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Unité du Développement des Lymphocytes (Centre National de la Recherche Scientifique, URA 1961), Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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L chain
and the TCR 
-chain, IgH allelic exclusion is the result of an
extremely stringent mechanism. We discuss two non-mutually exclusive
scenarios for the origin of IgH dual expressors: 1) IgH allelically
included cells arise when the first allele to rearrange productively is
unable to form a pre-BCR; dual expressors could be a subset of this
population in which, upon conventional L chain rearrangement, both IgH
are expressed at the surface; and 2) synchronous rearrangement of the
IgH alleles. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2) and 
L chain genes,
which, in addition, show isotypic exclusion (3) (as a
general rule, either or is expressed but not both).
Furthermore, the L chain undergoes subtypic exclusion
(4). Altogether, these mechanisms ensure lymphocyte
receptor monospecificity, a requisite for cells to function as the
selective units of the immune system. Several models have been proposed to explain allelic exclusion ever since it was first observed. The mainstream model relies on two key observations: 1) mature B cells carrying two productive VHDJH alleles (VHDJH+/VHDJH+) are rare (5, 6); 2) in mice carrying one wild-type IgH allele and another in which the membrane exon has been mutated by homologous recombination, allelic exclusion is incomplete (7). In other words, both alleles are thought to rearrange until the first to achieve a productive rearrangement expresses the corresponding protein in the membrane, signaling the shutdown of further somatic recombination in the locus.
In the last few years, it has been shown that the assembly of the pre-B
cell receptor (pre-BCR), composed of a µ-chain and of two invariant
chains, 5 and Vpre B (8, 9), is
required to achieve IgH allelic exclusion during early B cell
development (10).
Despite these recent findings, two questions remain unanswered: 1) What are the intermediate steps from the pre-BCR assembly to the loss of somatic recombination at the IgH locus? and 2) What is the extent of phenotypic IgH allelic exclusion? In the present study we focus on the latter question.
H chain phenotypic allelic inclusion has been described in BCR transgenic (11, 12) and gene-targeted animals (13), but it is presently not consensual whether IgH dual expressors (DEs) in normal mice can be detected and, if so, at what frequency. In an attempt to clarify this issue, we evaluated how tight allelic exclusion is and tried, by looking at the eventual exceptions, to gain insights into this mechanism.
We show that IgH DEs are present in normal mice at a frequency in the order of 1 in 104 splenic IgM+ B cells. Single cell (or clonal) analysis of the rearranged loci in DEs showed that we were, indeed, detecting truly allelically included cells at the genetic and phenotypic levels. The analysis of these rearrangements revealed that most cells carry a D-distal VH (notably of the J558 family), whereas the other allele is often a DH-proximal one. Interestingly, whereas the complementarity-determining region (CDR) 3 length in the population of DEs can be very heterogeneous, both IgH alleles for each of these cells tend to have a similar CDR3 length. These results show that phenotypic IgH allelic inclusion is extremely rare. We discuss two non-mutually exclusive hypotheses to explain the origin of IgH DE B cells in normal mice
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 and BALB/c animals were purchased from Iffa-Credo (L’Arbresle, France) and crossed in SPF-barrier-free conditions at the Pasteur Institute animal facilities. BC8 and CB20 congenic strains were crossed with C57BL/6 and BALB/c animals, respectively. Animals used in experiments were between 4 and 12 wk old.
Fluorescence surface staining, cell sorting, and flow cytometry
Abs used for FACS stainings and cell sorting were the following : FITC anti-IgMa (clone DS-1), PE anti-B220, anti-IgMb (clone AF6-78), biotin anti-B220, and streptavidin cychrome tricolor (all from PharMingen, San Diego, CA). For each animal, >107 spleen cells were stained. Cells were isolated in a FACStarPlus cell sorter (Becton Dickinson Immunocytochemistry Systems, Sunnyvale, CA). Dead cells in sorted samples were scored by propidium iodide (PI) and/or trypan blue exclusion. Fluorescence was measured with a FACScan flow cytometer (Becton Dickinson) using the Cell Quest 3.1 software (Becton Dickinson).
