somatic mutations within their immunoglobulin
variable (IgV) genes [63]. Naïve B cells represent 40–60%
of peripheral blood B cells and as discussed above can only
be distinguished from T3 cells by their ability to extrude
RH or MTG dyes due to their expression of the ABCB1
cotransporter [43]. Naïve B cells can therefore be gated as
CD27−IgD+CD10−RHhiCD45RBlo (Fig. 3A). Distinction by
dye extrusion is not possible using mass cytometry although
a metal tagged antibody to ABCB1 would theoretically allow
the distinction between T3 and naïve B cell, to our knowledge
this is not currently commercially available.
Naïve B cells are a heterogeneous population and a number
of naïve subsets have been identified including marginal zone
precursor cells (MZP), activated naïve (aNAV), and an IgMlo
anergic subset [26, 46, 47]. Naïve MZP cells are characterized
by high expression of CD45RB and also highly express IgM
and CD1c have been shown to undergo differentiation into
MZB like B cells following NOTCH ligation (Fig. 3G) [46].
A T3 population with a similar surface phenotype has also
been identified and is thought to be in developmental con-
tinuity with naïve MZP cells [9, 30]. Naïve B cells with high-
surface expression of CD45RB has been described as an early
memory population and found to have more somatic muta-
tions than CD45RB− naïve B cells [5]. The same study com-
mented on an anergic profile of naïve B cells as evidenced by
lower expression of intracellular transport proteins suggesting
Clinical and Experimental Immunology, 2022, Vol. 210, No. 3
that this subset would be less responsive to stimulation. Other
functional studies have, however, demonstrated robust prolif-
erative responses of naïve B cells to IgM crosslinking as well
as CD40 and TLR9 agonists [37, 44, 45, 112].
Naïve B cells with low IgM expression have been reported
as having anergic properties as evidence by lower intracel-
lular calcium influxes and expression of activation markers
following IgM crosslinking [47]. This subset was also found
to express higher levels of the inhibitory receptor CD22. The
identity of anergic B cells as IgMloIgD+ is supported by a
number of murine studies using the hen egg lysozyme model
in which the majority of B cells recognize this epitope. When
HEL is expression was induced endogenously the majority of
B cells adopted this phenotype, suggesting anergy can be in-
duced, and is a key tolerance mechanism [113–115].
ANAV are a subset of CD27−IgD+ naïve cells with low ex-
pression of CD21, CD23, CD24, and CD38 but high expres-
sion of FCRL5, CD11c, and T-bet [26, 27] (Fig. 3H). This
subset also does not express the ABCB1 cotransporter and is
therefore RH or MTG high following dye extrusion assays
[116]. ANAV were first identified in SLE as being clonally re-
lated to ASC populations and are hyperresponsive to TLR7
stimulation [26, 27]. The aNAV subset is therefore thought to
undergo extrafollicular maturation into ASC in SLE and are
primed by interferon and TLR7 agonists.
Inflammatory disorders in which extrafollicular B-cell
maturation is thought to occur include severe SLE and
COVID-19 and are associated with an increase in aNAV fre-
quency alongside increased frequencies of ASCs [26, 41, 51].
Conversely, MZP and naïve B cells are reduced in severe SLE
[9]. Furthermore, inhibition of BAFF with belimumab results
in a lower frequency of naïve B cells, suggesting the import-
ance of BAFF for their survival [117, 118]. Of note, numerous
studies of B-cell subset abnormalities in inflammatory disease
have gated naïve B cells as solely CD27− or CD27−IgD+ and
therefore included double negative (DN) and transitional B
cells, respectively in these gates [119–122].
MZB cells
The splenic marginal zone refers to a microanatomical area
on the periphery of B-cell follicles in the white pulp [123].
MZB cells are termed as such as they share similar surface
phenotype to the subset of cells found in the splenic marginal
zone that express low levels of IgD and high levels of CD1c
[52, 124]. In mice, MZB cells are confined to the spleen and
do not have IgV gene mutations and therefore arise independ-
ently of germinal centre responses. In humans, MZB cells
circulate in blood and lymphoid tissue and have IgV gene mu-
tations, although to a lower degree when compared to class-
switched memory (CSM) B cells [10, 52, 63, 125]. The finding
that MZB have IgV mutations in patients with CD40L defi-
ciency who lack germinal centres suggest they may mature in-
dependent of germinal centre responses as in mice [64, 125].
