By
From the Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom
The B cell antigen receptor, composed of membrane immunoglobulin (Ig) sheathed by the
Ig/Ig
heterodimer plays a critical role in mediating B cell development and responses to antigen. The cytoplasmic tails of Ig
and Ig
differ substantially but have been well conserved in
evolution. Transfection experiments have revealed that, while these tails share an esssential tyrosine-based activation motif (ITAM), they perform differently in some but not all assays and
have been proposed to recruit distinct downstream effectors. We have created transgenic
mouse lines expressing chimeric receptors comprising an IgM fused to the cytoplasmic domain
of each of the sheath polypeptides. IgM/
and IgM/
chimeras (but not an IgM/
with mutant ITAM) are each independently sufficient to mediate allelic exclusion, rescue B cell development in gene-targeted Igµ
mice that lack endogenous antigen receptors, as well as signal for B7 upregulation. While the (IgM/
) × (IgM/
) double-transgenic mouse revealed somewhat more efficient allelic exclusion, our data indicate that each of the sheath polypeptides is
sufficient to mediate many of the essential functions of the B cell antigen receptor, even if the
combination gives optimal activity.
The B cell antigen receptor (BCR)1, which is composed
of membrane immunoglobulin sheathed by the Ig Various chimeric receptors have been used to dissect the
relative contributions of the Ig Experiments to ascertain whether there is a division of
function between the Ig DNA Constructs, Transfectants and Transgenic Mice.
Plasmids driving the expression of the hen egg lysozyme (HEL)-specific receptors are based on pSV2gpt and pSV2neo. The
/
Ig
heterodimer (CD79
/CD79
), mediates the response
of the B cell to antigen by initiating transmembrane signaling and driving the internalization of antigen for presentation. The Ig
/Ig
sheath is not only necessary for allowing
the surface transport of membrane IgM but is also critical
for mediating the signaling of the BCR and endocytic activities (1, 2).
and Ig
cytoplasmic domains to BCR function. Because immunoreceptor tyrosine-based activation motifs (ITAMs) (3) present in both
sheath polypeptides are central to their signaling activity
(4), Ig
and Ig
may be functionally redundant. Indeed,
several transfection studies have failed to reveal significant
differences in the signaling activities of Ig
and Ig
(6, 7,
9). However, others have found that whereas chimeras
containing just the Ig
tail could mediate transmembrane
signaling in B cell transfectants, analogous Ig
chimeras
were impaired to variable extents (4, 10, 11, 12.). This, in
addition to the suggestion that the Ig
and Ig
cytoplasmic domains bind different downstream effectors (13) and exhibit differences in antigen-presenting activity (14), is consistent with the two chains fulfilling distinct functions.
and Ig
cytoplasmic domains reveal that in vivo both IgM/
and IgM/
chimeras are able
to induce the pro-B to pre-B transition and mediate allelic
exclusion (8, 15). Here, we extend the transgenic analysis
to the later stages of B cell development, to ask about the
triggering of B cell maturation and activation.
transcription unit, in which the V
segment of the mouse D1.3 mAb (16, 17) is linked to rat C
, was assembled by exchanging the SacI-XhoI
-globin insert of L
-
G (18) for an analogous PCR-generated SacI-XhoI fragment containing D1.3 V
. The heavy (H) chain
vectors derive from pSV-Vµ1 (19) but with the VNP VH segment
replaced by a PCR-generated PstI-BstEII D1.3 VH segment. For
the chimeric µ chains, the Cµ region was replaced by Cµ/
or
Cµ/
Y
L CH regions (20) or Cµ/
CH regions (20a) (Fig. 1 A).
Fig. 1.
Antigen induces IL-2 secretion from the A20 transfectants.
(A) Schematic representation of the transgene constructs and predicted structures of the receptors (R, EcoRI; C, ClaI; P, PstI; Bs, BstEII; K,
KpnI; X, XhoI; S, SacI). (B) Flow cytometric analysis of transfected receptors in A20 by staining for surface IgM; unfilled histograms depict the
unstained controls. (C) Mean levels of IL-2 secreted by A20 transfectants
after incubation with hen egg lysozyme (HEL) for 24 h. Standard errors
were below 0.5 U/ml except where indicated.
