Multiple Levels of Selection Responsive to Immunoglobulin Light Chain and Heavy Chain Structures Impede the Development of Dμ-Expressing B Cells

The School of Graduate Studies, Program in Molecular and Cellular Biology, State University of New York-Downstate Medical Center at Brooklyn, Brooklyn, NY 11203, USA.
The Journal of Immunology (Impact Factor: 4.92). 10/2008; 181(6):4098-106. DOI: 10.4049/jimmunol.181.6.4098
Source: PubMed
ABSTRACT
The truncated/V(H)-less mouse H chain Dmu forms precursor B cell receptors with the surrogate L chain complex that promotes allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be eliminated at the pre-B cell checkpoint. However, there is evidence that Dmu-lambda1 complexes can be made and are positively selected during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret Dmu's unusual structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that secretion-competent lambda1 L chains could associate with both full-length H chains and Dmu, whereas secretion-incompetent lambda1 L chains could only do so with full-length H chains. In contrast, Dmu could not form receptors with a panel of kappa L chains irrespective of their secretion properties. This was due to an incompatibility of Dmu with the kappa-joining and constant regions. Finally, the Dmu-lambda1 receptor was less active than the full-length mouse mu-lambda1 receptor in promoting growth under conditions of limiting IL-7. Thus, multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with Dmu H chain alleles to the repertoire.

Full-text

Available from: Betul Guloglu, Mar 18, 2014
Multiple Levels of Selection Responsive to Immunoglobulin
Light Chain and Heavy Chain Structures Impede the
Development of D
-Expressing B Cells
1
F. Betul Guloglu,
2
Brendan P. Smith, and Christopher A. J. Roman
3
The truncated/V
H
-less mouse H chain D
forms precursor B cell receptors with the surrogate L chain complex that promotes
allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be
eliminated at the pre-B cell checkpoint. However, there is evidence that D
-
1 complexes can be made and are positively selected
during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret D
’s unusual
structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that
secretion-competent
1 L chains could associate with both full-length H chains and D
, whereas secretion-incompetent
1L
chains could only do so with full-length H chains. In contrast, D
could not form receptors with a panel of
L chains irrespective
of their secretion properties. This was due to an incompatibility of D
with the
-joining and constant regions. Finally, the D
-
1
receptor was less active than the full-length mouse
-
1 receptor in promoting growth under conditions of limiting IL-7. Thus,
multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with D
H chain alleles to the repertoire. The Journal of Immunology, 2008, 181: 4098 4106.
B
cell development depends on the expression of structur-
ally sound Ig H chain and L chain proteins (reviewed in
Ref. 1). H chain and L chain proteins form signal trans-
duction complexes that are required to activate programs of dif-
ferentiation and cell growth. These receptors thereby serve as a
quality control mechanism to establish whether the V(D)J rear-
rangement process, necessary to assemble Ig genes from compo-
nent gene segments, created a functional Ig gene. For example,
before L chain rearrangement, progression from the progenitor
(pro) to precursor (pre) stage of development depends on the abil-
ity of the H chain to form a so called precursor B cell receptor
(preBCR)
4
complex with the surrogate L chain (SLC) components
5 and VpreB and signal transducers Ig
and Ig
(2– 4). B cells
that synthesize no H chains or H chains that fail to form signaling-
competent receptor complexes (either with or without the SLC)
due to intrinsic structural flaws are eliminated because of the ab-
sence of a preBCR signal. In this way, the SLC selects for H chains
with the best likelihood of forming BCRs with L chains that can be
regulated by Ag (5, 6).
Other SLC-dependent mechanisms prevent the emergence of B
cells expressing D
, a truncated mouse H chain that lacks a V
H
region (Ref. 7; reviewed in Ref. 8). D
can be synthesized in the
mouse before V
H
-to-DJ
H
joining when D
H
and J
H
are joined using
reading frame 2 (RF2) of D
H
.IfD
were innocuous, more than
half of all B cells should carry at least one such rearrangement;
however, D
H
-J
H
and V
H
-DJ
H
rearrangements using D
H
RF2 are
vastly underrepresented as early as the pre-B and later mature B
cell stages (9, 10). This is because D
associates with the SLC
complex to form an active but defective preBCR (reviewed in
Ref.11). Studies in vivo with D
-transgenic mice have shown that
D
, like most SLC-dependent full-length H chains, can enact al-
lelic exclusion (suppress V
H
-to-DJ
H
rearrangement), but in con-
trast it signals poorly if at all for survival, proliferation, or differ-
entiation of pro-B cells to small pre-B cells and possibly later
stages (12, 13). Thus, pro-B cells expressing D
could neither
developmentally progress nor continue IgH recombination to re-
place the D
rearrangement. A molecular correlate to the signaling
impairment is that D
-preBCRs fail to be transported out of the
endoplasmic reticulum (ER) to reach post-ER compartments and
the cell surface as efficiently as normal preBCRs with full-length
H chains (14–18). Mutational analysis of the SLC demonstrated
this was in part because VpreB requires a V
H
partner for this to
occur optimally (16).
There is also indication that L chain-dependent counterselective
mechanisms exist at later developmental stages to block the emer-
gence of D
B cells. Hypothetically, L chain partners that could
accommodate D
’s unusual structure and form receptors with it
might be able to allow D
signaling and promote the emergence of
B cells with D
alleles. However, there is still a bias against RF2
in mature B cells of
5-deficient mice (19). To help explain this,
biochemical studies have shown that D
could not associate with
two representative
L chains (15, 17), which was taken to suggest
that D
-
complexes could not be made. If this were categorically
The School of Graduate Studies, Program in Molecular and Cellular Biology, and The
Department of Microbiology and Immunology and the Morse Institute for Molecular
Genetics, State University of New York–Downstate Medical Center at Brooklyn,
Brooklyn, NY 11203
Received for publication November 29, 2007. Accepted for publication July 7, 2008.
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.
1
This work was supported in part by Research Project Grant 00-269-01-LBC from
the American Cancer Society, the New York City Council Speaker’s Fund of the New
York Academy of Medicine (C.A.J.R.), and the State University of New York.
2
Current address: Department of Molecular Microbiology and Immunology, Univer-
sity of Missouri, Columbia, MO 65212.
3
Address correspondence and reprint requests to Dr. Christopher Roman, State Uni-
versity of New York-Downstate Medical Center at Brooklyn, 450 Clarkson Avenue
Box 44, Brooklyn, NY11203. E-mail address: Christopher.Roman@Downstate.edu
4
Abbreviations used in this paper: preBCR, precursor B cell receptor; ER, endoplas-
mic reticulum; HEK, human embryonic kidney; IRES, internal ribosomal entry site;
pre-B, precursor B; pro-B, progenitor B; RF2, reading frame 2; SLC, surrogate light
chain; UR, unique region.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
www.jimmunol.org
Page 1
true, given that
is the favored L chain isotype in mice, this would
be a major barrier for emergence of D
-containing B cells.
