MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

Abstract

MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

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MS4a4B, a CD20 Homologue in T Cells, Inhibits T Cell
Propagation by Modulation of Cell Cycle
Hui Xu1*, Yaping Yan1, Mark S. Williams2, Gregory B. Carey2, Jingxian Yang1, Hongmei Li1, Guang-Xian
Zhang1, Abdolmohamad Rostami1
1Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America, 2Department of Microbiology and Immunology,
University of Maryland School of Medicine, Baltimore, Maryland, United States of America
Abstract
MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20,
FceRIb, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-
spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FceRIb and HTm4 have been found to
function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in
T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly
expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including
thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral
vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast,
knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation
by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with
upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle
progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a
modulator in the negative-feedback regulatory loop of activated T cells
Citation: Xu H, Yan Y, Williams MS, Carey GB, Yang J, et al. (2010) MS4a4B, a CD20 Homologue in T Cells, Inhibits T Cell Propagation by Modulation of Cell
Cycle. PLoS ONE 5(11): e13780. doi:10.1371/journal.pone.0013780
Editor: Derya Unutmaz, New York University, United States of America
Received June 9, 2010; Accepted October 6, 2010; Published November 1, 2010
Copyright: ? 2010 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the American Heart Association (0830072N) (H.X.) and from the National Institutes of Health (NS040085) (A.M.
R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hui.xu@jefferson.edu
Introduction
MS4a4B is a novel member of the MS4A gene family
(membrane-spanning 4-domain family, subfamily A, MS4As)
which is characterized by their structural features, with four
membrane-spanning domains, two extracellular domains and two
cytoplasmic regions [1]. The MS4A family includes CD20,
FceRIb, HTm4 and at least 26 novel members [2,3]. Chromo-
some mapping shows that the genes for human CD20, FceRIb,
HTm4 and 12 recently identified MS4A members are located in
chromosome 11q12-q13 [4,5], which is associated with increased
susceptibility to allergy and atopic asthma. The genes for mouse
CD20 and FceRIb are located in chromosome 19 [6,7]. The gene
clustering and the chromosomal localization of the MS4A family
may suggest their immunological relevance. So far, our knowledge
of the MS4A family is derived mainly from studies on CD20,
HTm4 and FceRIb. CD20 is a nonglycosylated, plasma-
membrane associated protein in B cells [7,8], which disappears
when B cells differentiate into plasma cells [9,10]. Early studies
show that CD20 functions in B cells as a Ca2+channel or Ca2+
channel regulator [11]. However an increasing body of data
suggests that CD20 is not only involved in calcium signaling but
also more extensively associated with B cell activation, differen-
tiation and apoptosis [12,13]. Moreover, CD20 has been used as
the target of anti-CD20 treatment for B cell lymphoma and
autoimmune diseases, which to date has been considered as the
most successful antibody-based therapeutics [14]. In comparison
with CD20, HTm4 is predominantly expressed on nuclear
membrane in hematopoietic lineages and is functionally associated
with differentiation of hematopoietic cells [15]. Unlike CD20 and
HTm4, FceRIb, as a part of the receptor complex for IgE Fc
fragment, contains an immunoreceptor tyrosine activation motif
(ITAM) in its C-terminal cytoplasmic domain that directly
contributes to IgE binding-mediated cell signalling [16,17,18].
The functions of other members remain largely unclear. Since we
cloned MS4a4B from the thymus of C57BL/6 mice, data from our
studies and others have shown that MS4a4B is highly expressed in
T cells and is closely related to the regulation of CD4+T cell-
mediated immune responses [1,19,20], suggesting its importance
in adaptive immunity.