[View Larger Version of this Image (29K GIF file)]
chimera, two founders positive for both H and L expression were identified but only the
first founder was used in subsequent studies as the second founder
segregated the H and L transgenes on breeding. With IgM/
Y
L
and IgM/
, only a single positive H+L founder was obtained in each case.
Flow Cytometry. Analysis was performed on Becton Dickinson FACScan® or FACScalibur® using LYSYS II or CELLQuest software. FITC-conjugated and biotinylated goat anti-IgM, and PE-conjugated rat anti-IgD were from Southern Biotechnology (Birmingham, AL); FITC-RA3-6B2 (rat anti-B220[CD45R]), PE-conjugated B3B4 (rat anti-CD23), biotinylated 53-2.1 (rat anti-Thy1.2[CD90]) and purified 7G6 (rat anti-CD21/35[CR2/1]) were from PharMingen (San Diego, CA); PE-conjugated RA36B2 and RED670-streptavidin were from GIBCO BRL (Paisley, UK); FITC-streptavidin was from Amersham (Amersham, UK); and PE-streptavidin was from Jackson ImmunoResearch (West Grove, PA). Cells making the E5.2 monoclonal anti-D1.3 idiotype antibody (22) were a gift from R. Poljak. The flow cytometric analyses illustrated are representative of multiple individual animals aged 2-4 mo old.
B Cell Activation. Production of IL-2 from A20 transfectants (105 cells in 200 µl) cultured for 24 h in medium containing various concentrations of HEL was monitored by providing the culture supernatant to IL-2-dependent HT2 cells (2.5 × 104 cells in 100 µl medium). The viability of the HT2 cells was determined after 24 h as previously described (20) and the assay calibrated with recombinant IL-2 standards.
To monitor proliferative responses, splenocytes that had been depleted of erythrocytes by hypotonic lysis were cultured in triplicate aliquots (2 × 105 in 200 µl) in RPMI, 10% FCS, 50 µM 2-ME in the presence of 1 µg/ml LPS (Sigma, Poole, UK) for 48 h before pulsing with 0.5 µCi [3H]thymidine for 15 h and scintillation counting. To monitor CTLA4 binding, spleen cells depleted of erythrocytes were cultured (106 cells/ml) in medium in the presence or absence of 10 µg/ml F(abBrdU Uptake. Mice were given two intraperitoneal injections 4 h apart of 1 mg BrdU (5-bromo-2-deoxyuridine) (Sigma) in PBS and the drinking water supplemented with 1 mg/ml BrdU for 72 h following the protocol of Torres et al. (23). Spleens were removed and, after staining for CD45R(B220) and either D1.3 idiotype or IgM, the cells were fixed and permeabilized with 70% ethanol and 1% paraformaldehyde, 0.01% Tween-20 in PBS, treated with DNaseI and stained with FITC-conjugated antiBrdU antibody (Becton Dickinson, San Jose, CA) for cytofluorimetric analysis.
To discriminate between the functions of Ig
and Ig
cytoplasmic domains, we constructed a set of plasmids encoding either wild-type or chimeric HEL-specific
receptors. The chimeras are composed of mouse µ and rat
Ig chains directly linked through a hydrophobic transmembrane segment to the cytoplasmic domains of either
Ig
, Ig
, or a mutated Ig
whose ITAM tyrosines are substituted by leucines (IgM/
, IgM/
, or IgM/
Y
L; Fig. 1
A). The transmembrane segment (which derives from the H-2Kb gene) confers sheath-independent surface transport
(24) and the receptors do not show detectable association
with endogenous Ig
or Ig
chains (20).
The plasmids encoding the various receptors were transfected into the A20 B cell lymphoma; the transfectants all
stained for IgM although there were some differences in
the brightness with the IgM/ chimera being the least well
transported to the cell surface (Fig. 1 B). The IgM/
and
IgM/
chimeras, as well as the wild-type IgM receptor,
were able to initiate signaling after antigen binding as
judged by the production of IL-2 from transfectants of the
A20 lymphoma (Fig. 1 C). However, the signaling activity was abolished by mutation of the ITAM tyrosines in the
Ig
cytoplasmic domain.