This is supported by studies that demonstrate little overlap
between MZB and CSM B-cell clones, suggesting that MZB
represent a distinct entity rather than just a precursor to CSM
[10, 61]. Germinal centre independent maturation of MZB is,
however, challenged by the finding that 20% of blood MZB
carry BCL6 gene mutations and that human MZB have been
reported to share clones with GC delivered IgG memory B
cells [126–128]. Similarly, whilst a specific MZB maturational
pathway exists in mice, the developmental origins of human
MZB remain incompletely understood. As described above,
207
human transitional and naïve MZP have an IgMhi gut homing
phenotype suggestive of a role of GALT in their induction [9].
Furthermore, MZB are observed to occupy a peri-germinal
centre niche and to diversify their BCR within GALT [10].
Functionally, MZB undergo T cell independent responses and
are important for immunity against encapsulated bacteria
[52, 129].
Uncertainty regarding the developmental origin and func-
tional status of MZB cells is reflected by the plethora of
names assigned to this subset in the literature. These include
unswitched memory [56, 130, 131], pre-switched memory
[58, 119, 132], non-switched [133, 134], natural effector
[64, 135], and IgM memory [136, 137] B cells. MZB have a
CD27+IgD+IgM+ surface phenotype and have high expression
of CD1c, CD21, and CD45RB (Fig. 4A) [63]. MZB have also
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been reported to have low levels of CD23 and high expres-
sion of the inhibitory receptor CD32b [138, 139]. Recently,
two distinct populations of MZB have been described that
differ in cell surface properties, transcriptional factors, and
distribution in lymphoid tissues [6]. These have been termed
MZB1 and MZB2, MZB1 having higher expression of β7 in-
tegrin, CCR7, CD24, and CD27 than MZB2 (Fig. 4B and C).
MZB1 and MZB2 were not found to be clonally related and
NOTCH induced genes were only upregulated in MZB2 sug-
gesting independent maturational pathways.
Significant changes in MZB frequency have been reported
in association with inflammatory diseases. As seen with MZP
subsets, severe depletion of MZB is observed in severe SLE
and this predominantly affects the MZB1 subset (Fig. 4D)
[6, 9]. As well as SLE, reduced frequencies of MZB have also
been reported in SS, SSc, and COVID-19 [9, 55, 56, 58–60,
135]. The factors that result in this depletion in blood are
not understood and could represent reduced cell survival or
genesis, altered tissue distribution or increased differentiation
into ASCs. The depletion of MZB alongside their putative
precursors in severe SLE suggests defective MZB differenti-
ation. The reciprocal reduction of MZB cells and the increase
of ASCs in SLE could also support their differentiation into
short-lived ASCs that are characteristic of SLE disease flares
[27]. Also, as MZB depletion in COVID and SLE has been
shown to be reversible following resolution of the infection
and with treatment respectively, inflammatory factors can re-
sult in significant plasticity in this population [55, 135]. The
functional significance of depletion in MZB is unknown but
could underly the increased susceptibility of patients to in-
fection with encapsulated bacteria or defective B regulatory
responses [140, 141].
CD27+ memory and IgM only B cells
Memory B cells are formed from mature naïve B cells that have
encountered antigen and undergone T dependent immune re-
sponses and germinal centre maturation [2, 142]. They can be
distinguished from naïve B cells by a much higher degree of
IgV gene mutations, the gain of expression of CD27, and the
loss of IgD expression [1, 63, 128, 143, 144]. Rounds of pro-
liferation and diversification within germinal centres create
clones of memory B cells that produce high-affinity immuno-
globulin and differentiate into long-lived ASCs [145–147].
Functionally, memory B cells proliferate rapidly in response
to stimulation with CD40L and interleukin (IL) 2 and IL21
[148, 149].
Distinct subsets of CD27+IgD− memory B cells can be
identified by the expression of IgM, IgA, or IgG (Fig. 5A).