Involvement of MS4A proteins in cell proliferation and cell
cycle regulation has been suggested by studies with CD20 and
HTm4 [13,15]. It has been shown that Epstein-Barr viral vector-
driven expression of CD20 in fibroblasts accelerates G1 progres-
sion in a Ca2+-dependent manner [21]. However surface cross-
linking of CD20 with different anti-CD20 monoclonal antibodies
generates the opposite results: cross-linking of CD20 with anti-B1a
antibody inhibits B cell progression into the S/G2+M stages of the
cell cycle [7,22] and drives B cells to undergo apoptosis [4,5] but
binding of anti-CD20 monoclonal antibody 1F5 to CD20 can
activate B cells and initiate cell cycle transition from G0 to G1
phase [23]. In contrast, overexpression of HTm4 in U937 cells
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inhibits the G1-S transition of cell cycle through interaction with
cyclin-dependent kinase-associated (CDK-associated) phospha-
tase-CDK2 (KAP-CDK2) complexes [15,24]. It remains unclear
whether other members of the MS4A family, including MS4a4B,
play roles in cell cycle and cell proliferation. Given that CD20 is
critical for cell proliferation and cell cycle regulation in B cells, and
serves as a target for anti-CD20-based immune therapeutics of B
cell-related diseases [25] we are encouraged to dissect the
biological function of MS4a4B in T cells.
In this report, we demonstrate that although MS4a4B is
expressed at high levels in mature T cells, its expression is silenced
in malignant T cells. We analyzed the impact of MS4a4B on T cell
proliferation by manipulating MS4a4B expression with MS4a4B-
expressing and –silencing approaches. We found that MS4a4B
negatively regulates T cell proliferation by interfering with cell
cycle progression from G0/G1 phase into S-G2/M phases
through inhibition of the Cdk2-Rb pathway, and that silence of
MS4a4B in thymoma is, at least partially, responsible for the
uncontrollable propagation of tumor T cells.
Results
MS4a4B is expressed in mature T cells but not in
malignant T cells
To define the expression of MS4a4B, we detected MS4a4B
protein in mouse cells, including normal T cells, non-T cells, and
malignant T cells, by using anti-MS4a4B antibodies and flow
cytometric analysis. Consistent with our previous findings [1],
MS4a4B was strongly expressed in naı ¨ve T cells but was not
expressed in B cells. In addition to T cells, we also examined
expression of MS4a4B in other cells. The results showed that
MS4a4B was also expressed at high levels in NK cells (marked by
NK1.1) and moderately expressed in macrophages (marked by
Mac1) (supplementary Fig. S1A). In bone marrow cells, only the
Mac1+population expressed low levels of MS4a4B, suggesting
MS4a4B expression in early hematopoietic progenitors (supple-
mentary Fig. S1A). Surprisingly, all malignant T cells, including
thymoma and T hybridoma cell lines that we have examined so
far, lose expression of MS4a4B (Table 1 and supplementary Fig.
S2). Lack of MS4a4B protein in thymoma cells led us to postulate
that MS4a4B may be required for appropriate functioning of
mature T cells and the absence of this protein may be, at least in
part, responsible for the uncontrollable growth of thymoma.
Western blotting with surface-biotin labeling of primary T cells
and histological studies with confocal microscopy of MS4a4B-
expressing retrovirus-infected EL4 thymoma cells showed that
MS4a4B was indeed expressed on cell surface (Fig. 1A and B),
suggesting that MS4a4B may potentially interact with other cell
membrane proteins in T cell regulation. Interestingly, expression
of MS4a4B is regulated not only during thymocyte development
[1] but also during primary T cell activation (Fig. 1C). Although
MS4a4B was constitutively expressed in primary naı ¨ve T cells,
levels of its expression were markedly increased upon stimulation
by mitogen concanavalin A (Con A). However, expression of
MS4a4B was gradiently decreased by 72 hour of activation. It is of
note that the down-regulation of MS4a4B was closely associated
with reduction of surface CD3 expression in T cells (Fig. 1D),
which led us to speculate whether this CD3low/MS4a4Blow
population might represent the cells undergoing apoptosis. We
further examined apoptosis of this population by Annexin V assay.
The CD3low/MS4a4Blowpopulation showed markedly high levels
of apoptotic cells (Fig. 1E). Thus, temporal expression of MS4a4B
in activated T cells and silence of the MS4a4B gene in malignant
T cells led us to hypothesize that MS4a4B may play a regulatory
role in propagation of T cells.