Culture conditions
Sorted dual (B220+IgMa+IgMb+) or single (B220+ IgM+) expressors were cultured at 37°C in an atmosphere of 5% CO2 for 4 or 5 days in OptiMEM (Life Technologies, Rockville, MD) medium supplemented with 10% FCS, 25 µg/ml LPS (Difco, Detroit, MI), 50 mM 2-ME, 50 U/ml penicillin-streptomycin in the presence of a feeder layer of 3000 rad irradiated S17 stromal cells. For limiting dilution conditions, cells were plated at 3, 1, 0.3, and 0.1 cells/well. Ninety-six replicates were plated for each cell concentration.
Intracytoplasmic stainings
Cells were cultured for 4 or 5 days and then fixed in 95% ethanol on slides according to standard procedures and incubated with FITC anti-mouse IgMa and biotin anti-mouse IgMb revealed by streptavidin-conjugated Texas Red (Southern Biotechnology Associates, Birmingham, AL). On an Axiophot Zeiss (Oberkochen, Germany) fluorescence microscope, we analyzed 0.05% p-phenylenediamine-buffered glycerol-mounted slides. For the estimation of the DE frequency in LPS cultures 103-2 x 104 splenocytes (per animal) were sorted as B220+IgMa+IgMb+. Background was standardized by staining cells from homozygous mice with both Abs. After culture, a minimum of 350 cells was scored per animal, and the total number of cells analyzed was >15,000. The number of cells analyzed from cultures of splenocytes sorted as B220+ cells was >5,000.
Estimation of the in vivo frequency of IgH DE (%DEin vivo)
The %DEin vivo was calculated based on
the frequency of ex vivo stained splenic cells in the dual DE gate
(%DEsort) and on the frequency of DE in cultures
of LPS-stimulated splenocytes sorted as DE
(DEcult), as estimated by intracytoplasmic
stainings. Formally, %DEin vivo =
DEsort x DEcult x
(fsing + 2 x
fdoub) x (100 - % dead cells in
DEsort) x
(1/DEpopf)/100, where fsing
(fdoub) is the fraction of singlets (doublets) in
the DE-sorted cells and DEpopf is the fraction of
the DE population FACS profile distribution included in the DE gate.
fsing and fdoub were
estimated by analyzing cells on the microscope after sorting; the
theoretical DE population distribution in the FACS profile was
calculated from the distributions of IgMa+ and
IgMb+ single expressors, assuming that both
alleles contribute equally for the membrane IgM density
(13), similar to that of single expressors; dead cells are
estimated by PI staining; since, in all experiments,
(fsing + 2 x
fdoub) x (100 - % dead cells in
DEsort)/100 x
(1/DEpopf) was estimated to be < 2, in
Table I
%DEin vivo
%DEsort x
DEcult.
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Supernatants from limiting dilution cultures were tested for the presence of IgM at day 10 of culture. Briefly, 96-well flat-bottom microtiter plates were coated overnight at 4°C at a concentration of 5 µg/ml with goat anti-total mouse µ-chain (Sigma, St. Louis, MO), anti-IgMa (RS3.1), or anti-IgMb (MB86). After blocking for 2 h with 1% BSA in PBS, wells were incubated with culture supernatants for 3 h at 37°C. Anti-µ HRP-labeled Ab (Jackson ImmunoResearch Immunotech, France) was then added, and plates were incubated for another hour at 37°C. Bound Abs were revealed with 2,2'-azino-bis(ethylbenzthiazoline-6-sulfonic acid (Sigma) in 50 mM phosphate citrate buffer containing 0.03% sodium perborate (Sigma). The absorbency was determined at 405/620 nm with an ELISA plate reader (Dynatech MR5000, Chantilly, VA). Washes between incubations were done with 0.1% Tween 20 in PBS. A well was considered to be positive when the absorbency measured was greater than the mean of the background plus three times the SD of the mean. Background staining for both anti-allotypic Abs was determined by repeating the ELISA reaction with sera from IgMa or IgMb mice.
VHDJH rearrangements by PCR and sequencing
DE double-sorted cells were diluted in 5% glucose and picked individually into Terasaky plates under a magnifying lens with a heat-extended capillar connected to a mouth piece. Alternatively, cells double sorted as DEs were set in limiting dilution conditions as previously described, the resulting clones being picked individually from Terasaky plates. In both cases, (VH)DJH rearrangements were amplified as previously described (6), with minor modifications. After a preliminary study, it was shown that the 5' VH primers that would account for most rearrangements were: VHA, 5'-GCGAAGCTTARGCCTGGGRCTTCAGTGAAG-3'; VHB, 5'-GCGAAGCTTCTCACAGAGCCTGTCCATCAC-3'; VHE, 5'-GCGAAGCTTGTGGAGTCTGGGGGAGGCTTA-3'; and VHF, 5'-GCGAAGCTTWCTGGAGGAGGCTTGGTGCAA-3'.
