Multiple Levels of Selection Responsive to Immunoglobulin
Light Chain and Heavy Chain Structures Impede the
Development of D?-Expressing B Cells1
F. Betul Guloglu,2Brendan P. Smith, and Christopher A. J. Roman3
The truncated/VH-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 ?1 L
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.
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)4complex 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
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-
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 VH
region (Ref. 7; reviewed in Ref. 8). D? can be synthesized in the
mouse before VH-to-DJHjoining when DHand JHare joined using
reading frame 2 (RF2) of DH. If D? were innocuous, more than
half of all B cells should carry at least one such rearrangement;
however, DH-JHand VH-DJHrearrangements using DHRF2 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 VH-to-DJHrearrangement), 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 VHpartner 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.
1This 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.
2Current address: Department of Molecular Microbiology and Immunology, Univer-
sity of Missouri, Columbia, MO 65212.
3Address 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
4Abbreviations 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
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 VH-to-DJHrecombination is profoundly im-
paired, productive ?1 and DH-to-JHrearrangements 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 VH
region with which a VLcould 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
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 His87in the V
domain of MOPC21? with Tyr by PCR mutagenesis. The VLof 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?5M 2-ME, and recombinant IL-7 (100 U/ml ? 5
ng/ml; Cell Sciences). Cells were seeded at a density of 0.5–2 ? 106
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
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
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 ? 106bone 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
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 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
4099 The Journal of Immunology
anti-mouse ? and ?1 from SouthernBiotech. All AP- and HRP-conjugated
secondary Abs were from Jackson ImmunoResearch Laboratories and
? L chains must be secretion competent to form surface
receptors with D?
Our previous studies showed that a secretion-competent ?1 L
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 VHregion, these results suggested that the
particular V? and the VpreB and ?5UR components of the SLC
required a complementary VHdomain 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 (VpreB?UR-
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 VLof ?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
F1:VpreB?UR-JC?5, F2:V?1-JC?5, and F3:VJ?1-C?5 L
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?5 or F1:
VpreB?UR-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
VpreB?UR-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:VpreB?UR-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:VpreB?UR-
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
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 VLdomain, 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 F2NS) and F4 (secreted; F4SEC) ? class L
chains because only a single amino acid differs between them.
Unlike HEK293 cells, the ?-F2NSreceptors were expressed at
?10-fold lower levels on the surface than were ?-F4SECand ?-?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
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.
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
4100SIGNALING IMPAIRMENTS OF THE D? H CHAIN
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?-F4SEC
and D?-?1 receptors were detected on the surface (Fig. 3B; ?1
stains), and no surface D?-F2NScomplexes 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
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, VL-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 ?
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
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, 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 VpreB?C-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).
BCR formation and L
4101The Journal of Immunology
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?, as was
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
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 ?-F4SECreceptors were the
most active, displaying similar levels of activity for each H chain,
whereas ?-F2NSreceptors were less active (Fig. 4A–C). This cor-
responded to the lower levels of mature and surface-expressed
?-F2NScomplexes 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?-F4SECcomplexes were less active than D?-?1, and differences
between ? and D? complexes in activating CD2 and CD22 ex-
pression were more pronounced with F4SECthan with ?1 (Fig.
4A–C). These differences paralleled the lower surface and matu-
ration levels of D? with F4SECcompared with ?1 (Fig. 3A–C).
Cells expressing D?-F2NSdid 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
4102 SIGNALING IMPAIRMENTS OF THE D? H CHAIN
receptor activity. Specifically, ?-?1 and ?-?2 complexes were ex-
pressed at similar or greater levels on the surface than was ?-F2NS
(Fig. 3A, IgM stains). They induced CD2 and CD22 expression as
well as ?-F2NS, but they were less active than ?-F2NSfor prolif-
eration (Fig. 4). Additionally, whereas the mouse ? H chain
showed about the same activity in association with ?1 and ?2 (Fig.
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.
?1 L chain’s autonomous folding ability correlates with an increase of surface BCR levels in association with full-length and D? H chains,
4103 The Journal of Immunology
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.
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 VLregion, 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.
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).
Activity of BCR re-
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
D?-?1 and TG.SA-?1 complexes are less active than ?-?1
4104 SIGNALING IMPAIRMENTS OF THE D? H CHAIN
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
? JCLsequence 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 ?1 CLregion 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 DHRF2 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.
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.
The authors have no financial conflicts of interest.
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4106SIGNALING IMPAIRMENTS OF THE D? H CHAIN