The transcription
units encoding the various receptors were introduced into
the germline of transgenic mice. Cytofluorimetric analysis
of spleen cells with anti-D1.3 idiotype antibody (Fig. 2 A)
as well as with anti-rat and labeled HEL (data not shown) revealed that they were all expressed on the B cell surface,
although the IgM/
staining was weaker than that of the
other receptors. With the mice bearing the chimeric receptors,
the receptors were also expressed on some CD45R(B220)
cells. These correspond to a subpopulation of T cells (Fig. 2 B), probably reflecting the expression pattern of the IgH
enhancer in transgenic mice (25). (The absence of surface
expression of the wild-type transgenic IgM receptor in this
subpopulation is consistent with the fact that wild-type
IgM, but not the IgM chimeras, requires endogenous Ig
/
Ig
for surface transport).
IgM/
Signals transmitted through membrane Ig are required
for B cell development; µMT mice that carry a targeted
disruption of the µ membrane exon are B cell deficient
(21). To see whether the individual Ig and Ig
cytoplasmic domains of the IgM chimeras were sufficient to signal
for B cell maturation, the various transgenic lines were bred
into a homozygous µMT background. It was immediately evident that the HEL-specific IgM BCR as well as the
IgM/
and IgM/
chimeras all had a significant effect on B
cell development; their presence led to a substantial (around
two log) increase in serum IgG levels as compared with
nontransgenic µMT controls. (Serum IgG is routinely detected in reconstituted µMT mice and is presumably encoded by Ig-transgenic B cells that also carry productive
VHDJH integrations on an endogenous µMT allele that has
switched to a downstream isotype). The reconstitution of
peripheral B cell development was confirmed by cytofluorimetric analysis of spleen cells: the presence of either the
IgM/
or the IgM/
chimera was sufficient to yield
splenic IgM+ B220+ cells with a functional ITAM being
essential for this activity (Fig. 3).
Phenotype of the Transgenic B Cells.
Although these analyses revealed that the IgM chimeras were able to drive the
maturation of a splenic B cell population, we found that
the reconstituted B cells proliferated extremely poorly on
in vitro culture with LPS (Fig. 4 A). Indeed, compared
with normal controls, the transgenic B cells exhibited a
two- to fivefold increase in cell death during a 24-h in vitro
culture. The diminished proliferative response is not unique
to the IgM chimeras: it is also evident in mice transgenic
for the wild-type anti-HEL IgM (Fig. 4 A). An altered
turnover of the transgenic B cells is also apparent in vivo:
BrdU labeling revealed that animals carrying the transgenic
receptors had an increased proportion of rapidly turning
over splenic B cells (Fig. 4 B). With regard to surface markers, splenic B cells from mice transgenic for both the wildtype IgM and the IgM chimeras revealed a shift to lower
expression of the mature B cell markers, CD21/35 and
CD23 (Fig. 4 C) as compared with normal mice, though
CD22 expression was unaffected. Thus, in view of their diminished proliferative response, increased in vivo turnover
and decreased expression of CD21/35 and CD23, it is evident that the splenic B cells whose development is driven by the transgenic anti-HEL receptors do not show the same
distribution of maturational development as splenic B cells in
normal mice. However, this is an effect of all the anti-HEL
transgenes and does not indicate compromised signaling by
either of the individual IgM chimeras.
Upregulation of B7 by the IgM/
To monitor signaling by the IgM chimeras in the transgenic B cells, we sought to measure the proliferative responses to anti-IgM and IL-4. However, as with LPS, the
responses were very poor; therefore, we used upregulation
of the B7 costimulatory molecules as our readout. Splenic
B cells from the transgenic mice were cultured for 24 h
with anti-IgM and upregulation of B7 was judged by staining with a CTLA4-Ig fusion protein (Fig. 5 A). Triggering the receptors with HEL or anti-D1.3 idiotype antibody
gave analogous results but additionally revealed that the
IgM/Y
L receptor was signaling defective (data not shown).