Inhibitory role of MS4a4B in T cell proliferation
T cell proliferation is a critical process for T cell-mediated
immunity, which is reciprocally regulated at multiple levels by
numerous activators and inhibitors [26,27,28]. Its regulation,
however, is still not clearly understood. Studies on CD20 suggest
that MS4A proteins may regulate cell proliferation [13,23]. To
determine the role of MS4a4B in T cell proliferation, we
constructed a MS4a4B-expressing retroviral vector, which co-
expresses GFP as a selection marker. We used the MS4a4B-
retroviral vector to manipulate expression of MS4a4B in primary
T cells, which were subsequently labeled with PHK-26, a red
fluorescent dye, and analyzed T cell proliferation upon CD3/
CD28 stimulation by flow cytometry. Over-expression of MS4a4B
by retroviral vector indeed inhibited proliferation of primary T
cells (Fig. 2A). Since primary T cells constitutively expressed high
levels of MS4a4B, we next used EL4 cells (MS4a4B2), a widely
used thymoma cell line [29,30,31], to confirm the inhibitory effect
of MS4a4B on T cell proliferation. We constructed a lentiviral
vector (LV) to express MS4a4B and a GFP marker in EL4 cells.
We sorted GFP+cells and generated a stable MS4a4B-expressing
EL4 cell line. To test proliferation of EL4 cells, the infected EL4
cells were labeled with PHK-26 and cell proliferation was analyzed
Table 1. Expression of MS4a4B in mouse T cells and non-T cells.
Primary T cell T cell lineMalignant T cellb
Non-T cell
Cell MS4a4BCell MS4a4BCell MS4a4BCell MS4a4B
CD4+
CD8+
+++a
+++
+++
T32
+++
+++
+++
EL4
2
B cell
2
AE7 TIB47
2
NK
+++
+
+
NKT 5CC7CRL1778
2
Bone marrow
HT2
2
2993
2
Macrophage
CTLL
2
T-180
2
58a
2
a28
2
aMS4a4B expression was determined by flow cytometric analysis with anti-MS4a4B intracellular staining. Negative is indicated as ‘‘2’’; positive is indicated by expression
levels based on fluorescent intensity as low (‘‘+’’), middle (‘‘++’’) and high (‘‘+++’’).
bEL4, TIB47, CRL1778, 2993 and T180 are thymoma cell lines; 58a and a28 cells are T hybridoma.
doi:10.1371/journal.pone.0013780.t001
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Figure 1. MS4a4B is expressed on cell surface and is potentially associated with activation of T cells. A, To determine whether MS4a4B
is expressed on cell surface, mouse spleen cells were surface-labeled with EZ-Link Sulfo-NHS-SS-Biotin according to the manufacturer’s protocol
(Pierce). Unlabeled spleen cells were used as control. Cells were lysed by lysis buffer containing 1% NP-40. Cell lysate was immunoprecipitated by
anti-MS4a4B-coupled protein A-beads and was separated on 12% SDS-PAGE, followed by blotting with streptavidin-HRP. MS4a4B was confirmed by
re-blotting with anti-MS4a4B antibody. B, EL4 cells, infected with either MS4a4B-expressing lentivirus (LV) vector or mock LV vector, were stained
with rabbit anti-MS4a4B antibody followed by labeling with anti-rabbit-IgG-Cy3 conjugate. Expression and localization of MS4a4B were observed by
confocal microscopy. Magnification, 640. C, Spleen cells were cultured for 24, 48 and 72 hrs in the presence or absence of Con A (5 mg/ml). Spleen
cells before culture were used as control (0 hr). Cells were first stained with anti-CD3-PE, and then intracellular stained with biotinylated anti-MS4a4B
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by flow cytometry. As predicted, forced expression of MS4a4B
inhibited EL4 thymoma cell proliferation (Fig. 2B).
Smallinterfering RNA(siRNA)-mediated gene silencing has been
shown to be a powerful approach for studying protein function [32].
Since forced expression of MS4a4B over physiologic levels in cells
may lead to artificial data interpretation in some cases, we next
proceeded to knockdown MS4a4B expression. To confirm the
inhibitory role of MS4a4B in T cell propagation, we used
synthesized siRNAs to silence expression of MS4a4B in T32 cells,
a T helper cell line derived from normal primary T cells, which
normally expresseshigh levels ofMS4a4Bprotein [1].Three siRNA
duplexes were designed to target cDNA regions encoding the N-
terminal region, the first extracellular domain, and the C-terminal
intracellular domain of MS4a4B respectively (supplementary Fig.