Only these VH primers were used in this analysis. The DJH-specific primers were: Q52, 5'-ACGTCGACGCGGACGACCACAGTGCAACTG-3'; and DFS, 5'-ACGTCGACTTTTGTSAAGGGATCTACTACTGT-3'. The Dye Terminator Sequencing (Perkin-Elmer, CA) kit was used to determine the sequence of (VH)DJH Gene Clean II Kit (Bio101, Vista, CA) purified PCR fragments, according to the instructions of the manufacturers. Sequences were obtained in a ABI 370A DNA sequencer (Applied Biosystems, Foster City, CA). The efficiency of amplification of a single allele was 0.28 when single cells were used and 0.50–0.75 (depending on the experiment) when clones were used. VH families were determined by BLAST search using partial VH sequences.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To estimate the frequency of DEs in mature B cells, we took
advantage of the IgH allotypic difference between C57BL/6 and BALB/c
mice. As a first attempt to determine this frequency, splenocytes from
C57BL/6 x BALB/c animals (B6CF1) were sorted as
IgMa+IgMb+B220+
(Fig. 1A). When the purity of
the sorted population was evaluated, it became clear that the
proportion of single expressors was still high (Fig. 1B). To
understand the failure in obtaining a pure population of DEs, 1:1
mixtures of BALB/c and C57BL/6 spleen cell populations that lack by
definition IgMa+IgMb+ cells
were analyzed. Fig. 1C shows the results of flow cytometry
analysis in which the frequency in the DE gate is plotted against the
FSC-H. As the upper limit of the forward scatter (FSC-H) increases,
single IgMa/single IgMb
doublets and other aggregates of more than two cells start being
included in the analysis, which could explain the increase in the
putative DE frequency as a function of FSC-H, observed with both B6CF1
and a 1:1 mixture of splenocytes. On the other hand, the increased
frequency of putative DEs in B6CF1 cells as compared with that of the
BALB/c and C57BL/6 cells mixture could be due to: 1) doublets that
would be in higher frequency in the B6CF1 suspension because cells came
from the same tissue; 2) nonspecific binding of serum IgM to the
surface of B lymphocytes; and 3) presence of real DEs.
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), and the results for B6CF1
splenocytes pooled from three independent experiments are shown in
Table I
. The initial percentage of
B220+IgM+ in the DE gate
(%DEsort) was then corrected by the frequency of
DEs in culture (%DEcult) based on the
intracytoplasmic stainings (see Table I and Materials and
Methods). We found that the frequency of phenotypic IgH
allelically included cells in normal mice is in the order of 1 in
104 IgM+ splenic B cells.
Finally, in a single independent experiment, IgH heterozygous mice with
the C57BL/6 (C57BL/6 x BC8) or BALB/c (BALB/c x CB20)
backgrounds were directly compared. We found that the frequency of DEs
is not statistically different in these two strains, showing no
influence of the mouse strain genetic background in this trait.
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It has been suggested that the frequency of an Ag responding cell
with a particular reactivity is in the order of 1 in
104. Thus, it could be argued that, instead of DE
cells, we were detecting IgMa or
IgMb single expressors reacting with a given
epitope on the anti-IgMb or
anti-IgMa Abs, respectively. It follows that
the formal demonstration that the cells we define as DEs are really
phenotypic allelically included relies on the analysis of
VHDJH rearrangements. After
sorting, single expressors still account for more than 90% of cells in
the population sorted as
IgMa+IgMb+, as shown in
Table I. Therefore, to characterize rearrangements at the single cell
level, further enrichment was necessary. Sorted cells were expanded by
LPS stimulation for 3.5 days, stained, and subjected to a second round
of cell sorting. Fig. 3 shows the
correspondent dot plots at each step. The frequency of cells in the DE
gate after sorting and culture was 6.8 ± 0.4 for cells previously
sorted as DEs and 1.3 ± 0.2 for cells sorted as
B220+ (in three independent experiments). The
value of 6.8% is above the one found by intracytoplasmic staining and
is likely to be an overestimation (see below). Nevertheless, it
suggested that, by two consecutive sortings, enrichment for DEs could
be achieved. As seen in Fig. 3B, cells initially sorted as
DEs using a broad gate resolve into three major populations of single
IgMa, IgMb, and DEs. The DE
population has a diagonal shape (representing around 1.5% of the
cells, which is consistent with the data from intracytoplasmic
stainings), indicating that cells with different levels of expression
for both alleles, if they are present after the first sorting,
represent a minority.