208 Velounias and Tull

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Figure 4: human MZB cells. (A) Flow cytometry plot showing a gating strategy used to identify MZB cells as CD27+IgD+ and histograms showing MFI
of markers that demonstrate that MZB cells are IgM+CD1c+CD45RB+CD21+. (B) Flow cytometry plots demonstrating the identification of two marginal
zone subsets, which can be distinguished based on expression of CCR7 and β7 integrin. MZB1 are CD27+IgD+IgM+CD1c+CCR7+β7hi and MZB2 are
CD27+IgD+IgM+CD1c+CCR7-β7lo. (C) SPADE on viSNE plot showing events exported from the MZB SPADE bubble in Fig. 1D. Higher expression of
CCR7 and β7 integrin is seen in MZB1 and low expression in MZB2. (D) Comparative flow cytometry plots showing the CD19+ compartment in a
HCD and a patient with SLE. Plots show reduction in CD27+IgD+ MZB cells in SLE compared to HCD [9]. (E) Quantification of flow cytometry data
demonstrating a significant reduction in CD27+IgD+ cells in SLE compared to HCD [9]

CD27+ memory cells also share high expression of CD24
and CD45RB and relatively low expression of CD38 (Fig.
5B). CD27 and CD38 are also important in distinguishing
memory B cells from ASCs (Fig. 5C). IgM only B cells are
CD27+IgD−IgM+ and have been identified to have a higher
frequency of IgV gene mutations than MZB cells and to share
clones with CSM B cells, suggesting that these cells are GC de-
rived [10, 61]. The functional differences of human IgM only
and CSM B cells in terms of recall responses and longevity
are not well understood in humans and other than IgM ex-
pression they share a similar surface phenotype (Fig. 5D)
[150]. Of interest is a recent study of BK polyoma virus re-
sponses demonstrated different antiviral specificities of IgM
and IgG memory B cells which suggests a distinct function
of IgM memory B cells [151]. CSM B cells consist of IgA+,
IgG+, and IgE+ subsets although the latter is a rare subset in
peripheral blood. CSM have a high frequency of IgV gene mu-
tations and differentiate into ASCs that produce high-affinity
Clinical and Experimental Immunology, 2022, Vol. 210, No. 3 209

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Figure 5: CD27+ memory B cells and IgM only B cells. (A) Flow cytometry plots showing a gating strategy used to identify memory (CD27+IgD−IgM−
and IgG+/IgA+) and IgM only (CD27+IgD−IgM+) B cells. Data are from a representative healthy control donor. (B) Comparative histogram plots from flow
cytometry data showing differences in expression of CD45RB, CD27, and CD38 in CD27+IgD− cells compared to naïve (CD27−IgD+) cells. (C) Histogram
plots from flow cytometry data showing lower expression of CD38 and CD27 and higher CD24 in CSM compared to ASCs. (D) Heatmap from mass
cytometry data from Fig. 1D showing median expression of lineage markers on naïve, CSM and IgM only B cells

immunoglobulin that are essential for humoral immunity and
the basis of effective vaccine responses [127]. Heterogeneity
within memory B-cell populations has been identified through
differential expression of CD73 and CD95. Memory B cells
can be CD73 positive or negative, its expression is inversely
correlated with IgM and is mainly seen in CSM B cells [5].
CD95 or Fas is a pro-apoptotic receptor which is mainly ex-
pressed by CSM, ASCs, and CD27− memory B cells which is
elevated in SLE and RA and thought to indicate an activated
memory B-cell phenotype [5, 152–154].
Memory B cells play a critical role in autoimmune, inflam-
matory and allergic diseases as they can differentiate into
210
ASCs that produce pathogenic autoantibodies. Autoreactive
memory B-cell clones arise due to checkpoint failure, where
autoreactive naïve B cells are allowed to undergo germinal
centre or extrafollicular responses. This checkpoint has been
shown to be defective in a variety of diseases including SLE
and SS, although the mechanisms that result in this are still
unclear [57, 155]. B cells in SLE are known to express greater
levels of CD40 and blockade of germinal centre responses with
CD40L (CD154) antagonists has therapeutic promise [156–
158]. Variable frequencies of CSM B cells have been observed
in SLE which is likely to represent heterogeneity in disease ac-
tivity, duration, and treatment [54, 65, 66]. Interestingly, un-
like naïve and MZB subsets, CSM frequency is not affected by
BAFF inhibition with belimumab [118]. Variable frequencies
of CSM B cells have also been observed in RA with a lower
proportion of CXCR3+CD27+ cells which correlated inversely
with disease activity [67, 68]. Increased frequency of switched
memory B cells have also been observed in other autoimmune
conditions including ANCA vasculitis [69]. B-cell depleting
agents such as rituximab have long-lasting effects of memory
B cells, which may underlie their efficacy in treating auto-
immune disease [159].
