Seminars in Immunology 17 (2005) 347–355
Bone marrow microenvironmental changes in aged mice compromise
V(D)J recombinase activity and B cell generation
Joseph E. Labrie III, Lisa Borghesi1, Rachel M. Gerstein∗
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, MA 01655, USA
that contribute to reduced B cell production in aged mice. Using in vivo labeling, we found that reduction in marrow pre-B cells reflects
increased attrition during passage from the pro-B to pre-B cell pool. Analyses of reciprocal bone marrow (BM) chimeras reveal that the
production rates of pre-B cells are controlled primarily by microenvironmental factors, rather than intrinsic events. To understand changes
in pro-B cells that could diminish production of pre-B cells, we evaluated rag2 expression and V(D)J recombinase activity in pro-B cells
at the single cell level. The percentage of pro-B cells that express rag2 is reduced in aged mice and is correlated with both a loss of V(D)J
recombinase activity in pro-B cells and reduced numbers of pre-B cells. Reciprocal BM chimeras revealed that the aged microenvironment
also determines rag2 expression and recombinase activity in pro-B cells. These observations suggest that extrinsic factors in the BM that
decline with age are largely responsible for less efficient V(D)J recombination in pro-B cells and diminished progression to the pre-B cell
stage. These extrinsic factors may include cytokines and chemokines derived from BM stromal cells that are essential to the development
of B cell precursors. The changes during aging within the BM hematopoietic microenvironment most likely are linked to the physiology of
aging bone. Bone degrades with age (osteoporosis) due to decreased formation of new bone by osteoblasts. Marrow stem cells (MSC) are
considered the progenitor of both adipocytes, osteoblasts and hematopoietic stromal cells and a controlled reciprocal regulation exists of
osteoblast versus adipocyte differentiation; with age adipocytes increase, and osteoblast decrease. It is possible that stromal cell generation
from MSC is compromised during aging. Currently, understanding of BM microenvironmental factors that regulate rag gene expression is
very limited. However, as early progenitors differentiate, it is increasing clear that a limited set of transcription factors (e.g. ikaros, PU.1, E2A,
EBF, pax5) regulate B-lineage specific genes, and that expression and stability of these factors is responsive to the microenvironment. Current
and future work by several groups will strive to understand mechanisms that regulate these factors and how aging impacts these regulatory
© 2005 Elsevier Ltd. All rights reserved.
Keywords: Aging; B cell; Immunoglobulin; V(D)J recombination; Bone marrow stromal cells; Osteoporosis
1. Humoral immunity, aging and B cell development
The renewal of the immune system in mice declines dur-
ing aging: B and T cell generation are reduced [1,2] and as
a result, old mice are more susceptible to pathogens . The
human immune system is also compromised during aging,
vaccine efficacy [2,4]. Given the critical role of humoral
∗Corresponding author. Tel.: +1 508 856 1044; fax: +1 508 856 5920.
E-mail address: Rachel.Gerstein@umassmed.edu (R.M. Gerstein).
1Present address: Department of Immunology, University of Pittsburgh
School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261, USA.
immunity for both vaccines and responses to infection, we
have used murine models to ask whether the generation of
the primary B cell repertoire is compromised during aging.
similar to that of young mice, however, the rate of turnover is
and reduced Ig diversity. Previous work has in fact shown
that Ig heavy chain gene VHto DJHjoining and Ig diversity
is reduced in aged mice [1–11].
1044-5323/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.
J.E. Labrie III et al. / Seminars in Immunology 17 (2005) 347–355
B cell development occurs continuously during life. In
potent progenitors (MPPs) derived from hematopoietic stem
phoid progenitors (CLPs) (reviewed in [12–14]). CLPs are
particularly efficient B cell progenitors [15–17] that progress
as pre-pro-B, pro-B, pre-B and immature B cells in the bone
marrow, and are then exported primarily to the spleen where
they progress through stages of immature transitional B cells
and develop into mature na¨ ıve B cells.
2. V(D)J recombination in B cell development
The generation of new B cells is completely dependent on
the assembly of Ig genes by V(D)J recombination. In each
B cell progenitor, a unique combination of VH, DHand JH
gene segments (one of each segment) are joined together to
encode the IgH variable region, and either V?and J?or V?
and J?gene segment are joined to encode the IgL variable
region. Each V, D and J segment is flanked by a conserved
recombination signal sequence (RSS) that are the targets of
the V(D)J recombinase (reviewed in [18,19]).
The V(D)J recombinase includes the products of the
recombinase activating genes, rag1 and rag2. During V(D)J
recombination, RAG1 and RAG2 bind to RSSs and assem-
ble a synaptic cleavage complex (SCC) that includes two
Ig gene segments (e.g. a DHand a JH). Following forma-
tion of the SCC, RAG1 and RAG2 introduce DNA nicks
and double-strand breaks between the RSS and the flanking
ing, non-homologous end joining (NHEJ) repairs the DNA
breaks. This repair requires several ubiquitously expressed
NHEJ proteins: Ku70, Ku80, DNA-PK, XRCC4, and DNA
Ligase IV. The two coding ends are ligated to form a coding
In most Ig recombination events, the signal joint forms a cir-
cular piece of DNA containing the region that was originally
between the two recombining gene segments and the coding
joint now connects two gene segments that encode part of a
new Ig variable region.
IgH and IgL genes are assembled sequentially during B
cell development. During the pro-B cell stage of develop-
ment, two recombination events in the IgH locus produce
a rearranged, intact IgH variable region gene. First DHto
JHrecombination occurs, followed by VHto DHJHrecom-
bination. During the pre-B cell stage, a light chain gene is
formed by V to J recombination of either V?to J?or V?to
J?. Expression of rag1 and rag2 are up-regulated both prior
to and during pro-B cell stage, when the IgH gene is assem-
bled, and are again up-regulated in the pre-B cell stage when
Ig light (IgL) chain gene is rearranged [20–23].
of the V(D)J recombinase and their coordinate expression
in the genome of all animals studied and are convergently
cis-elements control transcriptional regulation of rag1 and
rag2 [25–36]. In addition to core promoters, cis-acting ele-
ments 5?of rag2 are essential to expression of both rag1 and
rag2 and contribute to differential regulation in B and T cells
[23,37,38]. For example, the Erag element located 22kb 5?
of rag2 is essential for rag1 and rag2 expression in B but not
T lineage cells .