The IgM/
The ability to mediate
the feedback regulation of endogenous Ig gene rearrangement (allelic exclusion) provided another parameter for
comparing the signaling ability of the different receptors. Allelic exclusion in the transgenic mice was readily monitored by determining the proportion of D1.3 idiotype+ B
cells that coexpress IgD and gave similar conclusions to
staining with anti-µ allotype antibodies. It will be seen
(Fig. 5 B) that the exclusion mediated by the wild-type
HEL receptor is significantly greater than that mediated by
the IgM/ and IgM/
chimeras. The IgM/
Y
L receptor
is ineffective in exclusion with all D1.3 idiotype+ B cells
coexpressing an endogenous rearrangement. We crossed the IgM/
and IgM/
chimeras to see whether coexpression of the two receptors yielded more complete exclusion.
The double-transgenic mice exhibited greatly decreased
expression of endogenous Ig gene rearrangements (Fig. 5
B) despite the fact that there was no significant increase in
the abundance of transgenic IgM on the B cell surface (see
Fig. 2 A).
The results show that both the IgM/ and IgM/
chimeras can broadly perform many of the major in vivo functions of the complete BCR. Furthermore, the ability to
drive the maturation and activation of peripheral B cells is
dependent upon the ITAM; this parallels previous findings
on pre-B cell development (8, 15).
Transfection experiments using cell lines have revealed
that both Ig and Ig
are needed for surface transport of
membrane Ig (26, 27). Thus, mice carrying targeted disruptions of Ig
cannot express surface Ig and are B cell deficient (28). In contrast, both the IgM/
and IgM/
chimeras described here allow extensive B cell maturation
because, by virtue of their mutant transmembrane sequences, these chimeras can be transported to the B cell
surface without an attendant Ig
/Ig
sheath. The IgM/
chimera performs slightly better than the IgM/
chimera
in several of the assays but this may simply reflect the more
efficient surface transport of the IgM/
chimera.
Therefore, our results so far do not lend significant support to the idea that Ig and Ig
cytoplasmic domains perform distinct autonomous functions within the context of
the intact BCR. Nevertheless, it is clear that the chimeras are
not as effective as the complete BCR. Thus, the (IgM/
) × (IgM/
) double transgenic mouse is considerably more efficient than its single transgenic parents in effecting allelic
exclusion of endogenous Ig gene rearrangement. This is consistent with cell line transfection experiments indicating cooperativity between the two cytoplasmic domains with
the heterodimer giving a stronger signal than the component homodimers (29). Indeed, the structural conformation
of the heterodimer could differ substantially from that of
the homodimers and this could lead to differences in the
kinetics of phosphorylation or efficacy of effector protein
(e.g., Syk) recruitment, as well as in the sensitivity to antigen binding.
However, although the chimeric HEL-specific receptors
do not perform as well as the wild-type IgM BCR in driving the reconstitution of a splenic B cell compartment in
µMT mice, the difference is relatively small and the impaired B cell maturation is certainly not nearly as dramatic
as that observed by Torres et al. (23) in mice carrying a targeted disruption of the mb-1 gene that leads to the synthesis
of a BCR with a truncated Ig tail. The different performance of the various compromised BCRs in driving pre-B
and B cell development could well be accounted for by a
requirement for differing qualities of signal at the various
maturational checkpoints. It will obviously be interesting
to correlate the differentiative potential of the various mutant BCRs with their biochemical signaling activities.
Address correspondence to Dr. Michael Neuberger, Protein and Nucleic Acid Division, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
Received for publication 31 January 1997 and in revised form 13 March 1997.
1Abbreviations used in this paper: BCR, B cell antigen receptor; BrdU, 5-bromo-2-deoxyuridine; H, heavy; HEL, hen egg lysozyme; ITAM, immunoreceptor tyrosine-based activation motif; L, light.We thank S. Davies and T. Langford for animal handling, G. Williams for advice, V. Aluvihare for cells, A. Riddell for flow cytometry, the Howard Hughes Medical Institute for an international research scholars award, and R. Poljak, K. Rajewsky and P. Lane for provision of materials.
This work was supported by an international research scholars award from the Howard Hughes Medical Institute.
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