However, in vivo there is positive selection of in-frame
1 re-
arrangements in SCID mice, but not in Rag-deficient mice, which
has been attributed to the expression of D
H chains (20). Al-
though in SCID mice V
H
-to-DJ
H
recombination is profoundly im
-
paired, productive
1 and D
H
-to-J
H
rearrangements occur and thus
potentially produce D
-
1 receptors responsible for that selection
(20). In support of this model, we have shown that D
could form
D
-
1 receptors that drove proliferation and differentiation of
pro-B cells in some cases as well as full-length
-
1 complexes
(17). Even though in mice
L chain genes are rearranged later
during the pre-B cell stage and less frequently than
(21), this
level is sufficient to allow positive selection of IgM
1
B cells
in
L chain-deficient mice (22, 23). Nevertheless, mature B cells
expressing exclusively D
-
1 have not been reported in the adult
even when D
expression was enforced via a transgene (24), sug-
gesting that other levels of counterselection sensitive to H chain
structure might be responsible for preventing the appearance of
D
-
1 B cells.
The goals in this study were to determine what structural fea-
tures and properties of
and
L chains would be required for D
receptor formation and to gain further insight as to how D
struc-
ture affects receptor activity. One objective was to establish the
relationship of the ability of a L chain to fold autonomously and be
secreted to pairing with D
. This is because in our studies and in
those of others (15, 16), the representative
1 L chain was secre-
tion competent, whereas the
L chains were not and depended on
association with a full-length H chain for ER release and surface
expression. It was hypothesized that this may be an important pa-
rameter limiting L chain/D
association because D
lacks a V
H
region with which a V
L
could fold and pair. Given that in several
functional assays D
-
1 and
-
1 receptors exhibited similar ac-
tivity (17), another objective was to ask whether other, more strin-
gent conditions might reveal D
-
1 signaling impairments that
may help explain why these receptors are not found in the adult.
Herein, we show that secretion competency of
1 L chains was a
critical determinant controlling assembly of this class of L chains
with D
. In contrast, the J and C regions of
were the intrinsically
prohibitive elements irrespective of
L chain folding status. Stud-
ies in primary cells indicated that D
-containing receptor com-
plexes were on average less active in supporting cell growth under
conditions of limiting IL-7 than wild-type complexes. These re-
sults indicate that conventional L chains are in general structurally
and functionally incompatible with D
. Thus, they serve in syn-
ergy with the SLC to block the emergence of B cells expressing
this H chain first by severely limiting the frequency of D
-BCR
formation and then by restricting the signaling output of any rare
but fully assembled D
-BCR complexes.
Materials and Methods
Plasmids
The creation of cDNAs encoding the mouse
H chain 17.2.25, D
, the
human
H chain TG.SA (T),
1, MOPC
, and JC
have been described
previously (16). Briefly, JC
was made by replacing the leader and V
sequence of a MOPC21
L chain with the leader of
1.
1–
5 fusions were
created by PCR mutagenesis. Sec
was created by replacing His
87
in the V
domain of MOPC21
with Tyr by PCR mutagenesis. The V
L
of MPC11
was cloned from mouse genomic DNA using the published MPC11
se-
quence (25) and fused to the JC
region of MOPC21
by PCR. The
cDNAs were subcloned into either MiG (26), a murine retroviral construct
that contains the gene encoding the marker GFP linked to the cDNA via an
internal ribosomal entry site (IRES), or to MihCD4, a retroviral plasmid
that contains the marker gene encoding hCD4 similarly linked via an
IRES. MihCD4 was created by replacing IRES-GFP in MiG with the
IRES-hCD4 sequence from pMACS 4.1 (Miltenyi Biotec).
Cells and in vitro cell culture
Human embryonic kidney (HEK) epithelial 293 cells were grown in
DMEM supplemented with 10% FBS (Invitrogen), 1% penicillin-strepto-
mycin, and L -glutamine (PSG). Short-term primary IL-7-dependent pro-B
cell cultures were established by harvesting and plating total bone marrow
of 4 6-wk-old Rag1
/
5
/
mice (27, 28) in RPMI 1640 (Invitrogen)
supplemented with 10% FBS, antibiotics (1% penicillin-streptomycin,
L
-glutamine), 5 10
5
M 2-ME, and recombinant IL-7 (100 U/ml 5
ng/ml; Cell Sciences). Cells were seeded at a density of 0.5–2 10
6
cell/ml and maintained in culture for 2 days before retroviral infections (29,
30). The Rag1
/
5
/
v-abl-transformed cells have been described (17).
Mouse usage was reviewed and approved by the State University of New
York–Downstate Medical Center Institutional Animal Care and Use
Committee.
Transient transfections
HEK293 cells were cotransfected by calcium phosphate-mediated precip-
itation of MiG-L chain (4
g), pEBB-H chain (4
g), pEBB-Ig
(2
g),
and pEBB-Ig
(2
g) plasmids as described (16). Empty pEBB vector was
used to normalize the total amount of DNA introduced to cells. Two days
later, cells were harvested by incubation with PBS supplemented with
10 mM EDTA, followed by pipetting into single-cell suspensions. Ali-
quots were prepared for flow cytometry and Western blot analysis as
described (16).
Retroviral infections
Retroviruses were produced by calcium phosphate-mediated cotransfection
of HEK293 cells with retroviral plasmids plus p
ECO, which encodes
ecotropic helper functions (31). Primary cells were spin-infected with re-
covered supernatants as described (17). Briefly, viral supernatants and po-
lybrene (4 8
g/ml) were added to 0.5–2 10
6
bone marrow cells per
well in 12- or 24-well plates, followed by centrifugation at 2500 rpm at
25°C for 1.5 h. Supernatants were then replaced with fresh medium sup-
plemented with 100 U/ml IL-7 after infection. For double infections, the
spin infection was repeated twice with 1 day between infections. Double
infections of primary IL-7-dependent pro-B cells were done by first infect-
ing cells with the L chain-MiG viruses, then splitting the infected cells into
separate wells for infection with H chain viruses that did not contain a
marker gene. Consequently, relative amounts of H chain expression be-
tween infected cultures were determined by Western blot of infected cells.
Cells were analyzed 2– 4 days after infection by flow cytometry and West-
ern blot. Infections of v-abl-transformed cells were done as described (17).