Transit from the pro-B to the pre-B cell stage is regulated
the surrogate light chain (SLC) that itself is composed of ?5
and Vpre-B. Signaling through the pre-BCR is dependent
upon the presence of a rearranged functional IgH gene, and
therefore productive V(D)J recombination is a limiting step
essential to V(D)J recombination; therefore, in their absence
an intact IgH chain is not produced and B cell development
is blocked at the pro-B cell stage [40,41]. If a functional
heavy chain is made, signals through the pre-BCR promote
developmental changes in the B cell precursor. Expression
of rag1 and rag2 is turned off and the cell undergoes sev-
eral rounds of proliferation. This accelerates the degradation
of RAG1 and RAG2, as RAG2 is phosphorylated during
the cell cycle resulting in ubiquitin-dependent degradation
[42,43] and RAG1 half-life is shortened when associated
with RAG2 . These regulatory mechanisms stop further
recombination of IgH and thus contribute to allelic exclu-
sion. In addition, proliferation mediated by the pre-BCR
expands the number of pre-B cells, resulting in greater num-
bers of pre-B cells than pro-B cells (3:1 ratio) in young
3. B cell development is attenuated in aged mice due
to decreased pre-B cell generation
age-associated attenuation in mice: B cell generation is
markedly reduced [1,45], and fewer mature B cells are pro-
duced. However, the number of mature B cells is similar to
of mature cells [1,4]. Frequencies and numbers of B cell pro-
most noted change is the reduced frequency and number of
this occurs varies [45–54]. Initial reports indicated that num-
and others find that numbers of pro-B cells are significantly
reduced in aged mice [16,47,51]. The onset and severity of
reduced pre-B cell numbers can vary substantially between
mice, and interestingly, loss of pre-B cells within individuals
is well correlated with reductions in pro-B cells [51,55].
Our recent work sought to understand mechanism(s) that
contribute to the reduction in numbers of pre-B cells in aged
J.E. Labrie III et al. / Seminars in Immunology 17 (2005) 347–355
of pre-B cells, (2) reduced transit times of pre-B cells to the
immature B cell compartment and (3) reduced generation of
in aged mice.
To explore these questions, we examined the magnitude
and kinetics of each major B cell stage in aged versus young
adults. In accord with previous findings, the pre-B cell pool
of aged individuals is markedly diminished by ∼4-fold .
The kinetic changes underlying shifts in the size of B cell
versus aged mice and both ages produce ∼1 million cells per
day, with the number and production rate of pro-B cells only
kinetics within the pre-B cell pools of aged versus young
adult mice differ dramatically: young adults generate 9–13
million pre-B cells daily, whereas aged individuals produce
that the residency time within the pool is unchanged in aged
unchanged renewal rate corresponds well with the observed
four-fold diminution of the pre-B cell pool.
4. Microenvironment defects underlie reduced B cell
development with age
be due to either cell-intrinsic defects in the B cell precursors
themselves or alterations in the developmental microenvi-
ronment. Contact-mediated signals from bone marrow stro-
mal cells and soluble factors are required for commitment,
development, proliferation and survival of developing B cell
precursors (reviewed in [56–58]). Accordingly, stromal cell
cultures established from aged mice are less supportive of
proliferation and development of pro-B cells than cultures
established from younger mice [53,54]. A defect in the in
vivo microenvironment was indicated by the finding that the
number of pre-B cells in old mice receiving young marrow
was diminished .
To test whether age of the microenvironment con-
strains the generation of pre-B cells, aged→young and
young→aged bone marrow chimeras were generated and
the magnitude, turnover and renewal rates of each marrow
B cell compartment determined. When transferred to young
recipients, both aged and young donor marrow produced
newly formed B cell subpopulations of identical magnitude,
to aged recipients, young marrow yields a pre-B cell com-
partment different from that of young recipients, and which
mirrors the production and turnover rates observed in aged
tures of B lineage progenitors themselves, underlie altered
generation and attrition rates.
5. Age-associated reduction in pre-B cells is
correlated with a reduction in the percent of pro-B
cells that express rag2
The decreased generation of pre-B cells suggested to us
that the molecular defects in aged pro-B cells might include
sible for decreased efficiency of generating functional IgH to
serve as a component of the pre-BCR. When measured in the
total bone marrow of aged mice, rag1 and rag2 mRNA lev-
els decline dramatically [9,50,59]. However, the age-related
decline in rag expression could have been due to a global
loss of bone marrow cellularity, the decrease in the number
of pre-B cells that express rag and comprise ∼80% of the
rag2-expressing cells in the BM (Gerstein, unpublished), or
a decline in rag expression within pro-B cells.
To directly measure rag2 expression within pro-B cells
in the bone marrow of young and aged mice, we used the
NG strain of GFP transgenic rag2 reporter mice . This
When flow cytometric analysis of young and aged NG trans-
genic mice was used to determine the percent of pro-B cells
that express GFP as a reporter of rag2 expression, we found
that the percent of pro-B cells that are GFP+is significantly
finding that the reduction in pre-B cell numbers in aged mice
express rag2 supports the hypothesis that reduced numbers
of pre-B cells in aged mice is due to alterations that affect
expression of rag2.
We also analyzed B cell development in RAG2:GFP
knock-in (KI) mice . These KI mice have two advan-
tages compared to NG mice: (1) the GFP reporter is located
within the endogenous rag2 locus and (2) GFP is expressed
as a fusion protein with RAG2 and thus serves as a direct
reporter of cellular RAG2 protein levels. The RAG2–GFP
that the number of pre-B cells and the pre:pro ratio was sig-
nificantly lower in aged as compared to young KI mice. As
with the NG mice, the percent of pro-B cells that express
rag2 from the KI locus was significantly lower in aged mice
as compared to young KI mice. Because the KI reporter is
a RAG2–GFP fusion protein, the data suggest that protein
levels of RAG2 are lower in the pro-B cells of aged mice.