S3A and B). Knockdown of MS4a4B by selected MS4a4B-siRNAs
markedly reduced expression of MS4a4B at both RNA transcrip-
tion and protein levels (Fig. 2C, 2D and Supplementary Fig. S3C).
In comparison with RNA expression, siRNA-induced reduction of
MS4a4B protein was somewhat delayed, perhaps due to the longer
half-life of protein. To determine the impact of MS4a4B-
knockdown on cell proliferation, T32 cells pre-labeled with PHK-
26 were transfected with FAM-labeled either MS4a4B-specific
siRNA (siMS4a4B2) or negative control siRNA. Proliferation of the
transfected cells in culture was assessed by flow cytometry.
Consistent with the findings from over-expression studies, knock-
down of MS4a4B accelerated proliferation of T32 cells (Fig. 2E).
Similar results were observed in primary T cells from C57BL/6
mice when MS4a4B expression was knocked down by siRNA
approaches (Fig. 2F), suggesting that MS4a4B plays an inhibitory
role in T cell propagation. This appears not to be in line with our
previous observation in which overexpression of MS4a4B enhanced
IL-2 levels in activated T cells. It remains to be elucidated whether
MS4a4B plays a role in survival of differentiated Th1 cells, which
led to a prolonged IL-2 production in these cells. To test how
MS4a4B expression impacts IL-2 responsiveness of T cells, we
added serially diluted IL-2 in culture of T32 cells transduced with
either shMS4a4B lentiviruses (for MS4a4B knockdown) or shLuc
lentiviral vector as control and analyzed proliferation of these cells.
The results showed that knockdown of MS4a4B accelerated IL-2-
induced proliferation of T32 cells (Fig. 2G).
To confirm if MS4a4B does serve as a negative modulator for T
cell proliferation in vivo, EL4 cells infected with either MS4a4B-
expressing-lentivirus or mock control lentiviral vector were infused
into C57BL/6 recipients. EL4 cell propagation was assessed by
analyzing the percentage of infused EL4 cells in blood and spleen
with flow cytometry three days after cell transfer. As indicated by
the percentage of GFP-positive cells, MS4a4B-expression reduced
the propagation of EL4 cells in both peripheral blood (Fig. 3A and
B) and spleens (Fig. 3C and D). To exclude the possibility that
MS4a4B-infected EL4 cells may dominantly migrate into organs
other than spleen, we also examined brain, thymus, heart, lung,
liver, kidney and intestine. The results showed that there were no
infused EL4 cells (GFP+) detected in those organs except heart and
liver, in which the level of MS4a4B-infected EL4 cells was also
slightly lower than that of LV-infected EL4 cells (data not shown).
To further verify the inhibitory role of MS4a4B in solid thymoma
model, 16106MS4a4B-expressing lentivirus (or control vector)-
infected EL4 cells were inoculated subcutaneously in the right
flank of 8–10 week-old C57BL/6 mice. Tumor growth was
monitored daily after inoculation. Results showed that forced
expression of MS4a4B in EL4 cells significantly reduced tumor
growth in vivo (P,0.001) (Fig. 3E). This is unlikely due to host
specific cellular responses to MS4a4B protein given that spleen
cells from EL4-inoculated hosts did not respond to EL4 cells
expressing MS4a4B when they were cocultured with EL4-
MS4a4B cells in vitro (data not shown).