Flow cytometry to track surface marker expression and cell
growth
Single-cell suspensions were stained with the following Abs (directed
against mouse Ags except where noted) for flow cytometry by standard
protocols: anti-CD19-TRI and anti-mouse IgM-PE from Caltag Laborato-
ries; anti-
5-biotin (LM34), anti-CD2-PE, anti-CD22-PE, and streptavi-
din-PE from BD Pharmingen; and anti-human IgM-PE and anti-
1-PE
from SouthernBiotech. Analyses of CD2, CD22, and proliferation were
performed as described (17, 18). Briefly, the values shown for CD2 and
CD22 induction are the percentages of CD19
GFP
cells that were also
CD2
and CD22
4 days and 2 days after infection, respectively. Relative
growth was defined as the fold change in the percentge of CD19
GFP
cells in cultures after 24 or 48 h divided by the fold change in the per-
centage of CD19
GFP
cells in the same culture over the same time pe
-
riod. The fold change in CD19
GFP
cells in each sample was defined as
1 (no change in relative growth rate) in the bar graphs.
Comparison of the relative abilities of preBCRs/BCRs to
support growth over a concentration gradient of IL-7
Two days after infection, each sample was equally divided into six separate
cultures, and each was subcultured in different concentrations of IL-7 in
10-fold dilutions from 100 U/ml (5 ng/ml) to 0.01 U/ml (0.5 pg/ml). After
4 days, the growth of CD19
GFP
cells relative to the growth of
CD19
GFP
cells in each culture was calculated, as above. The numbers
plotted in the bar graph in Fig. 5 were calculated by dividing the relative
fold change in CD19
GFP
cells at 100 or 0.1 U/ml IL-7 to the relative
fold change in CD19
GFP
cells in the 100 U/ml IL-7 cultures.
Western blotting
Western blots were performed as described (17). The following Abs were
used for Western blot: rabbit and goat anti-mouse IgM,
H chain-specific,
hamster
globulin from Jackson ImmunoResearch Laboratories, and goat
4099The Journal of Immunology
Page 2
anti-mouse
and
1 from SouthernBiotech. All AP- and HRP-conjugated
secondary Abs were from Jackson ImmunoResearch Laboratories and
Caltag Laboratories.
Results
L chains must be secretion competent to form surface
receptors with D
Our previous studies showed that a secretion-competent
1L
chain could associate and form surface-expressed, signaling-com-
petent receptor complexes with D
, whereas a secretion-incom-
petent
L chain did not associate with D
(16, 17). Similarly,
JC
5, a truncated
5 molecule that lacks the UR and is secretion
competent, and JC
1, a secretion-competent and truncated
1
chain, could associate with D
and support D
surface expression
and, when tested, signaling (Refs. 16, 17 and data not shown).
Given that D
lacks a V
H
region, these results suggested that the
particular V
and the VpreB and
5UR components of the SLC
required a complementary V
H
domain from the H chain partner to
fold properly and thereby to allow efficient release of the resultant
receptor complexes from the ER. We therefore determined
whether secretion competence was a key and general property of L
chains indicative of their ability to form receptors with D
or if
there were other structural features of
L chains not related to
secretory status that were determining factors.
The sequences and properties of the
class of L chains (which
includes the SLC) that controlled receptor formation with D
were
determined by testing the ability of a panel of chimeric
L chain-
SLC fusion proteins constructed from VpreB,
5, and
1 chains to
form receptors with D
and
H chains. Fusion F1 (VpreBUR-
JC
5) was a single-chain version of the SLC that lacked the unique
regions (URs) of both VpreB and
5, also permitting the evalua-
tion of the URs to receptor formation (Fig. 1). Fusions F2, F3, and
F4 contained the JC or C regions of
5 linked to the V
L
of
1
rather than to VpreB (Fig. 1). The ability of these fusion proteins
to form receptors with D
or
H chains was first tested in a
nonlymphoid human embryonic kidney (HEK293) cell line. Ig
-
and Ig
-expression plasmids were cotransfected with those carry-
ing the L chain and/or H chain genes to complete the receptor
components (16).
F1:VpreBUR-JC
5, F2:V
1-JC
5, and F3:VJ
1-C
5L
chains were all comparable to
1 in escorting
to the cell surface
of transfected cells (Fig. 2A, top and bottom sets of panels, and B,
top panel; anti-IgM), indicating that these L chain fusion proteins
were structurally sound and compatible with a normal H chain. On
the other hand, only F3:VJ
1-C
5, but not F2:V
1-JC
5orF1:
VpreBUR-JC
5, could form a surface receptor complex with D
(Fig. 2A, top set of panels, and 2B; anti-IgM). Detection of surface
complexes with
1 and
5 Abs concurred (Fig. 2A, middle and
bottom sets of panels, and 2B, middle and bottom panels). Con-
sistent with the flow cytometry data, Western blot analysis of ex-
tracts from transfected cells showed similar amounts of the mature,
endo-H-resistant form of
in the presence of
1 or the
1–
5 and
VpreBUR-JC
5 fusion proteins (Fig. 2C). Similarly, the major
fraction of D
was endo-H resistant in D
- F3:VJ
1-C
5- and
D
-
1-expressing cells, but dramatically lower to undetectable in
D
-, D
plus F2:V
1-JC
5-, or F1:VpreBUR-JC
5-expressing
cells (Fig. 2C). In parallel with D
receptor formation capability,
F3:VJ
1-C
5 and
1, but not F2:V
1-JC
5 or F1:VpreBUR-
JC
5, were secreted into the medium, even though all of the L
chains were expressed at comparable levels within the cells (F2
and F3, Fig. 2D; F1, data not shown). Therefore, the ability of
these hybrid
L chains to form surface receptors with D
directly
correlated with their secretion competency in the nonlymphoid
cells.
Only three amino acids that differ between F2:V
1-JC
5 and
F3:VJ
1-C
5 could account for their differences in secretion com-
petency, with one lying at the junction of the V-J segments and two
within the different Js (Fig. 1). Indeed, replacement of the F2 Tyr
with Trp was sufficient to convert F2 into a secretion-competent L
chain (F4; Figs. 1 and 2D). This new L chain, referred to as F4:
V
1
-JC
5, behaved comparably to
1 with respect to forming
surface complexes with D
(Fig. 2). Therefore, for the
class of
L chains (
1 and
5), the barrier for D
receptor formation and
surface expression appeared to be imposed by the folding proper-
ties of the V
L
domain, either V
1 or VpreB.
We then asked whether these properties were also evident under
more physiological conditions, namely, in primary, IL-7-depen-
dent pro-B cells, which represent the first stage in B cell develop-
ment in which D
and L chains could hypothetically be coex-
pressed. cDNAs encoding H chain and L chain receptor
components were retrovirally transduced into H chain- and L
chain-deficient Rag1
/
5
/
pro-B cells, which express only the
VpreB component of the SLC (17). We focused on comparing D
and
complexes with secretion-incompetent F2 (nonsecreted;
herein referred to as F2
NS
) and F4 (secreted; F4
SEC
)
class L
chains because only a single amino acid differs between them.