Furthermore, we observed that the percent of pro-B cells that
express GFP was correlated with both the number of pre-
B cells and the pre:pro ratio. Taken together, our analyses
of both the NG transgenic and RAG2–GFP KI mice indicate
J.E. Labrie III et al. / Seminars in Immunology 17 (2005) 347–355
reduced rag2 expression in pro-B cells.
6. rag2 expression, V(D)J recombinase activity and
the pre:pro ratio are reduced in aged mice
Using H2-SVEX transgenic mice, we also found that
reduced expression of rag2 in aged mice yields a corre-
sponding decrease in recombinase activity . Cells that
undergo V(D)J recombination of this transgenic recombi-
nation substrate express the GFP variant VEX [61,62], are
easily detected by flow cytometry and are readily resolved
from cells expressing conventional GFP [61,62].
a population reflects the level of rag2 expression [60,63].
Thus, we crossed NG mice to H2-SVEX mice to generate
double transgenic mice in which rag2 expression could be
measured in conjunction with V(D)J recombinase activity.
We used adoptive transfers to evaluate rag2 expression and
recombinase activity in young versus aged recipient mice as
percent of donor-derived pro-B cells that express rag2 was
significantly lower in aged recipients as compared to young
recipients, and V(D)J recombinase activity, as indicated by
the percent of pro-B cells that are VEX+, was also signifi-
cantly lower in pro-B cells from aged recipients as compared
to young recipients. This suggests that the reduction in rag2
expression in pro-B cells of aged mice is sufficient to result
numbers in the young and aged recipients. The donor pre:pro
ratio was significantly lower in the aged compared to the
young recipient mice. The age-associated reduction in rag2
expression, recombinase activity and pre:pro ratio might be
interrelated; and, using statistical analyses we found that all
three parameters were correlated.
We demonstrated that recombinase activity is reduced in
pro-B cells that develop in aged mice. This supports the
hypothesis that reduced rag2 expression in pro-B cells is of
sufficient magnitude to reduce recombinase activity, poten-
tially limiting heavy chain rearrangement, pre-BCR expres-
sion and subsequent transit to the pre-B cell stage. Our
findings of fewer pro-B cells and reduced rag2 expression
and recombinase activity in pro-B cells from aged mice sup-
ports a model in which reduced recombination of IgH might
compromise both the pro-B cell compartment and the pro-
B to pre-B cell transition. Attrition of pro-B cells during
aging was first noted by van der Put et al.  and pro-B
cell reduction was also demonstrated by Miller and Allman
. We also observed a reduced percent of CD24highpro-B
cell cells in aged mice, and decreases in the number of pro-B
cells . Failure to rearrange IgH is predicted to deplete
Fr. C?CD24highpro-B cells and limit proliferation of pre-
BCR+cells, as Fr. C?CD24highpro-B cells and large pre-B
and aged mice in each fraction of marrow development .
Thus, since the C?and D fractions show similar proportions
of cells in S and G2/M phases, the proliferative burst associ-
ated with fraction C?does occur in aged mice, and therefore
decreased proliferation does not underlie reduced pre-B cell
Our results indicate that the fewer cells in the pro-B cell
stage of development have an active V(D)J recombinase in
cleaved DFL16.1 signal ends and compromised VHto DJH
joining in pro-B cells from aged mice. Thus, reduced recom-
binase activity could contribute to reduced diversity of IgH
chains and thus compromise the Ig repertoire in aged mice.
Interestingly, lower enzymatic activity of RAG in rag “core-
only” knock-in mice resulted in reduced VHto DJHjoining
and an altered repertoire [64–66]. Alternatively, the reduced
rag2 expression and V(D)J recombinase activity could sup-
port formation of a normal repertoire, but with decreased
efficiency in the generation of pre-B cells.
7. The bone marrow microenvironment also controls
rag2 expression and V(D)J recombinase activity in
To determine if these age-associated alterations are
tive transfers. We observed that young donor-derived pro-B
into aged as compared to young recipient mice . This
indicates that age-associated alterations specific to the bone
marrow microenvironment are sufficient to produce these
defects in B cell development. To determine if cell-intrinsic
defects also affect rag2 expression in pro-B cells and the
pre:pro ratio, bone marrow from aged and young NG trans-
genic donor mice was transferred into young recipient mice.
Thus, progenitors from young and aged mice were evaluated
after differentiation within the same microenvironment; the
reconstitution of young mice, we found that rag2 expression
by the age of the bone marrow donors, indicating that the
reduction in rag2 expression in pro-B cells and diminished
pre-B cell numbers of aged mice is not likely to be the result
of defects that are intrinsic to the developing cells.
8. Potential bone marrow microenvironment defects
The bone marrow microenvironment includes factors
derived from bone marrow stromal cells that are essential
to the development of B cell precursors. As specific extrinsic
J.E. Labrie III et al. / Seminars in Immunology 17 (2005) 347–355
age-related decrease in rag2 expression in pro-B cells might
reflect attenuation of yet unknown inductive signals. The
nature of these changes remains to be determined, although
rapid progress is being made in understanding the control of
B cell development by stromal-derived factors.
IL-7 is produced by stromal cells and is essential for pro-
liferation and differentiation of B cell progenitors in vivo
[67,68]. Signaling through IL-7R is necessary for commit-
ment to the B cell lineage as IL-7R? and ?c-deficient mice
have normal frequencies of CLPs but a block in produc-
tion of pre–pro-B cells and further B cell development in
the bone marrow . Interestingly, expression of IL-7R
is similar between precursors from young and aged mice
although the response to IL-7 is blunted in pro-B from aged
mice [46,48,54]. This could indicate that intrinsic signaling
in response to IL-7 is defective in B cell precursors in aged
mice. Most of the evidence supporting cell-intrinsic, age-
the cellular defects observed in culture are due to changes
that are “imprinted” on developing B cells by changes in the
in vivo microenvironment. Consistent with this is the obser-
vation that CLPs from IL-7-deficient mice are reduced in
old mice have a significant reduction in CLPs  are also
consistent with a potential defect in signals like IL-7 that are
needed to sustain the CLP population.