MS4a4B regulates T cell proliferation by interfering cell
cycle progression
It has been documented that CD20 regulates B cell proliferation
by impacting cell cycle progression in these cells [22,33]. HTm4
was found to prevent cell cycle progression from G0/G1 phase
into S-G2/M phase in hematopoeotic cells [15,24]. To get some
insight into how MS4a4B modulates T cell proliferation, we tested
if MS4a4B protein regulates cell cycle progression by using
MS4a4B-lentivirus-infected EL4 cells. EL4 cells stably infected by
MS4a4B-lentivirus (or mock lentiviral vector as control) were
synchronized by two cycle treatment with 2.5 mM thymidine and
serum starvation. Synchronized cells then were stimulated with
complete medium containing 15% FCS. Cell samples were
collected at serial time points after stimulation and cell cycle was
analyzed by flow cytometry with propidium iodide staining. After
8 hours of stimulation, 68.5% of control cells entered S-G2/M
phase. In contrast, the percentage of cells that entered S-G2/M
phase was markedly reduced in MS4a4B-expressing vector-
infected EL4 cells (Fig. 4A). These results suggest that MS4a4B
protein inhibits cell proliferation by preventing cell cycle
progression from G0/G1 phase into S-G2/M phases.
To further confirm the role of MS4a4B in cell cycle regulation
by knockdown approaches, we constructed a lentiviral vector
expressing short hairpin RNA (shRNA) for MS4a4B (MS4a4B-
shRNA), which was designed based on the targeting sequence
(siMS4a4B2) selected by siRNA knockdown experiments described
above. Knockdown by MS4a4B-shRNA vector markedly reduced
MS4a4B mRNA (supplementary Fig. S4) and protein expression
(Fig. 4B and C). To determine the impact of MS4a4B-knockdown
on cell cycle, shRNA-targeting vector-infected T32 cells were
isolated by flow cytometric cell sorting to achieve .99% of GFP
expression. The purified T32 cells were synchronized as described
for EL4 cells above. The cells then were stimulated by complete
medium containing 15% FCS. Cell samples from culture were
analyzed for cell cycle by propidium iodide staining and flow
cytometric analysis. Consistent with the observation from over-
expression experiments, knockdown of MS4a4B protein promoted
cell cycle progression from G0/G1 phase into S-G2/M phase
(Fig. 4D), indicating that reduction of MS4a4B protein released
cells from MS4a4B-mediated suppression of cell cycle.
MS4a4B-mediated cell cycle inhibition is correlated with
enhanced expression of cell cycle inhibitors and reduced
Cdk2 activity
Cell cycle progression is driven by the temporal induction and
activation of cyclin-dependent kinases (CDKs) [34]. Activation of
CDKs requires the formation of CDK-cyclin complexes and is
antibody (blue line) or biotinylated Ig control (red line), followed by labeling with streptavidin-Red 670. For flow cytometric analysis, cells were first
gated on CD3, and then were analyzed for MS4a4B expression. D, Spleen cells pre- or 48 hr post Con A stimulation were co-immunostained with
anti-CD3 and anti-MS4a4B antibody. Cells were analyzed by flow cytometry. Note the CD3low/MS4a4Blowpopulation. E, Spleen cells were stimulated
with Con A for 48 hr and were co-stained with Annexin V, anti-CD3 and anti-MS4a4B antibodies. CD3low/MS4a4Blowand CD3high/MS4a4Bhigh
populations were gated for assessment of apoptosis indicated by Annexin V binding.
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Figure 2. MS4a4B negatively regulates T cell proliferation. A, Retrovirus-driven over-expression of MS4a4B inhibits proliferation of primary T
cells. Primary CD4+T cells isolated from spleen with anti-CD4 magnetic beads were stimulated by anti-CD3/ anti-CD28 antibodies and then were
infected with MS4a4B-retroviral vector or mock vector. After infection, cells were labeled with PKH-26 Red Fluorescent linker kit. Proliferation of the
tested cells was analyzed by flow cytometry (FL2). Data shown are representative of three repeat experiments. B, Forced expression of MS4a4B by LV-
vector reduced proliferation of EL4 cells. Purified EL4 cells with stable lentiviral infection were labeled with PKH-26 Red and then cultured in complete
RPMI 1640 medium. Cells were collected on the days indicated. Cell proliferation was assessed by flow cytometry. Data shown are representative of
three repeat experiments. The numbers in histograms are mean fluorescence intensity (MFI) 6 SD. ***, p,0.001. C, Knockdown of MS4a4B by siRNA.
T32 cells were transfected with either FAM-labeled negative control siRNA or FAM-labeled MS4a4B-specific siRNAs (siMS4a4B1 and siMS4a4B2). Cells
MS4a4B Regulates T Cells
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