Unlike HEK293 cells, the
-F2
NS
receptors were expressed at
10-fold lower levels on the surface than were
-F4
SEC
and
-
1
receptors (Fig. 3A, IgM; Fig. 3B,
1). Western analysis of infected
cells showed that this was not due to differences in total H chain
expression but rather corresponded to a proportional decrease in
FIGURE 1. Ig L Chains used in this study. Shown on the left is an alignment of the amino acid sequences of the CDR3 region of the naturally occurring
and engineered surrogate and conventional L chain molecules used in this study. The amino acids in boldface type in the V regions (Y and W,
chains;
H and Y,
chains) are those targeted for site-directed mutation that are different among the matched pairs of
(top set)or
L chains (bottom set), as
discussed in the text. Boldface amino acids in the
J regions are naturally occurring but are different between
1 and
5 chains. Adjacent is a schematic
of the natural and engineered L chains (leader (L), V, J, and C regions, not drawn to scale) with the underlined section designating the location of the amino
acid sequences elaborated above.
4100 SIGNALING IMPAIRMENTS OF THE D
H CHAIN
Page 3
the relative amounts of mature, trans-Golgi-modified
H chains
(Fig. 3C), indicating this was at the level of ER export. These
differences between L chains were also evident with the human
H chain TG.SA, which can form BCRs with mouse
and
L
chains that support B cell development in mice (Fig. 3, A and B,
referred to as “T”) (16, 17, 32). As in HEK293 cells, D
-F4
SEC
and D
-
1 receptors were detected on the surface (Fig. 3B;
1
stains), and no surface D
-F2
NS
complexes could be detected (Fig.
3, A and B). This corresponded to the appearance of the trans-
Golgi-modified D
species only in D
-F4- and D
-
1-expressing
cells (Fig. 3, C and D). However, relative surface levels of these
D
receptors were much less than the corresponding mouse and
human
receptors (Fig. 3, A and B), being barely detectable with
the anti-IgM Abs, and despite being expressed comparably to
receptors in HEK293 cells. These results imply that there are dif-
ferences in the folding, assembly, and transport of H chains and L
chains within the secretory pathway of these two cell types; pro-B
cells were more restrictive such that receptor biosynthesis was
more sensitive to L chain-secretion competency and H chain
structure.
Intrinsic incompatibility of
sequences with
D
prohibits D
-
L chain surface receptor formation
Previous studies from our laboratory and those of others have
shown that D
cannot productively associate with two represen-
tative
L chains (15, 16). The full-length
L chains used in those
studies were secretion incompetent, implying that they required a
H chain partner to fold properly. Following the
1 paradigm, it
would have been predicted that on this basis those
L chains
would not form a receptor with D
. However, D
was not able to
form a receptor complex with JC
, the truncated, V
L
-less coun
-
terpart of JC
5 (17). However, it was unclear whether JC
could
not associate with D
because it could not fold and be secreted
like JC
5, or because there were incompatibilities with
sequences.
Therefore, the truncated JC
and a panel of
L chains were
tested for their secretion competency and ability to form surface
D
-
receptors. The secretion-incompetent
1:MOPC21
we
used previously (16) contained a His residue within the V region
that is a Tyr or Phe in most
L chains. Substitution of this His
FIGURE 2. BCR formation and L
chain secretion in nonlymphoid cells.
A, Dot plots of surface IgM,
1, and
5 epitope expression (y-axis) by
HEK293 cells cotransfected with the
indicated H chain and L chain pairs.
Transfected cells express the GFP
protein (x-axis). B, Bar graph repre-
sentation of surface stains plotting
relative mean fluorescence from A.L
chains are indicated below each bar;
D
and mouse
H chains are indi-
cated in each panel. C, Western blot
of D
and
H chains in HEK293
cells cotransfected with the indicated
H chain and L chain constructs. Top
panel, Untreated extract; bottom
panel, extracts treated with endogly-
cosidase Hf (Endo-Hf), which cleaves
only high mannose N-linked polysac-
charides present on immature H
chains in the ER (lower band of dou-
blet); mature H chains contain com-
plex, trans-Golgi-derived N-linked
polysaccharides that are insensitive
and are responsible for its slower mi-
gration (upper band of doublet). D,
Western blot of
and
1 L chains in
the medium and in total cell lysates of
transiently transfected HEK293 cells
(upper panels) or infected v-abl pro-B
cells (lower panel) with the indicated
H chain and
or
1 L chain expres-
sion constructs. The VpreBC-JC
5
fusion protein F1 cannot be detected
with the anti-
1 Ab; however, it was
detected in the total lysate but not in
the medium with rat anti-mouse Ab
against
5 (data not shown).
4101The Journal of Immunology
Page 4
residue in the V region of a nonsecreted VJ
-C
1 fusion protein
into Phe or Tyr converted this fusion L chain from a nonsecreted
into a secreted form (33). Based on this, the His in
1 was con-
verted to Tyr to create
3:sec
. Also tested was the V region from
a secreted
L chain from a MPC11 myeloma cell line (
2:
MPC11
; Fig. 1) (25).
As shown in Fig. 2D,JC
,
2:MPC11
, and
3:sec
, but not
1:MOPC21
, were detected in the medium of transfected
HEK293 and infected v-abl transformed pro-B cells, even though
all were expressed at comparable levels intracellularly. In primary
pro-B cells, these
L chains all formed complexes with the mouse
H chain that were expressed on the surface, with
-
2:sec
and
-
3:MPC11
BCRs at higher surface
levels than
-
1:
MOPC21
or
-JC
(Fig. 3A; the
-JC
complexes contain en-
dogenous VpreB, while the
-
complexes do not; see Ref. 17).
Western blot analysis indicated this directly correlated with dif-
ferences in the relative amounts of mature, endo-H-resistant
pro-
teins (Fig. 3D). The human
H chain TG.SA (T) also could form
surface receptors with the full-length
L chains, although it was
not able to form a surface receptor complex with JC
,aswas
shown previously (Fig. 3A) (17). There was no detectable surface
H chain detected by IgM staining on the surface of cells expressing
D
with any of the
L chains irrespective of their secretion com-
petency. Correspondingly, only the immature form of D
was de-
tected by Western blot in all cases (Fig. 3D). These results support
the idea that
L chains are categorically incapable of productively
associating with D
to form surface receptors.