The BM microenvironment can also modulate IL-7 avail-
ability, as heparan sulfate proteoglycans (HSPG) bind IL-7
and regulate its bioactivity, HS on pro-B cells appears to
be required for efficient responsiveness to IL-7  and
growth factor interaction with HSPGs have previously been
suggested to organize the hematopoietic microenvironment
[71,72]. IL-7 binding by pro-B cells is low in pax5−/− cells
and then is restored by transduction with the PAX5-target
BLNK, and interestingly, in this system, pre-BCR signal-
ing leads to expression of the enzyme 3-OST that modifies
ulated by WNT5a, as we found ablation of WNT5a results
in increased pro-B cell production and more rapid entry into
cell cycle in response to IL-7 . IL-7 production may in
fact be impaired during aging as IL-7 mRNA is low in fresh
BM cells from old mice  and bone marrow cultures from
old mice produce less IL-7 [54,76].
Reduced numbers of either stromal cells or specific cellu-
reduced pre-B cell generation rates. In aged mice the percent
of pro-B cells that expresses rag2 is reduced. However, we
noticed in the pro-B cells that do express rag2, the level of
This could indicate that fewer pro-B cells receive signals and
factors from the stromal microenvironment, but those that do
receive the required factors develop normally.
The changes during aging within the BM hematopoietic
microenvironment most likely are linked to the physiology
of aging bone. Bone degrades with age (osteoporosis) due to
decreased formation of new bone by osteoblasts and “neg-
ative balance” of remodeling by osteoclasts. Both increased
activity of osteoclasts and decreased life span of osteoblasts
have been noted (reviewed in [78,79]). Marrow stem cells
(MSC) are considered the progenitor of both adipocytes,
osteoblasts and hematopoietic stromal cells (reviewed in
[80–82]). Evidence exists for a controlled reciprocal regula-
tion of osteoblast versus adipocyte differentiation from bone
osteoclasts decrease: these changes may be related to age-
associated increases in the PPAR?2 transcription factor in
of osteoblasts that also promotes adipocytes) . Adipoge-
nesis is held in check by WNT signaling [85–87]. Therefore,
WNT production by stromal cells  and by B cell pro-
genitors themselves  would be interesting to characterize
mal cells is compromised during aging, leading to decreased
stromal cell production. Consistent with this, marrow stro-
mal cells from old human donors had decreased life-span in
culture and accelerated senescence .
Bone marrow stromal stem cells (BMSSCs) examined
1 (SDF-1). SDF-1 expression was rapidly down-regulated
when BMSSCs were cultured under osteoinductive condi-
displayed an increased capacity for cellular growth and pro-
tection against interleukin-4-induced apoptosis. Therefore,
SDF-1 may be important for the maintenance of the imma-
ture BMSSC population . It is intriguing to note that
SDF-1 is essential for B cell development, particularly at
the Lin−CD19−c-kit+IL-7R?+AA4.1+(CLP) stage of fetal
B cell development , as deletion of either SDF-1 or its
receptor CXCR4 is embryonic lethal and these mice have
very few pro-B cells in fetal liver at day 18 [92,93]. Thus, its
tempting to speculate that decreased SDF-1 expression with
aging could both reflect and cause impaired regeneration of
BMSSC, and therefore contribute to the impaired generation
of pro- and pre-B cells.
9. Transcription factors regulate B-lineage specific
genes, including rag1 and rag2
Currently, understanding of BM microenvironmental fac-
tors that regulate rag gene expression is very limited. How-
ever, as early progenitors differentiate, it is increasing clear
that a limited set of transcription factors regulates B-lineage
specific genes. Key transcription factors implicated in con-
trolling early stages of lymphopoiesis include ikaros, PU.1,
E2A, EBF and pax5 [94–98]. Of these, E2A, EBF and pax5
are required for commitment to the B lineage (reviewed in
). A transcriptional hierarchy has been defined in which
E2A appears to regulate EBF expression which in turn reg-
ulates pax5 [100,101]. Together, these genes coordinately
J.E. Labrie III et al. / Seminars in Immunology 17 (2005) 347–355
regulate the lineage-specific set of proteins that are required
for B cell development and function including CD19, IgL,
mb-1, B29, ?5, VpreB and IL7R. Loss of any one of these
transcription factors is sufficient to ablate B lineage develop-
The E2A gene products E47 and E12 are of partic-
ular interest as mediators of rag gene expression. E2A
is expressed at both mRNA and protein levels in LSKs
[103,104], the progenitor subset in which rag expression is
of-function technology suggest that E2A genes may regulate
rag expression. Ectopic expression of E12 simultaneously
promotes expression of rag1 and EBF in the 70Z/3 pre-B
cell line . Similarly, retroviral reconstitution of E47 in
long-term cultured E2A-deficient hematopoietic progenitors
restores both rag1 and rag2 expression .
Because E2A-deficient and E47-deficient mice have a
the absence of detectable IgH rearrangements in these ani-
mals reflects a V(D)J recombination defect or a develop-
mental defect. Both whole bone marrow and fetal liver from
mice deficient in E2A appear to lack rag transcripts as well
as detectable D–JHrearrangements [94,102,107]. However,
long-term culture of E2A-deficient lin−hematopoietic pro-
genitors is sufficient to restore detectable D–JH (but not
V–DJH) joints in vitro .
To identify which hematopoietic progenitor subsets are
still intact in E47-deficient animals, and to determine the
role of this transcription factor in regulating initiation of
RAG activity in vivo, we characterized H2-SVEX trans-
genic mice that were E47-null or E47-heterozygous .
