Cutting Edge: BLyS Enables Survival of
Transitional and Mature B Cells Through Distinct
Benjamin L. Hsu,2* Susan M. Harless,2* R. Coleman Lindsley,*
David M. Hilbert,†and Michael P. Cancro3*
These studies characterize BLyS responsiveness and receptor
expression among transitional and mature peripheral B cells.
The results show a maturation-associated increase in BLyS
binding capacity that reflects differential expression patterns
of the three BLyS receptors. Accordingly, BLyS administra-
tion enlarges only late transitional and mature peripheral B
(MB) cell compartments. Furthermore, bromodeoxyuridine la-
beling and cell cycle analyses show these effects are mediated
through enhanced proportional survival of cells traversing the
T2, T3, and MB cell stages, rather than by causing prolifera-
tion or slowing transit within these subsets. Despite similar
effects on survival, BLyS up-regulates the antiapoptotic genes
A1and bcl-xLin MB cells but not immature B cells. Together,
these findings show that, while BLyS influences B cell survival
in several peripheral differentiation subsets, the downstream
mediators differ, thus providing the first direct evidence for an
established B lineage survival system whose intermediates
change as B cells mature. The Journal of Immunology, 2002,
vive (1, 2). Deletion of self-reactive cells contributes to attrition (3,
4) and immature B cells are differentially susceptible to induced
cell death in vitro (5, 6), but shifts in B lineage survival pathways
during normal B cell maturation have not yet been described. In
contrast to negative selection events, some cell losses reflect fail-
ure to meet minimal BcR signaling requisites, suggesting that
specificity-dependent positive selection also plays a key role (7–9).
Once mature, B cells have an average life span of 80–120 days,
lymphocytes transit several differentiation stages as they
leave the bone marrow to mature in the periphery, and
only a fraction of these marrow e ´migre ´s ultimately sur-
but clonotypic longevity varies, subject to relative fitness in com-
petition for viability-promoting cues (10, 11).
B lymphocyte stimulator protein (BLyS; trademark, Human Ge-
nome Sciences) (4, 5) profoundly influences peripheral B cell ho-
meostasis and selection (12–18), but the relative roles of expan-
sion, survival, and differentiation rates in these activities, as well as
whether BLyS acts similarly on newly formed and mature periph-
eral B cells, remain unknown. Therefore, we have examined BLyS
binding, receptor expression, and activity in each peripheral mat-
Materials and Methods
Mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All
procedures were conducted in accord with the Animal Welfare Act.
Abs and flow cytometry
Cytofluorometric analyses were conducted as described (1, 2). The allo-
phycocyanin-conjugated anti-AA4.1 was provided by Dr. D. Allman (Uni-
versity of Pennsylvania, Philadelphia, PA).
Mice were treated with 10 ?g rBLyS s.c. daily. After 4 days of BLyS
treatment, mice also received i.p. injections of 0.5 mg bromodeoxyuridine
(BrdU4; Sigma-Aldrich, St. Louis, MO) twice daily, and splenocytes were
analyzed at successive intervals thereafter as described (1).
Cell cycle analysis
Mice received 10 ?g rBLyS i.p. daily for 8 days. Splenic B cell subsets
were sorted directly into cold 95% ethanol and kept at ?20°C for ?24 h.
Flow cytometric analysis for DNA content was performed following a
30-min incubation in PI buffer (0.1% glucose in PBS, 100 U RNase, and 1
?g/ml propidium iodide). Doublets were excluded based on size.
B cell subset isolation and culture
Immature B cells were prepared from irradiated autoreconstituting mice as
described (1). RBC-depleted splenocytes were treated with 100 ?g/ml
DNase for 10 min, washed in DMEM, then magnetically depleted of
CD43?. This yielded ?80% B cells, of which ?95% were of the immature
phenotype. Mature splenic B cells were prepared from normal mice by
magnetic selection for CD23?splenocytes. Isolated cells were typically
?90% CD23?B cells. T1, T2, T3, and mature splenic B cell subsets were
isolated by FACS from untreated mice. Cells were cultured in RPMI 1640
medium with 10% FBS (HyClone Laboratories, Logan, UT), 2 mM glu-
tamine, 15 mg/ml 1% oxaloacetic acid, 5 mg/ml sodium pyruvate, 20 U/ml
insulin, 1% nonessential amino acids, 50 ?M 2-ME, and 100 U/ml peni-
cillin/streptomycin. Immature B or mature B (MB) cells were cultured at
4 ? 106cells/ml in 24-well plates with or without 100 ng/ml rBLyS.