D
-L chain and
-L chain receptor activity generally but not
absolutely correlates with relative levels of ER export and
surface expression
The signaling competency of
and D
complexes was evaluated
by how well they promoted preBCR or BCR-dependent growth
and differentiation of primary Rag1
/
5
/
pro-B cells. In these
cells, de novo receptor expression via retroviral transduction of
missing receptor components induces CD2 and CD22 expression
and promotes proliferation and survival (17). Among the mouse
and human
-
BCRs, the
-
1 and
-F4
SEC
receptors were the
most active, displaying similar levels of activity for each H chain,
whereas
-F2
NS
receptors were less active (Fig. 4
A–C). This cor-
responded to the lower levels of mature and surface-expressed
-F2
NS
complexes in pro-B cells compared with the other
-
BCRs (Fig. 3A–C). However, whereas
-
1 and D
-
1 complexes
exhibited comparable levels of activity in the proliferation assay,
D
-F4
SEC
complexes were less active than D
-
1, and differences
between
and D
complexes in activating CD2 and CD22 ex-
pression were more pronounced with F4
SEC
than with
1 (Fig.
4A–C). These differences paralleled the lower surface and matu-
ration levels of D
with F4
SEC
compared with
1 (Fig. 3A–C).
Cells expressing D
-F2
NS
did not induce CD2 or CD22 or out
-
grow Rag1
/
5
/
pro-B cells, being indistinguishable from
Rag
/
5
/
pro-B cells infected with GFP-only, H chain, or L
chain viruses alone, and consistent with no ER export or surface
expression of this complex.
Overall,
-
BCR complexes were also active for signaling in a
manner that paralleled relative post-ER H chain maturation and
surface receptor expression levels, with
-
3 complexes being the
most active and on par with
-
1 in all cases (Fig. 4D–F). One
difference between the mouse
and human
H chains was with
JC
, which only productively associated with the mouse H chain.
Similarly, no signaling activity above BCR
controls was detected
in cells coexpressing any of the
L chains in conjunction with D
(Fig. 4D–F), consistent with the observation that none of the
L
chains was able to promote ER export of D
(Fig. 3D).
Interestingly, differences in surface expression and H chain mat-
uration did not always account for some observed differences in
FIGURE 2. (continued)
4102 SIGNALING IMPAIRMENTS OF THE D
H CHAIN
Page 5
receptor activity. Specifically,
-
1 and
-
2 complexes were ex-
pressed at similar or greater levels on the surface than was
-F2
NS
(Fig. 3A, IgM stains). They induced CD2 and CD22 expression as
well as
-F2
NS
, but they were less active than
-F2
NS
for prolif
-
eration (Fig. 4). Additionally, whereas the mouse
H chain
showed about the same activity in association with
1 and
2 (Fig.
FIGURE 3.
1 L chain’s autonomous folding ability correlates with an increase of surface BCR levels in association with full-length and D
H chains,
whereas
L chains can associate only with the full-length H chains in primary pro-B cells. A and B, Flow cytometry analyses of surface BCR expression
by Rag1
/
5
/
pro-B cells expressing the indicated H chain and/or L chain via retroviruses, as detected either with Abs against IgM (A)or
1(B). Shown
are representative panels of dot plots, with GFP expression plotted on the x-axis marking infected cells, and bar graphs plotting the average relative IgM
or
1 surface staining of GFP
cells for each sample set (n 3, with SE bars shown). In A, IgM stain values of human TG.SA (T) are shown in black
in the bar graph to indicate that the human and mouse IgM stains cannot be directly compared with each other because different detecting Abs were required
for each. C and D, Immunoblots of mouse
and D
H chain protein expression in primary Rag1
/
5
/
cells double-infected with the indicated H
chain-expressing retrovirus plus (C) control or the indicated
L chain or (D) control or the indicated
L chain.
4103The Journal of Immunology
Page 6
4D–F), the human
H chain T-
1 receptor was less able to induce
CD2 and CD22 expression than T-
2, even though surface expres-
sion levels of the two complexes were equivalent (Fig. 3A, filled
bars). Therefore, the clonotypic structure of the Ig components
may also influence BCR activity.
D
-
1 is less able to synergize with the IL-7R than the mouse
-
1 at low concentrations of IL-7
The above and previous findings indicated that D
-
1 and the
mouse and human
-
1 complexes exhibited comparable abilities
to support Rag1
/
5
/
B cell growth, which was surprising
considering that mature B cells coexpressing exclusively D
and
1 were not reported in mice when D
was expressed from a
transgene (13, 24). The experiments found in Ref. 17 and in Fig.
4 were performed in the presence of 100 U/ml IL-7, a high con-
centration that supports robust pro-B and pre-B cell growth. At
lower amounts (0.1 U/ml range) there is significant cell loss in both
preBCR
and preBCR
cells, but pre-B cell growth and survival
are more strongly favored due to synergy between the preBCR and
IL-7R signaling pathways (29, 30, 34), conditions thought to better
represent the physiological environment. Under these more strin-
gent conditions, the D
-
1 receptor was less active than the mouse
and more comparable to the human, a profile resembling their
relative activities in inducing CD2 and CD22 expression.
Discussion
D
provides a unique example of how Ig H chain and surrogate
and conventional L chain structure influence receptor signaling
and selection of the adult Ig H chain repertoire. At the pre-B cell
stage, the D
-preBCR signaling impairment appears to primarily
affect expression of maturation markers and proliferation whereas
allelic exclusion is relatively intact. The findings in this study now
suggest that even if D
-preBCRs up-regulate L chain germline
transcription and rearrangement (12, 13), D
appears to be struc-
turally incompatible with any
and secretion-incompetent
L
chains. Moreover, the data also imply that not only is the D
H
chain impaired to utilize a broad spectrum of L chains, but even if
compatible
L chains were made, the resulting D
-
receptors
would be less able to support development and growth than
-
receptors. Thus, multiple mechanisms appear to impede the emer-
gence of D
alleles in the mature B cell repertoire.
Interestingly, the mechanisms restricting D
-BCR formation
were different for the
and
L chains. The restrictive entity in
chains was the V
L
region, which had to maintain secretion com
-
petence to allow receptor formation with D
. In contrast, secretion
competency was irrelevant for the
chains, and the inability to
form D
-
complexes was due to general incompatibility between
and D
. By comparison, secretion competence of full-length
L chains enhanced their ability to form BCRs with both mouse and
human full-length H chains, particularly in primary pro-B cells.
FIGURE 4. Activity of BCR re-
ceptor complexes in primary pro-B
cells. Rag1
/
5
/
pro-B cells were
double-infected with retroviruses ex-
pressing the indicated H chains alone
or plus
(A–C)or
(D–F) L chains,
and infected cells (GFP
) were eval
-
uated after the appropriate culture pe-
riod for CD2 expression (48 h later, A
and D), CD22 expression (24 h later,
B and E), and relative fold increase in
population compared with GFP
cells (48 h later, C and F). The H and
L chains expressed in each sample are
indicated below each bar (n 6 for
all except
3:sec
, for which n 3;
SE bars are shown).