We show that loss of the transcription factor E47 completely
ablates V(D)J recombination in CLPs in vivo. E47-deficient
mice also have a 70–90% decrease in recombinase activity
in downstream pre-pro B cells. By contrast, loss of either
E47, or its cis-acting target Erag (enhancer of rag transcrip-
tion), has relatively little effect on recombinase activity in
thymic T lineage progenitors. Thus, these results indicate
that E47 is required for initiating B lineage-specific patterns
Erag also regulates rag expression and activity in pro-B cells
[39,60] and E47 is dramatically up-regulated in pro-B cells
[104,109], corresponding to up-regulation of rag expression
and activity , it seems likely that E47 is also required for
rag expression at the pro-B cell stage.
Although it is not yet clear how E47 up-regulation in pro-
consider defects in E47 as a prime candidate for the recom-
binase defect in aging B cell progenitors. B cell precursors
from bone marrow of aged mice have decreased E2A protein
turnover [111,112]. Depressed E47 levels during aging may
affect other targets of E47 needed for the pro-B to pre-B cell
transition such as ?5 [47,113]. It is intriguing to note that IL-
7 stimulated pro-B cells cultured with stem cell factor have
greatly increased E47 turn-over, whereas these pro-B cells
retained high levels of E47 protein when cultured in Flt3L
. Pro-B cells express both receptors and proliferate in
response to IL-7 combined with either SCF or FL. The two
cytokine receptors are highly homologous and thought be
redundant in support of generation of pro-B cells (reviewed
cates that signaling pathways for the receptors differ, and
microenvironment might translate to differential control of
E47 targets such as rag gene expression.
We thank Alex Sah, David Allman, Michael Cancro
(UPenn) for their invaluable contribution to our collabora-
(UMMS) as well as Bonnie Blomberg and Richard Riley
in part by NIH grants AI043534 and AG19042 to R.M.G.,
and NIDDK 5 P30 DK32520 to the UMMS Diabetes and
 Kline GH, Hayden TA, Klinman NR. B cell maintenance in aged
mice reflects both increased B cell longevity and decreased B cell
generation. J Immunol 1999;162:3342–9.
 Miller RA. The aging immune system: primer and prospectus. Sci-
 Sambhara S, Kurichh A, Miranda R, James O, Underdown B, Klein
M, et al. Severe impairment of primary but not memory responses
to influenza viral antigens in aged mice: costimulation in vivo par-
tially reverses impaired primary immune responses. Cell Immunol
 Ghia P, Melchers F, Rolink AG. Age-dependent changes in
B lymphocyte development in man and mouse. Exp Gerontol
 Ghia P, ten Boekel E, Rolink AG, Melchers F. B-cell develop-
ment: a comparison between mouse and man. Immunol Today
 Callahan JE, Kappler JW, Marrack P. Unexpected expansions of
CD8-bearing cells in old mice. J Immunol 1993;151:6657–69.
 Zheng B, Han S, Takahashi Y, Kelsoe G. Immunosenescence and
germinal center reaction. Immunol Rev 1997;160:63–77.
 Riley SC, Froscher BG, Linton PJ, Zharhary D, Marcu K, Klin-
man NR. Altered VH gene segment utilization in the response to
phosphorylcholine by aged mice. J Immunol 1989;143:3798–805.
 LeMaoult J, Szabo P, Weksler ME. Effect of age on humoral immu-
nity, selection of the B-cell repertoire and B-cell development.
Immunol Rev 1997;160:115–26.
 Szabo P, Shen S, Telford W, Weksler ME. Impaired rearrangement
of IgH V to DJ segments in bone marrow pro-B cells from old
mice. Cell Immunol 2003;222:78–87.
 Li F, Jin F, Freitas A, Szabo P, Weksler ME. Impaired regeneration
of the peripheral B cell repertoire from bone marrow following
lymphopenia in old mice. Eur J Immunol 2001;31:500–5.
 Hardy RR. B-cell commitment: deciding on the players. Curr Opin
 Allman D, Miller JP. Common lymphoid progenitors, early B-
lineage precursors, and IL-7: characterizing the trophic and instruc-
J.E. Labrie III et al. / Seminars in Immunology 17 (2005) 347–355
tive signals underlying early B cell development. Immunol Res
 Baba Y, Pelayo R, Kincade PW. Relationships between hematopoi-
etic stem cells and lymphocyte progenitors. Trends Immunol
 Izon D, Rudd K, DeMuth W, Pear WS, Clendenin C, Lindsley
RC, et al. A common pathway for dendritic cell and early B cell
development. J Immunol 2001;167:1387–92.
 Miller JP, Izon D, DeMuth W, Gerstein R, Bhandoola A, Allman
D. The earliest step in B lineage differentiation from common
lymphoid progenitors is critically dependent upon interleukin 7. J
Exp Med 2002;196:705–11.
 Allman D, Sambandam A, Kim S, Miller JP, Pagan A, Well D, et
al. Thymopoiesis independent of common lymphoid progenitors.
Nat Immunol 2003;4:168–74.
 Swanson PC. The bounty of RAGs: recombination signal com-
plexes and reaction outcomes. Immunol Rev 2004;200:90–114.
 Schatz DG. V(D)J recombination. Immunol Rev 2004;200:5–11.
 Oettinger MA, Schatz DG, Gorka C, Baltimore D. RAG-1 and
RAG-2, adjacent genes that synergistically activate V(D)J recom-
bination. Science 1990;248:1517–23.
 Schatz DG, Oettinger MA, Baltimore D. The V(D)J recombination
activating gene, RAG-1. Cell 1989;59:1035–48.
 Grawunder U, Leu TM, Schatz DG, Werner A, Rolink AG, Melch-
ers F, et al. Down-regulation of RAG1 and RAG2 gene expression
in preB cells after functional immunoglobulin heavy chain rear-
rangement. Immunity 1995;3:601–8.
 Yu W, Misulovin Z, Suh H, Hardy RR, Jankovic M, Yannoutsos
N, et al. Coordinate regulation of RAG1 and RAG2 by cell type-
specific DNA elements 5?of RAG2. Science 1999;285:1080–4.