*Department of Pathology and Laboratory Medicine, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104; and†Human Genome Sciences, Inc.,
Rockville, MD 20850
Received for publication March 22, 2002. Accepted for publication April 25, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by U.S. Public Health Service Grant AI420990 (to
M.P.C.), an Arthritis Foundation Postdoctoral Fellowship (to B.L.H.), and funds from
U.S. Public Health Service Training Grant CA09140 (to S.M.H.).
2B.L.H. and S.M.H. contributed equally to the findings reported in this work.
3Address correspondence and reprint requests to Dr. Michael P. Cancro, 284 John
Morgan Building, University of Pennsylvania School of Medicine, 36th and Hamilton
Walk, Philadelphia, PA 19104-6082. E-mail address: firstname.lastname@example.org
4Abbreviations used in this paper: BrdU, bromodeoxyuridine; MB, mature B;
BCMA, B cell maturation Ag; TACI, transmembrane activator and cAML interactor.
The Journal of Immunology
Copyright © 2002 by The American Association of Immunologists, Inc.0022-1767/02/$02.00
Semiquantitative RT-PCR gene expression analysis
RNA was isolated using TRIzol reagent (Life Technologies, Rockville,
MD). RNA (1 ?g) was pretreated with RNase-free DNase I, then reverse
transcribed using random hexamers (250 ng) and Superscript II reverse
transcriptase (Life Technologies). Each RT-PCR sample consisted of 1/20
of template reverse transcriptase reaction mixture in a 50-?l PCR with Taq
polymerase (1.5 U; Roche, Basel, Switzerland) and 0.4 ?M gene-specific
primers. As an endogenous reference standard for comparing starting tem-
plate cDNA, 18S ribosomal RNA was coamplified with transmembrane
activator and cAML interactor (TACI), A1, or bax using a QuantumRNA
18S kit (Ambion, Austin, TX). Aliquots (6 ?l) were collected at successive
cycles, analyzed by agarose gel electrophoresis, stained with SYBR Green
I (Molecular Probes, Eugene, OR), densitometrically imaged, and analyzed
with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Semi-
quantitative RT-PCR was graphed as cycle number vs log (density), and
the linear portions of the curves were compared and normalized to an 18S
ribosomal RNA internal standard. Densitometric values of other gene-spe-
cific RT-PCR were multiplied by correction factors derived from the 18S
rRNA RT-PCR results, and in turn plotted as cycle number vs log (adjusted
density) for comparison.
PCR primers had the following sequences: murine B cell maturation Ag
(BCMA)-1 and BCMA-3 primers were as reported by Madry et al. (19);
murine TACI sense 5?-gcgcacctgtacagacttc-3?, TACI antisense 5?-gcct
caatcctggaccatg-3?; murine BR3 sense 5?-gcccagactcggaactgtccca-3?, BR3
antisense 5?-gcccagtagagatccctgggttcc-3? (18); bcl-2 and A1 primers as re-
ported for semiquantitative RT-PCR (20); bcl-x sense 5?-taagtgagcaggt
gttttggac-3?, antisense 5?-gggaggtgagaggtgagtgg-3?; bax primers and “clas-
sic” 18S primers were purchased as a relative RT-PCR kit (Ambion).
Results and Discussion
BLyS binding capacity and receptor expression shift with
Marrow B lineage subsets were resolved according to Hardy et al.
(21) and analyzed for BLyS binding. No appreciable binding was
observed in fractions A through D, but fraction E (IgM?AA4.1?
B220low) displayed clear BLyS binding (Fig. 1A). Within fraction
E, a small population of CD23?cells bound BLyS with greater
average intensity than CD23?fraction E cells. The basis for this is
presently unclear but might suggest alternative maturation path-
ways that diverge within this fraction. Mature recirculating B lym-
phocytes (fraction F) displayed bright BLyS binding comparable
to that seen in mature splenic B cells (see below, Fig. 1B).
Splenic maturation stages were divided according to Allman et
al. (2), yielding three transitional subsets: T1 (CD23?IgMhigh
AA4.1?), T2 (CD23?IgMhighAA4.1?), and T3 (CD23?IgMlow
AA4.1?). BLyS binding was demonstrable in all transitional sub-
sets. Although the average intensity was somewhat greater in the
T2 and T3 subsets, all distributions were dispersed, suggesting
considerable heterogeneity in BLyS binding characteristics within
these pools. BLyS binding intensity was highest and tightly dis-
tributed among MB cells (Fig. 1B).