FIGURE 5. D
-
1 and TG.SA-
1 complexes are less active than
-
1
at promoting survival at low IL-7 concentrations. Relative growth of
GFP
CD19
primary Rag1
/
5
/
pro-B cells double-infected with H
chain and/or
1 retroviruses at high (100 U/ml) and low (0.1 U/ml) IL-7
concentrations. Relative fold increase in GFP
cell number in 4 days was
calculated as indicated in Materials and Methods. n 5, SE bars are
shown.
4104 SIGNALING IMPAIRMENTS OF THE D
H CHAIN
Page 7
Moreover, JC
only formed receptors with the mouse but not hu-
man full-length H chain and not with D
, despite being secretion
competent, whereas JC
5 and JC
1 were able to do so with all H
chains (this study and data not shown). It therefore appears that the
JC
L
sequence endows the
1 L chain with the ability to be more
accommodating of H chain structure than
L chains. This is con-
sistent with the greater flexibility of
vs
L chains at “elbows” at
the J-C junctional sequences (35).
L chain usage is a character-
istic frequently associated with edited B cells (36). We speculate
that this property may reflect the imperative of B cells undergoing
editing to self-rescue by L chain replacement with L chains that
have the best chance of pairing with whatever H chain is present
when the
locus is exhausted.
Our studies also support the model that the structure of Ig mol-
ecules can affect the activity of surface receptors, because ob-
served differences in surface expression could not always account
for differences in BCR activity. For example, the ability of
-
1:
MOPC21
and
-
2:MPC11
complexes to promote proliferation
was less than
-F2:V
1JC
5 even though they were all expressed
at similar surface levels (Fig. 3A). Similarly, although the surface
expression profiles of mouse and human
BCR complexes with
different full-length L chains were nearly indistinguishable, the
human BCRs were not always as active as the corresponding
mouse BCRs, with activity frequently more comparable to D
-
BCRs. We speculate that the impaired signaling properties of D
complexes may therefore be due to the combined actions of im-
paired release of D
complexes from the ER and manifestations of
structural defects of surface D
-
complexes. Indeed, this has
been shown for
1 BCRs from the SLJ strain of mice, which con-
tain an amino acid polymorphism in the
1C
L
region that renders
the surface
1 BCR complexes signaling impaired (37).
Although the data support the idea that D
-
1 complexes would
not promote adult mature B cell survival or differentiation as well
as normal H chains, they were not inert nor did they cause deletion
in our tissue culture models. This partial activity may therefore
explain why H chain alleles using D
H
RF2 are underrepresented
rather than completely absent. Why then might exclusively D
-
expressing B cells not be found when D
expression is enforced
(24)? In addition to structural flaws in D
that impair signaling,
1
BCRs in general may be more restricted in their ability to support
B cell homeostasis compared with
, as there is age-dependent loss
of
1-expressing B cells in
1-transgenic mice via selection of
cells that have silenced the transgene and expressed endogenous
L chains (38). In D
-transgenic Rag
B cells, D
and endogenous
H chains were coexpressed, but the D
protein remained in an
immature form, consistent with it not pairing with a compatible L
chain like
1 (13, 24). Nevertheless, if D
-
1 receptors are
formed, we speculate that their signaling properties may only be
compatible with particular B cell populations. One example is fetal
B cell progenitors, in which D
-
1 complexes may be a force for
positive selection (20); another may be marginal zone B cells,
which remained intact in D
-transgenic mice (24). Our tissue cul-
ture system can show whether any given clonotypic preBCR or
BCR forms an active signal transduction complex. However, its
readouts for receptor activity represent only a subset of changes
characteristic of the pro- to pre-B cell transition. The in vitro sys-
tem thus provides an important starting point for comparison to in
vivo systems in which more elaborate and physiological execution
of programs of B cell differentiation, proliferation, survival, allelic
exclusion, and activation of the underlying signal transduction
pathways can be used as parameters to compare the activity of
receptors like D
-
1 and
-
1.
Acknowledgments
We thank all members of the Roman Laboratory for support, and Dr. S.
Gottesman (Pathology, State University of New York-Downstate) and
Dr. L. Eckhardt (Hunter College, City University of New York) for critical
reading of the manuscript.
Disclosures
The authors have no financial conflicts of interest.
References
1. Meffre, E., R. Casellas, and M. C. Nussenzweig. 2000. Antibody regulation of B
cell development. Nat. Immunol. 1: 379 –385.
2. Clark, M. R., A. B. Cooper, L. D. Wang, and I. Aifantis. 2005. The pre-B cell
receptor in B cell development: recent advances, persistent questions and con-
served mechanisms. Curr. Top. Microbiol. Immunol. 290: 87–103.
3. Vettermann, C., K. Herrmann, and H. M. Jack. 2006. Powered by pairing: the
surrogate light chain amplifies immunoglobulin heavy chain signaling and pre-
selects the antibody repertoire. Semin. Immunol. 18: 44 –55.
4. Geier, J. K., and M. S. Schlissel. 2006. Pre-BCR signals and the control of Ig
gene rearrangements. Semin. Immunol. 18: 31–39.
5. Melchers, F. 1999. Fit for life in the immune system? Surrogate L chain tests H
chains that test L chains. Proc. Natl. Acad. Sci. USA 96: 2571–2573.
6. Minegishi, Y., and M. E. Conley. 2001. Negative selection at the pre-BCR check-
point elicited by human mu heavy chains with unusual CDR3 regions. Immunity
14: 631– 641.
7. Reth, M. G., and F. W. Alt. 1984. Novel immunoglobulin heavy chains are
produced from DJH gene segment rearrangements in lymphoid cells. Nature 312:
418 423.
8. Raaphorst, F. M., C. S. Raman, B. T. Nall, and J. M. Teale. 1997. Molecular
mechanisms governing reading frame choice of immunoglobulin diversity genes.
Immunol. Today 18: 37– 43.
9. Gu, H., D. Kitamura, and K. Rajewsky. 1991. DH reading frame bias: evolu-
tionary selection, antigen selection or both? Evolutionary selection. Immunol.
Today 12: 420 421.
10. Haasner, D., A. Rolink, and F. Melchers. 1994. Influence of surrogate L chain on
DHJH-reading frame 2 suppression in mouse precursor B cells. Int. Immunol. 6:
21–30.
11. Hendriks, R. W., and S. Middendorp. 2004. The pre-BCR checkpoint as a cell-
autonomous proliferation switch. Trends Immunol. 25: 249–256.
12. Malynn, B. A., A. C. Shaw, F. Young, V. Stewart, and F. W. Alt. 2002. Truncated
immunoglobulin D
causes incomplete developmental progression of RAG-de-
ficient pro-B cells. Mol. Immunol. 38: 547–556.