 Schlissel MS. Regulating antigen-receptor gene assembly. Nat Rev
 Lauring J, Schlissel MS. Distinct factors regulate the murine
RAG-2 promoter in B- and T-cell lines. Mol Cell Biol 1999;19:
 Jin ZX, Kishi H, Wei XC, Matsuda T, Saito S, Muraguchi
A. Lymphoid enhancer-binding factor-1 binds and activates the
recombination-activating gene-2 promoter together with c-Myb and
Pax-5 in immature B cells. J Immunol 2002;169:3783–92.
 Miranda GA, Villalvazo M, Galic Z, Alva J, Abrines R, Yates
Y, et al. Combinatorial regulation of the murine RAG-2 promoter
by Sp1 and distinct lymphocyte-specific transcription factors. Mol
 Kitagawa T, Mori K, Kishi H, Tagoh H, Nagata T, Kurioka H,
et al. Chromatin structure and transcriptional regulation of human
RAG-1 gene. Blood 1996;88:3785–91.
 Kishi H, Wei XC, Jin ZX, Fujishiro Y, Nagata T, Matsuda T, et al.
Lineage-specific regulation of the murine RAG-2 promoter: GATA-
3 in T cells and Pax-5 in B cells. Blood 2000;95:3845–52.
 Kishi H, Jin ZX, Wei XC, Nagata T, Matsuda T, Saito S, et al.
Cooperative binding of c-Myb and Pax-5 activates the RAG-2 pro-
moter in immature B cells. Blood 2002;99:576–83.
 Wang QF, Lauring J, Schlissel MS. c-Myb binds to a sequence in
the proximal region of the RAG-2 promoter and is essential for pro-
moter activity in T-lineage cells. Mol Cell Biol 2000;20:9203–11.
 Fong IC, Zarrin AA, Wu GE, Berinstein NL. Functional analy-
sis of the human RAG 2 promoter. Mol Immunol 2000;37:391–
 Fuller K, Storb U. Identification and characterization of the murine
Rag1 promoter. Mol Immunol 1997;34:939–54.
 Zarrin AA, Fong I, Malkin L, Marsden PA, Berinstein NL.
Cloning and characterization of the human recombination acti-
vating gene 1 (RAG1) and RAG2 promoter regions. J Immunol
 Brown ST, Miranda GA, Galic Z, Hartman IZ, Lyon CJ, Aguilera
RJ. Regulation of the RAG-1 promoter by the NF-Y transcription
factor. J Immunol 1997;158:5071–4.
 Kurioka H, Kishi H, Isshiki H, Tagoh H, Mori K, Kitagawa T, et
al. Isolation and characterization of a TATA-less promoter for the
human RAG-1 gene. Mol Immunol 1996;33:1059–66.
 Monroe RJ, Seidl KJ, Gaertner F, Han S, Chen F, Sekiguchi J,
et al. RAG2:GFP knockin mice reveal novel aspects of RAG2
expression in primary and peripheral lymphoid tissues. Immunity
 Yannoutsos N, Barreto V, Misulovin Z, Gazumyan A, Yu W, Rajew-
sky N, et al. A cis element in the recombination activating gene
locus regulates gene expression by counteracting a distant silencer.
Nat Immunol 2004;5:443–50.
 Hsu LY, Lauring J, Liang HE, Greenbaum S, Cado D, Zhuang Y,
et al. A conserved transcriptional enhancer regulates RAG gene
expression in developing B cells. Immunity 2003;19:105–17.
 Mombaerts P, Iacomini J, Johnson RS, Herrup K, Tonegawa S,
Papaioannou VE. RAG-1-deficient mice have no mature B and T
lymphocytes. Cell 1992;68:869–77.
 Shinkai Y, Rathbun G, Lam KP, Oltz EM, Stewart V, Mendelsohn
M, et al. RAG-2-deficient mice lack mature lymphocytes owing
to inability to initiate V(D)J rearrangement. Cell 1992;68:855–
 Li Z, Dordai DI, Lee J, Desiderio S. A conserved degradation signal
regulates RAG-2 accumulation during cell division and links V(D)J
recombination to the cell cycle. Immunity 1996;5:575–89.
 Lee J, Desiderio S. Cyclin A/CDK2 regulates V(D)J recombination
by coordinating RAG-2 accumulation and DNA repair. Immunity
 Grawunder U, Schatz DG, Leu TM, Rolink A, Melchers F. The
half-life of RAG-1 protein in precursor B cells is increased in the
absence of RAG-2 expression. J Exp Med 1996;183:1731–7.
 Riley RL, Kruger MG, Elia J. B cell precursors are decreased in
senescent BALB/c mice, but retain normal mitotic activity in vivo
and in vitro. Clin Immunol Immunopathol 1991;59:301–13.
 Miller JP, Allman D. The decline in B lymphopoiesis in aged
mice reflects loss of very early B-lineage precursors. J Immunol
 Sherwood EM, Blomberg BB, Xu W, Warner CA, Riley RL.
Senescent BALB/c mice exhibit decreased expression of lambda5
surrogate light chains and reduced development within the pre-B
cell compartment. J Immunol 1998;161:4472–5.
 Stephan RP, Sanders VM, Witte PL. Stage-specific alterations in
murine B lymphopoiesis with age. Int Immunol 1996;8:509–18.
 Weksler ME, Szabo P. The effect of age on the B-cell repertoire.
J Clin Immunol 2000;20:240–9.
 Ben-Yehuda A, Szabo P, Dyall R, Weksler ME. Bone mar-
row declines as a site of B-cell precursor differentiation with
age: relationship to thymus involution. Proc Natl Acad Sci USA
 van der Put E, Sherwood EM, Blomberg BB, Riley RL. Aged mice
exhibit distinct B cell precursor phenotypes differing in activation,
proliferation and apoptosis. Exp Gerontol 2003;38:1137–47.
 Weksler ME, Goodhardt M, Szabo P. The effect of age on
B cell development and humoral immunity. Springer Semin
 Stephan RP, Lill-Elghanian DA, Witte PL. Development of B cells
in aged mice: decline in the ability of pro-B cells to respond to
IL-7 but not to other growth factors. J Immunol 1997;158:1598–
 Stephan RP, Reilly CR, Witte PL. Impaired ability of bone mar-
row stromal cells to support B-lymphopoiesis with age. Blood
 Labrie III JE, Sah AP, Allman DM, Cancro MP, Gerstein RM.