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LPS culture does not bias the determination of DE frequency
The estimation of the frequency of DEs was based on the postulate that dual and single expressors respond equally to LPS stimulation. We, therefore, directly assessed the frequency of LPS responders and the clonal size after double sorting of B220+IgM+ and B220+ IgMa+IgMb+ cells. The frequency of DE responders was found to be 1 in 1.5 cells, similar to the one of single expressors (1 in 1.5). This frequency was 30% lower than that of the respective starting populations after one round of sorting, found to be 1 in 1 (14). This is probably due to the fact that, when cells were sorted for the second time, they were less able to proliferate than ex vivo sorted cells, because they had already been in culture for 3.5 days. Clone size of LPS-stimulated cells from both double sorted populations was analyzed by seeding one cell per well in Terasaky plates and scoring numbers of cells from each clone after 4 days. No statistically significant differences were observed since the clone size was 31.5 ± 24.9 and 33.0 ± 27.5 for dual and single expressor populations, respectively, in more than 50 clones scored for each population.
Double-sorted population as DEs is enriched for genetic allelically included cells
The results described above prompted us to characterize the VHDJH rearrangements on cells from the population double sorted as DE. IgH-rearranged alleles were PCR amplified directly from micromanipulated double sorted single cells or from double sorted derived clones that were cultured in limiting dilution conditions. The set of primers used recognizes all DHJH, and the majority of VHDJH rearrangements with VH genes from the 7183, Q52, S107, X24, 3660, J606, MRL-DNA4, and J558 murine VH families and were previously described (6). Amplified products were sequenced directly. Even if technical limitations did not allow the detection of both rearrangements in many cells, the analysis of the simple VHDJH sequences already suggested that, although not pure, the population double sorted as IgH DEs had a high frequency of real DEs. Phenotypic allelically included cells have necessarily two VHDJH+ rearrangements, whereas single producers can be VHDJH+/VHDJH- or VHDJH+/DJH. For any given VH family, VHDJH+ alleles should have identical chances to be PCR amplified over VHDJH- alleles. The finding that 8 out of 11 VHDJH sequences with D-proximal VH (Q52 and 7183) were VHDJH+ and that all 23 VHDJH with VH other than D-proximal ones were VHDJH+, can be explained only if a large proportion of cells in the population double sorted as IgMa+IgMb+ were VHDJH+/VHDJH+ (in a population of single expressors, considering all VHDJH, 30% are expected to be VHDJH-).
The formal demonstration that we were dealing with genetic and
phenotypic allelically included cells comes from the observation, shown
in Table II, that 13 out of the 22 cells
(clones) double sorted as DEs, for which both alleles were successfully
amplified by PCR, are allelically included at the genetic level, i.e.,
they carry two productive
VHDJH rearrangements (the
remaining cells were 5
VHDJH+/VHDJH-
and 4
VHDJH+/DJH).
The fact that we found 9 single expressors out of 22 cells (41%) after
a second sorting for DEs rules out the possibility that, once in
culture after the first sorting, DEs would down-regulate one of the
alleles and become phenotypic allelically excluded.
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Similar CDR3 length for IgH alleles from single DEs
The VH usage in DEs is summarized in Table II. A J558 VH is typically used in one of the
alleles whereas in the other allele any VH can be
used, including D-proximal VH in six of the
cells.
With respect to N and P additions and D element usage, DEs do not
appear to have any particular feature. However, 3 (possibly only 2) out
of the 19 alleles (16%; possibly only 11%) for which a D element
reading frame (RF) can be assigned use RF2, which suggests an increase
in the usage of this RF, which is around 3% immature lymphocytes
(17, 18), although a larger sample would be of interest
here. The bias for JH4 usage is likely to result
from a technical artifact since PCR products from rearrangements using
JH4 are smaller and thus easier to amplify.