13. Tornberg, U. C., I. Bergqvist, M. Haury, and D. Holmberg. 1998. Regulation of
B lymphocyte development by the truncated immunoglobulin heavy chain protein
D
. J. Exp. Med. 187: 703–709.
14. Tsubata, T., R. Tsubata, and M. Reth. 1991. Cell surface expression of the short
immunoglobulin
chain (D
protein) in murine pre-B cells is differently regu-
lated from that of the intact
chain. Eur. J. Immunol. 21: 1359 –1363.
15. Horne, M. C., P. E. Roth, and A. L. DeFranco. 1996. Assembly of the truncated
immunoglobulin heavy chain D
into antigen receptor-like complexes in pre-B
cells but not in B cells. Immunity 4: 145–158.
16. Fang, T., B. P. Smith, and C. A. Roman. 2001. Conventional and surrogate light
chains differentially regulate Ig
and D
heavy chain maturation and surface
expression. J. Immunol 167: 3846 –3857.
17. Guloglu, F. B., E. Bajor, B. P. Smith, and C. A. Roman. 2005. The unique region
of surrogate light chain component
5 is a heavy chain-specific regulator of
precursor B cell receptor signaling. J. Immunol. 175: 358 –366.
18. Guloglu, F. B., and C. A. Roman. 2006. Precursor B cell receptor signaling
activity can be uncoupled from surface expression. J. Immunol. 176: 6862– 6872.
19. Loffert, D., A. Ehlich, W. Muller, and K. Rajewsky. 1996. Surrogate light chain
expression is required to establish immunoglobulin heavy chain allelic exclusion
during early B cell development. Immunity 4: 133–144.
20. Ruetsch, N. R., G. C. Bosma, and M. J. Bosma. 2000. Unexpected rearrangement
and expression of the immunoglobulin
1 locus in scid mice. J. Exp. Med. 191:
1933–1943.
21. Takemori, T., and K. Rajewsky. 1981.
chain expression at different stages of
ontogeny in C57BL/6, BALB/c, and SJL mice. Eur. J. Immunol. 11: 618 625.
22. Zou, Y. R., S. Takeda, and K. Rajewsky. 1993. Gene targeting in the Ig
locus:
efficient generation of
chain-expressing B cells, independent of gene rearrange-
ments in Ig
. EMBO J. 12: 811– 820.
23. Sanchez, P., D. Rueff-Juy, P. Boudinot, S. Hachemi-Rachedi, and
P. A. Cazenave. 1996. The
B cell repertoire of
-deficient mice. Int. Rev.
Immunol. 13: 357–368.
24. Wikstrom, I., I. Bergqvist, D. Holmberg, and J. Forssell. 2006. D
expression
causes enrichment of MZ B cells, but is non permissive for B cell maturation in
Rag2
/
mice even if combined with Bcl-2. Mol. Immunol. 43: 1316 –1324.
25. Smith, G. P. 1978. Sequence of the full-length immunoglobulin
-chain of mouse
myeloma MPC 11. Biochem. J. 171: 337–347.
26. Yang, L., X. F. Qin, D. Baltimore, and L. Van Parijs. 2002. Generation of func-
tional antigen-specific T cells in defined genetic backgrounds by retrovirus-me-
diated expression of TCR cDNAs in hematopoietic precursor cells. Proc. Natl.
Acad. Sci. USA 99: 6204 6209.
27. Spanopoulou, E., C. A. Roman, L. M. Corcoran, M. S. Schlissel, D. P. Silver,
D. Nemazee, M. C. Nussenzweig, S. A. Shinton, R. R. Hardy, and D. Baltimore.
4105The Journal of Immunology
Page 8
1994. Functional immunoglobulin transgenes guide ordered B-cell differentiation
in Rag-1-deficient mice. Genes Dev. 8: 1030–1042.
28. Kitamura, D., A. Kudo, S. Schaal, W. Muller, F. Melchers, and K. Rajewsky.
1992. A critical role of lambda 5 protein in B cell development. Cell 69:
823– 831.
29. Marshall, A. J., H. E. Fleming, G. E. Wu, and C. J. Paige. 1998. Modulation of
the IL-7 dose-response threshold during pro-B cell differentiation is dependent on
pre-B cell receptor expression. J. Immunol. 161: 6038 6045.
30. Fleming, H. E., and C. J. Paige. 2001. Pre-B cell receptor signaling mediates
selective response to IL-7 at the pro-B to pre-B cell transition via an ERK/MAP
kinase-dependent pathway. Immunity 15: 521–531.
31. Pear, W. S., G. P. Nolan, M. L. Scott, and D. Baltimore. 1993. Production of
high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci.
USA 90: 8392– 8396.
32. Nussenzweig, M. C., A. C. Shaw, E. Sinn, D. B. Danner, K. L. Holmes,
H. C. Morse III, and P. Leder. 1987. Allelic exclusion in transgenic mice that
express the membrane form of immunoglobulin mu. Science 236: 816 819.
33. Dul, J. L., O. R. Burrone, and Y. Argon. 1992. A conditional secretory mutant in
an Ig L chain is caused by replacement of tyrosine/phenylalanine 87 with histi-
dine. J. Immunol. 149: 1927–1933.
34. Hess, J., A. Werner, T. Wirth, F. Melchers, H. M. Jack, and T. H. Winkler. 2001.
Induction of pre-B cell proliferation after de novo synthesis of the pre-B cell
receptor. Proc. Natl. Acad. Sci. USA 98: 1745–1750.
35. Stanfield, R. L., A. Zemla, I. A. Wilson, and B. Rupp. 2006. Antibody elbow
angles are influenced by their light chain class. J. Mol. Biol. 357: 1566 –1574.
36. Retter, M. W., and D. Nemazee. 1998. Receptor editing occurs frequently during
normal B cell development. J. Exp. Med. 188: 1231–1238.
37. Sun, T., M. R. Clark, and U. Storb. 2002. A point mutation in the constant region
of Ig
1 prevents normal B cell development due to defective BCR signaling.
Immunity 16: 245–255.
38. Neuberger, M. S., H. M. Caskey, S. Pettersson, G. T. Williams, and M. A. Surani.
1989. Isotype exclusion and transgene down-regulation in immunoglobulin-
transgenic mice. Nature 338: 350 –352.
4106 SIGNALING IMPAIRMENTS OF THE D
H CHAIN
Page 9
  • [Show abstract] [Hide abstract] ABSTRACT: B cell development is a process tightly regulated by the orchestrated signaling of cytokine receptors, the pre-B cell receptor (BCR) and the B cell receptor (BCR). It commences with common lymphoid progenitors (CLP) up-regulating the expression of B cell-related genes and committing to the B cell lineage. Cytokine signaling (IL-7, stem cell factor, FLT3-L) is essential at this stage of development as it suppresses cell death, sustains proliferation and facilitates heavy chain rearrangements. As a result of heavy chain recombination, the pre-BCR is expressed, which then becomes the primary determiner of survival, cell cycle entry and allelic exclusion. In this review, we discuss the mechanisms of B cell lineage commitment and describe the signaling pathways that are initiated by the pre-BCR. Finally, we compare pre-BCR and pre-TCR structure, signal transduction and function, drawing parallels between early pre-B and pre-T cell development.