DN memory B cells
CD27− memory B cells were first identified as IgG+ cells in
blood with a lower level of somatic IgV gene mutations than
CSM [70]. DN B cells expressing IgM, IgA, and IgA were then
identified and found to be expanded in SLE [71]. They are
termed DN B cells as they lack both CD27 and IgD expres-
sion. Their accumulation in chronic infections, autoimmune
diseases and with advanced age has also led to them being
termed exhausted memory B cells and age associated B cells
(ABC) [160–162]. ABC share features of DN cells such as
upregulation of T-bet and low expression of CD21 but have
been described using a variety of markers and likely repre-
sent a heterogeneous population which are not entirely syn-
onymous with DN cells [34, 162].
Since their first description a number of DN B-cell subsets
have been identified. Initially DN B cells were split into DN1
(CD21+CXCR5+) and DN2 B cells (CD21−CXCR5−) [26].
Similar to aNAV, DN2 also have low expression of CD24
(Fig. 6A). DN2 cells were found to be a rare subset in health
but are found in much higher frequencies in SLE [26]. DN2
B cells also highly express CD11c as well as the transcription
factor T-bet but lack FCRL5 expression (Fig. 6B and C) [26,
116]. Functionally, DN2 cells are hyperresponsive to TLR7
signalling and can undergo rapid ASC differentiation in re-
sponse to IL21 and IFN-γ and are therefore felt to be pivotal
in extrafollicular B-cell responses [116, 163]. DN2 cells have
been identified to be clonally related to aNAV B cells and ASC
with a lower level of IgV gene mutations and are enriched in
autoreactive cells [27]. More recently DN3 and DN4 subsets
have been reported, the DN3 cluster being CD11c−CXCR5−
and the DN4 subset being enriched in IgE expressing cells and
to highly express transcripts for IL4R and CD40 [72, 73].
SLE disease flares are associated with dramatic increases
in ASCs that secrete pathogenic autoantibodies that are de-
rived from extrafollicular B-cell responses [27]. This is asso-
ciated with expansion of aNAV and DN2 B-cell populations
(Fig. 6D) which are proposed to differentiate into ASCs via
extrafollicular B-cell responses [116]. DN2 B cells are more
frequent in SLE patients of African–American ethnicity and
Velounias and Tull
with lupus nephritis in whom they have been identified within
the nephrotic kidney [26, 163]. Extrafollicular B-cell matur-
ation has been correlated with the severity of COVID-19 and
similar to severe SLE, increased frequencies of aNAV, DN2
cells are observed alongside depletion of MZB cells [9, 41,
60, 78, 116]. The correlation between the severity of infec-
tion and B-cell subset abnormalities suggests a pivotal role of
cytokines such as Type II interferon. CD11c positive B cells
have been reported in patients with MS, RA, and SSc and
to accumulate with age in female patients with RA [164].
Autoreactive CD27−CD19hiCD21lo B cells akin to DN2 cells
have also been reported in SS [165]. Increased frequency of
DN B cells has also been correlated with improved clinical
responses to rituximab of patients with RA whilst reduced
numbers have been reported to predict response to IL-6 in-
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hibition with tocilizumab [77, 166]. Further work is needed
to investigate the clinical significance of these findings. DN
cells may be of pathogenic significance through the produc-
tion of inflammatory cytokines or differentiation into ASCs.
Alternatively, altered DN cell frequency may be indicative of
certain inflammatory environments that affect treatment out-
comes.
Conclusion
The evolution of high dimensional phenotypic tools has
greatly advanced our understanding of B-cell subsets in blood
and the surface markers and transcriptomic properties that
define them. However, significant gaps remain in our under-
standing of the functional roles of these subsets, their develop-
mental origins and pathogenic roles in disease. By addressing
these areas of uncertainty, we will advance our knowledge
of human B-cell development and the role these cells play in
normal homeostasis and in the pathogenesis of autoimmune
and inflammatory disease.
Materials and methods for data used in
figures and figure generation
Experimental subjects
Blood samples were donated by adult HCD and patients with
SLE with regional ethics committee (REC) approval and in-
formed consent (REC reference 11/LO/1433).
Isolation and storage of PBMCs
PBMCs were obtained from whole blood by Ficoll-Hypaque
density gradient centrifugation as previously described [9].