Interestingly, whereas the CDR3 length (in base pairs) can be very
heterogeneous (43.4 ± 6.2), IgH alleles from the same cell tend
to have a similar CDR3 size, which is illustrated by the CDR3 length
correlation that was found for the pairs of
VHDJH+/VHDJH+
(Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(19, 20) and 
(21) chains and the Ig (2, 22, 23, 24) and
(4) L chains. The finding that phenotypic allelically included cells do exist but are kept at very low frequencies raises some questions concerning the selective pressures that shaped allelic exclusion. It is now clear that allelically included cells can go through B cell development and mature (our unpublished observations; Ref. 13). Nevertheless, one question remains: would the vertebrate immune system be less efficient if a high proportion of cells would be allelically included? Assuming that the probability of negative selection of a newly formed lymphocyte (p) is a function of the number of receptors the cell can display (for example, if p for IgH single expressors is x, for IgH DEs it will be 2x), negative selection would be more severe on a DE population. However, in terms of the efficiency of lymphocyte output and in the absence of an estimation of x, it is difficult to argue that this difference is, indeed, relevant. At the level of B cell responsiveness, although some observations indicate that DEs become single expressors during the course of an affinity maturation response (3, 25), it is not known whether a single expressor would initially be recruited preferentially over a phenotypic allelically included cell. Additionally, we lack data on the efficiency of triggering in cells with a 2-fold (or higher) decrease in membrane receptor density. In contrast with the idea that allelic exclusion was selected per se, one could still consider it as an indirect consequence of other selective pressures. For instance, somatic rearrangement and extensive diversification at the junctional level in the IgH locus lead to two nonproductive rearrangements in a substantial fraction of B cell precursors (7, 17). In the absence of simultaneous allelic rearrangement, the selective pressure to recruit cells for further differentiation as soon as one productive rearrangement occurs could account for the allelic exclusion we observe.
Although the frequency of phenotypic IgH allelically included cells is extremely low, given that an adult mouse has 108 B cells, at any time the animal has to cope with 104 DEs. These cells express two different IgH variable domains from which one could be self reactive, inducing, once activated, an autoimmune condition. This is unlikely because, as previously discussed, before achieving maturation, DEs should be checked for both receptors. Presumably, negative selection is ensured even if only half of the receptors in one cell are self reactive. If a parallel with T cell physiology is allowed, this rather simplistic view does not entertain the possibility of some autoimmune hazard resulting from the activation, once in the periphery, of passenger autoreactive receptors in DEs that may have escaped deletion due to low levels of surface expression (26).
Presently, several non-mutually exclusive hypotheses can be envisaged to explain the origin of IgH DEs. One very simple interpretation is to consider these cells as rare escapees that would leak through the tight mechanism of allelic exclusion. More elaborate scenarios include: 1) de novo rearrangement in the periphery; and 2) synchronous VH to (VH)DJH rearrangement on both alleles occurring in a small fraction of cells. The former scenario is unlikely to explain our results. In fact, although B cell somatic recombination activity has been reported in germinal centers (27), it remains to be formally shown that a silenced IgH allele can rearrange and be expressed in the periphery; furthermore, B cells in germinal centers lose IgM surface expression and would not be detected in our assay. On the other hand, the synchronous rearrangement hypothesis would conciliate the existence of DEs with the mainstream feedback model of allelic exclusion. By "synchronous," we mean that both alleles rearrange within the time window defined by the first rearrangement and the decay in the potential of recombination which would compromise rearrangement in the second allele. The length of the N region has been interpreted as a measure of the activity of TdT at the time of rearrangement (28). Assuming that TdT levels change in ontogeny, similar N region lengths in both alleles could be interpreted as a signature of synchronous rearrangement. In the described DEs, this is not observed, either in D-JH or in V-DJH junctions. What about the D RF usage? The Dµ protein (transcribed and translated from a RF2 DJH, for all D elements except Q52) is thought to be involved in the shutdown of somatic recombination (29, 30), consequently driving the cell to an endpoint. This explains the RF2 counter selection observed in mature B cells. However, it is conceivable that, if cells rearrange D to JH at the same time and immediately after VH rearranges in both alleles, the cell would not have time to accumulate Dµ, and RF2 counter selection should be diminished. Although 16% of D segments were found to be in RF2 (as compared with 3% (17) previously found for single expressors), we do not valorize this finding due to the low number of sequences (2) and therefore we lack altogether support for the synchronous rearrangement hypothesis.