    No preview · Article · Feb 2005 · Current topics in microbiology and immunology
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: Progenitor B lymphocytes that successfully assemble a heavy chain gene encoding an immunoglobulin capable of pairing with surrogate light chain proteins trigger their own further differentiation by signaling via the pre-BCR complex. The pre-BCR signals several rounds of proliferation and, in this expanded population, directs a complex, B cell-specific set of epigenetic changes resulting in allelic exclusion of the heavy chain locus and activation of the light chain loci for V(D)J recombination.
    Preview · Article · Mar 2006 · Seminars in Immunology
  • [Show abstract] [Hide abstract] ABSTRACT: DHJH rearrangements start in progenitor and precursor B cells and occur in three reading frames (rf). A strong bias for rf I has been noticed in murine and chicken antibodies, while the representation of rf II has been found suppressed both in peripheral as well as in precursor B cells. H chain gene loci DHJH rearranged in rf II are potentially capable of expressing a truncated DHJHC mu protein on the cell surface. Mice incapable of expressing this protein on the surface have previously been shown to have all reading frames represented in near equal frequency, suggesting that membrane-bound DHJHC mu protein is involved in the suppression of rf II. In this paper we show that suppression of rf II is not yet established in c-kit+ CD43+ IL-7/stromal cell-reactive pre-B I cells of fetal liver at day 15 of gestation, but becomes established when such precursor cell populations are expanded in vitro on stromal cells in the presence of IL-7. H chain gene loci using the DQ52 segment for rearrangements (which contains a stop codon in rf II, thus being unable to make DHJHC mu protein) do not show rf II suppression under these conditions. The same type of fetal liver-derived pre B-I cells from lambda 5 deficient mice also do not show rf II suppression after in vitro expansion. Bone marrow-derived pre B-I cells from normal mice assayed ex vivo and expanded in vivo show rf II suppression, while the corresponding pre-B I cells from lambda 5T mice do not. Collectively these experiments suggest that surrogate L chain is involved in rf II suppression. This may happen by inhibition of proliferation of pre-B cells expressing a complex of DHJHC mu protein and surrogate L chain.
    No preview · Article · Feb 1994 · International Immunology
  • [Show abstract] [Hide abstract] ABSTRACT: Early in B-cell development, productive V(D)J recombination leads to synthesis of the membrane Ig heavy (H)-chain protein μ, which associates with the surrogate light (L)-chain proteins λ5 and VpreB to form the pre-B-cell receptor (pre-BCR). Pre-BCR expression serves as a checkpoint that monitors for functional Ig H-chain rearrangement and triggers clonal expansion and developmental progression of Igμ+ pre-B cells. Recent intriguing observations have shed new light on the apparently constitutive ligand-independent signalling capacity of the pre-BCR and the unexpected roles of the downstream signalling molecules SLP-65 and Btk, which limit pre-B-cell proliferation and thereby act as tumour suppressors. Taken together, these observations indicate that the pre-BCR checkpoint functions as a cell-autonomous proliferation switch.
    No preview · Article · Jun 2004 · Trends in Immunology
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: The development of B lymphocytes from progenitor cells is dependent on the expression of a pre-B cell-specific receptor made up by a mu heavy chain associated with the surrogate light chains, immunoglobulin (Ig)alpha, and Igbeta. A variant pre-B cell receptor can be formed in which the mu heavy chain is exchanged for a truncated mu chain denoted Dmu. To investigate the role of this receptor in the development of B cells, we have generated transgenic mice that express the Dmu protein in cells of the B lineage. Analysis of these mice reveal that Dmu expression leads to a partial block in B cell development at the early pre-B cell stage, probably by inhibiting VH to DHJH rearrangement. Furthermore, we provide evidence that Dmu induces VL to JL rearrangements.
    Full-text · Article · Apr 1998 · Journal of Experimental Medicine
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: The production of lambda chain-expressing B cells was studied in mice in which either the gene encoding the constant region of the kappa chain (C kappa) or the intron enhancer in the Ig kappa locus was inactivated by insertion of a neomycin resistance gene. The two mutants have similar phenotypes: in heterozygous mutant mice the fraction of lambda chain-bearing B cells is twice that in the wildtype. Homozygous mutants produce approximately 7 times more lambda-expressing B cells (and about 2.3 times fewer total B cells) in the bone marrow than their normal counterparts, suggesting that B cell progenitors can differentiate into either kappa- or lambda-producing cells and do the latter in the mutants. Whereas gene rearrangements in the Ig kappa locus are blocked in the case of enhancer inactivation, they still occur in that of the C kappa mutant, although in this mutant RS rearrangement is lower than in the wildtype. This indicates that gene rearrangements in the Ig lambda locus can occur in the absence of a putative positive signal resulting from gene rearrangements in Ig kappa, including RS recombination. Complementing these results, we also present data indicating that in normal B cell development kappa chain rearrangement can be preceded by lambda chain rearrangement and that the frequency of kappa/lambda double producers is small and insufficient to explain the massive production of lambda chain-expressing B cells in the mutants.
    Preview · Article · Apr 1993 · The EMBO Journal
  • [Show abstract] [Hide abstract] ABSTRACT: Analysis of the B cell repertoire is complicated by the huge diversity inherent in the germ line determined combinatory. Making use of knockout technology, K-deficient mice have been obtained. They constitute a shrewd model to follow the expression of an Ig minilocus, such as the λ one, in the normal condition compared with classical transgenic models. Indeed, in contrast to wild type mice, in which only 5% of λ B cells are produced, these mutant mice exclusively produce λ positive B cells. Although, the λ locus is well characterized and has a relatively simple organization, the mechanistic and selective pressures that govern its utilization are still poorly understood. The analysis of the λ B cell repertoire in K-deficient mice, should therefore bring more conclusive informations. Here we present the λ subtype distribution in the various cellular compartments of the K-deficient mice, and discuss the rules that can be responsible for this distribution. Our recent data indicate that the λ subtype proportions in the bone marrow and the spleen result, for the major part, from mechanistic processes (i.e., recombinase accessibility, production of V-J functional joint and H/L pairings) while the λ proportions found in the peritoneal cavity ensue from selective processes. Finally, the capacity to respond to various antigens is discussed from such a generated λ B cell repertoire.
    No preview · Article · Jul 2009 · International Reviews Of Immunology
Show more