Together, these data indicate that BLyS binding activity ensues
concomitant with surface IgM expression in the bone marrow and
increases with maturation. These results could indicate generally
increasing levels of all three BLyS receptors with maturation, or
might instead reflect the composite of disparate, individually reg-
ulated receptor expression patterns. Therefore, we determined the
expression patterns of BCMA, TACI, and Bcmd/BR3 in sorted B
cell subsets using semiquantitative RT-PCR (Fig. 2). After nor-
malization, BCMA transcripts were most prominent in the T1 sub-
set, with lowest expression in the MB cell subset. In contrast,
TACI displayed a reciprocal expression pattern, whereby MB cells
had nearly 10-fold as much TACI as the T1 subset. While BR3/
Bcmd transcripts were detectable in all subsets, the T1 subset ex-
hibited significantly lower levels than all later differentiative
These maturation-associated variations in BLyS binding and re-
ceptor expression suggested BLyS might influence immature B
and MB cells differently. For example, Bcmd/BR3 may be the
principal receptor required for recruitment and maintenance of the
tiation subsets. Bone marrow (A) or splenocytes (B) were harvested and
stained as previously described (17). Subsets were resolved as shown in the
left plots of each panel, and the surface binding of biotinylated BLyS was
assessed in each subset (center histograms of each panel). Immature mar-
row B cells (fraction E) were further resolved into CD23?and CD23?
groups, and their corresponding BLyS binding is shown in the right his-
tograms. CD23?splenic transitional B cells were further resolved as T2
and T3 subsets by IgMhighvs IgMlowcriteria, and their BLyS binding is
depicted in the right histograms. Data are representative of five experi-
ments. Negative controls shown are identically stained B220?splenocytes.
In addition, fluorochrome-coupled streptavidin without biotinylated BLyS
yielded similar negative control histograms, and preincubation with excess
unlabeled BLyS competitively inhibited biotinylated BLyS staining (data
BLyS binding in bone marrow and splenic B cell differen-
mature peripheral B cell subsets. RNA from FACS-sorted subsets was
subjected to RT-PCR for the BCMA, TACI, and Bcmd/BR3 transcripts.
Three replicates of each subset yielded similar results. Gel images (A) were
subjected to densitometric analysis and adjusted for amount of 18S RNA
amplified relative to other subset samples, and the log10of adjusted density
was plotted (B).
Patterns of BLyS receptor expression within immature and
5994CUTTING EDGE: BLyS ENABLES TRANSITIONAL B AND MB CELL SURVIVAL DIFFERENTLY
follicular B cell pool, because mice lacking this receptor have a
severe follicular B cell deficiency (18, 22–24); whereas signals via
TACI and BCMA may play dominant roles in earlier or alternative
maturation subsets. Furthermore, BLyS-induced effects on these
subsets might proceed through different downstream mediators. To
assess these possibilities, we determined the magnitude, produc-
tion rate, turnover rate, and mitotic activity of each peripheral dif-
ferentiation subset during exogenous BLyS treatment.
BLyS enhances survival among late immature and mature
peripheral B cells
Despite their BLyS binding capacity, neither immature bone mar-
row B cells (data not shown) nor the peripheral T1 subset (Fig. 3A)
changed appreciably during exogenous BLyS administration.
Marked increases were observed in both the T3 and MB cell sub-
sets (p ? 0.01), and a milder but reproducible effect (p ? 0.05)
was seen in the T2 subset (Fig. 3A).
The basis for these increases was established by in vivo BrdU
labeling. These analyses revealed significant increases in the num-
ber of labeled cells per day entering the T2 (p ? 0.05), T3 (p ?
0.01), and mature (p ? 0.01) peripheral populations during BLyS
administration (Fig. 3B). In contrast to these increased absolute
labeling (production) rates, no significant differences in the pro-
portional labeling (renewal) rates were observed (Fig. 3C), indi-
cating that BLyS does not extend residency time in any of the
transitional subsets. While we have also included the short-term
proportional labeling plot of mature peripheral B cells to
strengthen our argument against increased mitotic activity (below),
this is too short a time frame to assess MB cell turnover, because
the average life span of MB cells is ?80 days. In fact, we previ-
ously showed that BLyS receptor mutations increase MB cell turn-
over (22), indicating that MB cell life span is indeed influenced
Because transitional B and MB peripheral subsets are quiescent
(2), enhanced transit from each subset’s predecessor pool was
likely responsible for enhanced production rates. Nonetheless, be-
cause BLyS has been reported to facilitate B cell proliferation in
vitro, it remained possible that these increases reflected prolifera-
tion. We directly addressed this possibility by examining the effect
of BLyS on the proliferative activity of splenic B cell subsets.