One last remaining possibility is based on a report in which genetic allelically included cells were described (18). It was proposed that these allelically included cells arise when the second allele is given a chance to recombine because, although the rearrangement in the first allele was productive, the correspondent H chain failed to associate with the surrogate L chain. Furthermore, it has been shown that the pre-BCR mediates a shift in the B cell VH gene repertoire, usage representation (31). Normally, pre-BCR formation is essential for B cell progression. In the c-kit+, cytoplasmic µH chain+ pre-B cell compartment, D-proximal VH genes are preferentially expressed, but, notably, most µH from the Q52 family and the 81X gene fail to form a pre-BCR. Consequently, these elements are underrepresented in the VHDJH+ pool from large pre-BII cells, which necessarily express the pre-BCR.
Could these pre-B genetic (but not phenotypic) allelically included cells correspond to a premature developmental stage of the mature DEs? The fact that close to one third of the DEs we found in the periphery use one Q52 VH in one of the alleles argues in that direction. In fact, this observation indicates that these cells failed allelic exclusion because the chain from the first allele to rearrange VHDJH+ failed to form a pre-BCR. If the second allele rearranges its VH productively and the correspondent µ-chain associates with the surrogate L chain, then these cells can progress in differentiation.
We, therefore, propose a scenario where, upon conventional L chain
expression, the µ-chain that did not pair to form the pre-BCR will be
"rescued" in a fraction of genotypic allelically included cells and
expressed at the surface, just like the µ-chain that first paired
with the surrogate L chain. This fraction is likely to be very small,
following the notion that µ-chains that first failed to associate
with the surrogate L chain are less prone to pair with conventional L
chains (32, 33). This would largely explain the difference
between the frequency we estimated for phenotypic allelically included
cells and the 
2.5% (18) calculated for genetic
allelically included B lymphocytes. Although based on only 2
VDJ+/VDJ+ cells out of 39
carrying two VDJ rearrangements, this value suggests that the frequency
of cells carrying two productively rearranged VDJ alleles is higher
than the one for DEs. In light of this hypothesis, the CDR3 length
correlation would be interpreted as an additional physical constraint
imposed by the conventional L chain, to achieve IgH surface expression
of both alleles.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Vasco C. Barreto, Unité du Développement des Lymphocytes, Institut Pasteur, 28 rue du Docteur Roux, 75 724 Paris Cedex 15, France. E-mail address:
3 Abbreviations used in this paper: IgH, Ig H chain; DEs, dual expressors; RF, reading frame; CDR, complementarity-determining region; pre-BCR, pre-B cell receptor; FSC-H, forward scatter.
Received for publication August 24, 1999. Accepted for publication October 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M.-G. Velez, M. Kane, S. Liu, S. B. Gauld, J. C. Cambier, R. M. Torres, and R. Pelanda Ig Allotypic Inclusion Does Not Prevent B Cell Development or Response J. Immunol., July 15, 2007; 179(2): 1049 - 1057. [Abstract] [Full Text] [PDF]
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 C. Sirac, C. Carrion, S. Duchez, I. Comte, and M. Cogne Light chain inclusion permits terminal B cell differentiation and does not necessarily result in autoreactivity PNAS, May 16, 2006; 103(20): 7747 - 7752. [Abstract] [Full Text] [PDF]
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L. Heltemes-Harris, X. Liu, and T. Manser An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion Int. Immunol., November 1, 2005; 17(11): 1447 - 1461. [Abstract] [Full Text] [PDF]
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S. Liu, M.-G. Velez, J. Humann, S. Rowland, F. J. Conrad, R. Halverson, R. M. Torres, and R. Pelanda Receptor Editing Can Lead to Allelic Inclusion and Development of B Cells That Retain Antibodies Reacting with High Avidity Autoantigens J. Immunol., October 15, 2005; 175(8): 5067 - 5076. [Abstract] [Full Text] [PDF]
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G. Kalmanovich and R. Mehr Models for Antigen Receptor Gene Rearrangement. III. Heavy and Light Chain Allelic Exclusion J. Immunol., January 1, 2003; 170(1): 182 - 193. [Abstract] [Full Text] [PDF]
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V. Barreto, T. Meo, and A. Cumano Mice Triallelic for the Ig Heavy Chain Locus: Implications for VHDJH Recombination J. Immunol., May 1, 2001; 166(9): 5638 - 5645. [Abstract] [Full Text] [PDF]
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