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
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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