Isolated PBMCs were then centrifuged and cryopreserved in
ice-cold freezing medium (FCS with 10% DMSO) to give a
cell concentration of 5–10 × 106 cells/ml.
Mass cytometry staining and analysis
The staining protocol and antibody panel for the mass
cytometry data displayed in Figs. 1 and 2 is described by Tull
TJ et al. [9]. FCS files were normalized using Nolan Laboratory
Software (https://github.com/nolanlab/beadnormalization).
Single live B cells from 10 HCD (5 male and 5 female, mean
age 31.8 years) are gated as displayed in Fig. 1B. ViSNE plots
displayed in Fig. 1C are generated using Cytobank software
(https://mrc.cytobank.org/) using equal numbers of B cells
(n = 35 000) from 10 HCD. SPADE was then run on the
viSNE coordinates and nodes corresponding to B-cell subsets
are grouped as displayed in Fig. 1D. Transitional B cells were
Clinical and Experimental Immunology, 2022, Vol. 210, No. 3 211

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Figure 6. DN B cells. (A) Flow cytometry plots demonstrating a gating strategy used to identify DN1 and DN2 DN B cells. DN1 cells are
CD27−IgD−CD21+CD24+ and DN2 cells are CD27−IgD−CD21−CD24−. Data are from a representative HCD. (B) SPADE on viSNE plot from mass
cytometry data showing expression of CD21, CD11c, and CXCR5 used to identify DN1 (CD21+CD11c−CXCR5+), DN2 (CD21−CD11c+CXCR5−), DN3
(CD21−CD11c−CXCR5−), and DN4 (CD21+CD11c+CXCR5+). (C) Heatmap demonstrating marker expression by DN B-cell subsets from mass cytometry
data. (D) Comparative SPADE on viSNE plots demonstrating expansion of DN2 cells in SLE compared to health (Tull TJ et al. [9])

identified as CD27−IgD+CD24+++/++CD38+++/++. Events within
this transitional cell bubble were then exported and a fur-
ther viSNE run using equal events (n = 3535) and all panel
markers except CD45, CD3, CD14, and class-switched
isotypes IgA and IgG, which are not expressed by transitional
B cells. The SPADE plots in Fig. 1C are generated from these
viSNE coordinates. SPADE on viSNE plots in Fig. 4C are gen-
erated by exporting events within the MZB SPADE bubble in
Fig. 1D and re-clustered using CD45RB, IgD, CD21, integrin
beta 7, CD27, CD24, IgM, HLA-DR, and CCR7. The DN
SPADE on viSNE was generated using data from 8 HCD and
8 patients with active severe SLE as described by Tull TJ et al.
[9]. CD19+ cells were gated and SPADE on viSNE plots cre-
ated as in Fig. 1D. Events within the DN SPADE bubble were
then exported and DN nodes were re-clustered using CD11c,
CD21, CXCR5, and CD24 to identify DN subsets.
Flow cytometry staining and analysis
Cryopreserved PBMCs were stained with the fluorochrome-
conjugated antibodies as previously described [9]. The fol-
lowing antibodies clones were used: CD27 APC (M-T271),
IgD APC-Cy7 (IA6-2), CD19 PerCP-Cy5.5 (HIB19), CD10
BV421 (HI10a), CD24 BV605 (ML5), IgM BV711 (MHM-
88), CD1c PE (L161), and CD45RB PE-TexasRed (MEM-
55). CD21 BUV737 (HB7) and CD38 BUV395 (B-ly4). DAPI
was used for live dead staining. Flow cytometry data were
obtained by running samples on a Fortessa flow cytometer.
FCS files obtained were analysed using FlowJo software.
212
Acknowledgements
Ms Louise Nel, Prof. David D’Cruz, and Dr Mike Robson
for sample collection. The Guy’s and St Thomas’ NHS Trust
BRC flow and mass cytometry cores for sample processing
and data generation.
Conflict of interest
None declared.
Funding
This work was funded by the Medical Research Council of
Great Britain (MR/R000964/1) and the St Thomas’ Lupus
Trust.
Data availability
Raw mass cytometry data can be requested from the corres-
ponding author.
Author contributions
T.J.T.: Manuscript generation and figure review. R.L.V.:
Figure generation and manuscript review.
Permission to reproduce
Granted within terms set out by the licensing agreement.
Clinical trial registration
Not applicable.
The animal research adheres to the ARRIVE
guidelines
Not applicable.
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