Bone marrow microenvironmental changes underlie reduced RAG-
mediated recombination and B cell generation in aged mice. J Exp
 Peault B. In-vitro models of stroma-dependent lymphopoiesis.
Semin Immunol 1995;7:169–75.
J.E. Labrie III et al. / Seminars in Immunology 17 (2005) 347–355
 Dorshkind K, Narayanan R, Landreth KS. Regulatory cells and
cytokines involved in primary B lymphocyte production. Adv Exp
Med Biol 1992;323:119–23.
 Baird AM, Gerstein RM, Berg LJ. The role of cytokine recep-
tor signaling in lymphocyte development. Curr Opin Immunol
 Szabo P, Zhao K, Kirman I, Le Maoult J, Dyall R, Cruik-
shank W, et al. Maturation of B cell precursors is impaired in
thymic-deprived nude and old mice. J Immunol 1998;161:2248–
 Borghesi L, Hsu L-Y, Miller JP, Anderson M, Herzenberg LA,
Schlissel MS, et al. B lineage-specific regulation of V(D)J recom-
binase activity is established in common lymphoid progenitors. J
Exp Med 2004;199(4):491–502.
 Anderson MT, Baumgarth N, Haugland RP, Gerstein RM, Tjioe
T, Herzenberg LA. Pairs of violet-light-excited fluorochromes for
flow cytometric analysis. Cytometry 1998;33:435–44.
 Anderson MT, Tjioe IM, Lorincz MC, Parks DR, Herzenberg LA,
Nolan GP. Simultaneous fluorescence-activated cell sorter analysis
of two distinct transcriptional elements within a single cell using
engineered green fluorescent proteins. Proc Natl Acad Sci USA
 Borghesi L, Gerstein RM. Developmental separation of V(D)J
recombinase expression and initiation of IgH recombination in B
lineage progenitors in vivo. J Exp Med 2004;199:483–9.
 Dudley DD, Sekiguchi J, Zhu C, Sadofsky MJ, Whitlow S,
DeVido J, et al. Impaired V(D)J recombination and lympho-
cyte development in core RAG1-expressing mice. J Exp Med
 Akamatsu Y, Monroe R, Dudley DD, Elkin SK, Gartner F,
Talukder SR, et al. Deletion of the RAG2 C terminus leads to
impaired lymphoid development in mice. Proc Natl Acad Sci USA
 Liang HE, Hsu LY, Cado D, Cowell LG, Kelsoe G, Schlissel MS.
The “dispensable” portion of RAG2 is necessary for efficient V-
to-DJ rearrangement during B and T cell development. Immunity
 von Freeden-Jeffry U, Vieira P, Lucian LA, McNeil T, Burdach
SE, Murray R. Lymphopenia in interleukin (IL)-7 gene-deleted
mice identifies IL-7 as a nonredundant cytokine. J Exp Med
 Goodwin RG, Friend D, Ziegler SF, Jerzy R, Falk BA, Gimpel S,
et al. Cloning of the human and murine interleukin-7 receptors:
demonstration of a soluble form and homology to a new receptor
superfamily. Cell 1990;60:941–51.
 Dias S, Silva Jr H, Cumano A, Vieira P. Interleukin-7 is necessary
to maintain the B cell potential in common lymphoid progenitors.
J Exp Med 2005;201:971–9.
 Borghesi LA, Yamashita Y, Kincade PW. Heparan sulfate proteo-
glycans mediate interleukin-7-dependent B lymphopoiesis. Blood
 Roberts R, Gallagher J, Spooncer E, Allen TD, Bloomfield F, Dex-
ter TM. Heparan sulphate bound growth factors: a mechanism for
stromal cell mediated haemopoiesis. Nature 1988;332:376–8.
 Gordon MY, Riley GP, Watt SM, Greaves MF. Compartmen-
talization of a haematopoietic growth factor (GM-CSF) by gly-
cosaminoglycans in the bone marrow microenvironment. Nature
 Schebesta M, Pfeffer PL, Busslinger M. Control of pre-BCR sig-
naling by Pax5-dependent activation of the BLNK gene. Immunity
 Liang H, Chen Q, Coles AH, Anderson SJ, Pihan G, Bradley
A, et al. Wnt5a inhibits B cell proliferation and functions as a
tumor suppressor in hematopoietic tissue. Cancer Cell 2003;4:349–
 Tsuboi I, Morimoto K, Hirabayashi Y, Li GX, Aizawa S, Mori
KJ, et al. Senescent B lymphopoiesis is balanced in suppressive
homeostasis: decrease in interleukin-7 and transforming growth
factor-beta levels in stromal cells of senescence-accelerated mice.
Exp Biol Med (Maywood) 2004;229:494–502.
 Updyke LW, Cocke KS, Wierda D. Age-related changes in
production of interleukin-7 (IL-7) by murine long-term bone
marrow cultures (LTBMC). Mech Ageing Dev 1993;69:109–
 Tokoyoda K, Egawa T, Sugiyama T, Choi BI, Nagasawa T. Cellu-
lar niches controlling B lymphocyte behavior within bone marrow
during development. Immunity 2004;20:707–18.
 Seeman E. Invited review: pathogenesis of osteoporosis. J Appl
 Seeman E. Reduced bone formation and increased bone resorption:
rational targets for the treatment of osteoporosis. Osteoporos Int
 Kassem M. Mesenchymal stem cells: biological characteristics and
potential clinical applications. Cloning Stem Cells 2004;6:369–74.
 Edwards RG. Stem cells today: B1. Bone marrow stem cells.
Reprod Biomed Online 2004;9:541–83.
 Grove JE, Bruscia E, Krause DS. Plasticity of bone marrow-derived
stem cells. Stem Cells 2004;22:487–500.