Following 8 days of continuous BLyS treatment, transitional and
mature splenic B cells were isolated by cell sorting and stained for
DNA content (Fig. 3D). Negligible proportions (?0.5%) of cells
were observed in the G2? M gate among all transitional subsets
of untreated control mice, in accord with Allman et al. (2). The
proportion of cells in cycle was not significantly altered by
exogenous BLyS administration (Fig. 3D). Moreover, enhanced
division within these populations should have yielded increased
short-term proportional BrdU labeling, which was not observed
Together, these findings suggest that enhanced survival is a pri-
mary activity of BLyS in vivo. Accordingly, we favor the notion
that BLyS regulates peripheral B cell numbers in two ways: by
varying the proportion of cells lost to death during late transitional
B cell development, as shown here, and by serving as the primary
determinant of mature follicular B cell survival, as evidenced by
our studies in the B cell-deficient A/WySnJ mouse (18, 20).
Only MB cells up-regulate Bcl-xLand A1 in response to BLyS
Members of the Bcl-2 family influence lymphocyte survival (25),
and a relationship between BLyS-mediated survival and bcl-2 fam-
ily member expression has been suggested (26, 27). Therefore, we
investigated how BLyS affects A1, bcl-2, bcl-xL, and bax expres-
sion in immature and mature peripheral B cells in vitro.
Among MB cells, the expression of A1 and bcl-xLincreased 2-
to 7-fold in the presence of BLyS, whereas bcl-2 and bax transcript
mature peripheral subsets during in vivo BLyS treat-
ment. A, Spleens were harvested from mice that had
received either no treatment (E) or at least 8 days of
continuous rBLyS (10 ?g/day i.p.; ‚). The B cells were
determined by multiplying the proportion of IgM?cells
by the total number of splenocytes. Each point repre-
sents one mouse and means are indicated as a solid line.
B and C, Mice were either untreated (E) or received 10
?g of BLyS per day i.p. (‚). Beginning on day 4 of
BLyS treatment, all mice received 0.5 mg BrdU i.p.
twice daily. Spleens were harvested daily following on-
set of BrdU administration and the magnitude and pro-
portion of labeled cells in each peripheral subset was
determined. D, B cell subsets were sorted from mice that
had received no treatment (solid line) or 8 days of BLyS
(dashed line). DNA content was determined by pro-
pidium iodide staining. Thymocytes used for validation
(data not shown) routinely yielded ?6% cells in the G2
? M gate. Plots are representative of three separate
Magnitude and kinetics of immature and
cells treated with BLyS in vitro. Semiquantitative RT-PCR was used to
determine the relative change in gene expression of A1, bcl-xL, bcl-2, and
bax in B cells cultured with or without BLyS. Transitional and mature
peripheral B cells were cultured for 3–7 h either in medium alone or with
rBLyS (100 ng/ml). No trend between culture duration and degree of gene
induction was observed within this time frame. Each symbol represents an
independent experiment involving immature (E) or mature (?) resting B
cells. Fold induction value is the amount of gene-specific RNA in BLyS-
treated cells divided by the amount in cells cultured in medium alone.
Bcl-2 family member expression in immature B and MB
5995The Journal of Immunology
levels did not change. In contrast, none of the bcl-2 family mem-
bers examined were up-regulated when total transitional B cells
(T1–T3) were cultured with BLyS (Fig. 4). We have further de-
termined that the T2/T3 fraction up-regulates A1 and bcl-xL?3-
fold in the presence of BLyS (data not shown).
These results are consistent with our previous studies that
showed A1 is up-regulated as developing B cells enter the mature
peripheral pool, and are in general accord with reports that BLyS
can activate Bcl-2 family members. Moreover, these findings pro-
vide the first demonstration of a B lineage-specific survival system
whose receptors and downstream mediators correlate with matu-
ration subset. Because transitional B cells are targets of specificity-
based selection and are differentially sensitive to death via BcR
ligation, it is tempting to speculate that BLyS-mediated survival
mechanisms are integral to these processes. For example, alterna-
tive differentiation and survival pathways for emerging B cells
might be determined by independently controlling BCMA, TACI,
and Bcmd/BR3 expression through adaptive vs innate immune re-
ceptors. Determining the nature and extent of these relationships
will likely prove key to understanding the survival, selection, and
sorting processes active during peripheral B cell maturation.
We thank Drs. David Allman and Avinash Bhandoola for insightful dis-
cussions and critical review of the manuscript.
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5996CUTTING EDGE: BLyS ENABLES TRANSITIONAL B AND MB CELL SURVIVAL DIFFERENTLY