 Ahdjoudj S, Fromigue O, Marie PJ. Plasticity and regulation of
human bone marrow stromal osteoprogenitor cells: potential impli-
cation in the treatment of age-related bone loss. Histol Histopathol
 Moerman EJ, Teng K, Lipschitz DA, Lecka-Czernik B. Aging
activates adipogenic and suppresses osteogenic programs in mes-
enchymal marrow stroma/stem cells: the role of PPAR-gamma2
transcription factor and TGF-beta/BMP signaling pathways. Aging
 Kennell JA, Macdougald OA. Wnt signaling inhibits adipogene-
sis through beta-catenin dependent and independent mechanisms. J
Biol Chem 2005 [Epub ahead of print].
 Bennett CN, Ross SE, Longo KA, Bajnok L, Hemati N, Johnson
KW, et al. Regulation of Wnt signaling during adipogenesis. J Biol
 Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson
RL, et al. Inhibition of adipogenesis by Wnt signaling. Science
 Reya T, O’Riordan M, Okamura R, Devaney E, Willert K, Nusse R,
et al. Wnt signaling regulates B lymphocyte proliferation through
a LEF-1 dependent mechanism. Immunity 2000;13:15–24.
 Stenderup K, Justesen J, Clausen C, Kassem M. Aging is associated
with decreased maximal life span and accelerated senescence of
bone marrow stromal cells. Bone 2003;33:919–26.
 Kortesidis A, Zannettino A, Isenmann S, Shi S, Lapidot T, Gron-
thos S. Stromal derived factor-1 promotes the growth, survival and
development of human bone marrow stromal stem cells. Blood
 Egawa T, Kawabata K, Kawamoto H, Amada K, Okamoto
R, Fujii N, et al. The earliest stages of B cell development
require a chemokine stromal cell-derived factor/pre-B cell growth-
stimulating factor. Immunity 2001;15:323–34.
 Nagasawa T, Kikutani H, Kishimoto T. Molecular cloning and
structure of a pre-B-cell growth-stimulating factor. Proc Natl Acad
Sci USA 1994;91:2305–9.
 Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4
is required for the retention of B lineage and granulocytic pre-
cursors within the bone marrow microenvironment. Immunity
 Bain G, Maandag EC, Izon DJ, Amsen D, Kruisbeek AM, Wein-
traub BC, et al. E2A proteins are required for proper B cell
development and initiation of immunoglobulin gene rearrangements
[see comments]. Cell 1994;79:885–92.
 DeKoter RP, Lee HJ, Singh H. PU.1 regulates expression of
the interleukin-7 receptor in lymphoid progenitors. Immunity
J.E. Labrie III et al. / Seminars in Immunology 17 (2005) 347–355 Download full-text
 Lin H, Grosschedl R. Failure of B-cell differentiation in mice lack-
ing the transcription factor EBF. Nature 1995;376:263–7.
 Nichogiannopoulou A, Trevisan M, Neben S, Friedrich C, Geor-
gopoulos K. Defects in hemopoietic stem cell activity in Ikaros
mutant mice. J Exp Med 1999;190:1201–14.
 Nutt SL, Heavey B, Rolink AG, Busslinger M. Commitment to
the B-lymphoid lineage depends on the transcription factor Pax5.
 Singh H, Medina KL, Pongubala JM. Contingent gene regulatory
networks and B cell fate specification. Proc Natl Acad Sci USA
 Medina KL, Pongubala JM, Reddy KL, Lancki DW, Dekoter R,
Kieslinger M, et al. Assembling a gene regulatory network for
specification of the B cell fate. Dev Cell 2004;7:607–17.
 Seet CS, Brumbaugh RL, Kee BL. Early B cell factor promotes
B lymphopoiesis with reduced interleukin 7 responsiveness in the
absence of E2A. J Exp Med 2004;199:1689–700.
 Bain G, Robanus Maandag EC, te Riele HP, Feeney AJ, Sheehy
A, Schlissel M, et al. Both E12 and E47 allow commitment to the
B cell lineage. Immunity 1997;6:145–54.
 Igarashi H, Gregory SC, Yokota T, Sakaguchi N, Kincade PW.
Transcription from the RAG1 locus marks the earliest lymphocyte
progenitors in bone marrow. Immunity 2002;17:117–30.
 Zhuang Y, Jackson A, Pan L, Shen K, Dai M. Regulation of
E2A gene expression in B-lymphocyte development. Mol Immunol
 Kee BL, Murre C. Induction of early B cell factor (EBF) and
multiple B lineage genes by the basic helix–loop–helix transcription
factor E12. J Exp Med 1998;188:699–713.
 Ikawa T, Kawamoto H, Wright LY, Murre C. Long-term cul-
tured E2A-deficient hematopoietic progenitor cells are pluripotent.
 Zhuang Y, Soriano P, Weintraub H. The helix–loop–helix gene E2A
is required for B cell formation. Cell 1994;79:875–84.
 Borghesi L, Gerstein RM. E47 is required for recombinase activity
in common lymphoid progenitors (submitted for publication).
 Quong MW, Martensson A, Langerak AW, Rivera RR, Nemazee D,
Murre C. Receptor editing and marginal zone B cell development
are regulated by the helix–loop–helix protein, E2A. J Exp Med
 Frasca D, Nguyen D, Riley RL, Blomberg BB. Decreased E12
and/or E47 transcription factor activity in the bone marrow as well
as in the spleen of aged mice. J Immunol 2003;170:719–26.
 Frasca D, van der Put E, Riley RL, Blomberg BB. Age-related
differences in the E2A-encoded transcription factor E47 in bone
marrow-derived B cell precursors and in splenic B cells. Exp
 van der Put E, Frasca D, King AM, Blomberg BB, Riley RL.
Decreased E47 in senescent B cell precursors is stage specific
and regulated posttranslationally by protein turnover. J Immunol
 Sherwood EM, Xu W, King AM, Blomberg BB, Riley RL. The
reduced expression of surrogate light chains in B cell precursors
from senescent BALB/c mice is associated with decreased E2A
proteins. Mech Ageing Dev 2000;118:45–59.
 Riley RL, Knowles J, King AM. Levels of E2A protein expression
in B cell precursors are stage-dependent and inhibited by stem cell
factor (c-kit ligand). Exp Hematol 2002;30:1412–8.