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J Repertoire in Human Fetal Spleen: Evidence for Positive Selection and Extensive Receptor Editing1
Department of Internal Medicine and Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75235
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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J rearrangements obtained from genomic DNA of individual
IgM+ B cells from human fetal spleen were analyzed. A
nonrandom pattern of gene rearrangements that differed from the
adult V repertoire was found. The V distal genes 8A and 4B were
absent from the nonproductive fetal repertoire, whereas 2E and 3L were
overrepresented and 1B was underrepresented in the productive fetal
repertoire. Positive selection of the V gene, 2E, along with V
rearrangements employing homologous VJ joins were observed in the
fetal, but not in the adult V repertoire. Overrepresentation of J
distal cluster C genes rearranging to the V distal J segment, J7,
in both productive and nonproductive fetal repertoires suggested that
receptor editing/replacement was more active in the fetus than in
adults. Numerous identical VJ junctions were observed in both the
productive and nonproductive repertoire of the fetus and adult, but
were significantly more frequent in the productive repertoire of the
fetus, suggesting expansion of B cells expressing particular -light
chains in both stages of development, with more profound expansion in
the fetal repertoire. Notably, B cells expressing identical -light
chains expressed diverse heavy chains. These data demonstrate that
three mechanisms strongly influence the shaping of the human fetal
-chain repertoire that are less evident in the adult: positive
selection, receptor editing, and expansion of B cells expressing
specific -light chains. These events imply that the expressed fetal
repertoire is shaped by exposure to self Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12 wk of gestation,
production of B lineage cells shifts to the bone marrow, which then
assumes the major role throughout life (1). B cell development in bone marrow requires successful rearrangement of the Ig H and L chain gene loci and the surface expression of the B cell Ag receptor (BCR)4 (2). These properties define the stages of B cell maturation during bone marrow development. At the pre-B cell stage, cells have successfully completed functional rearrangement of the heavy chain and it pairs with surrogate light chain to express the pre-B cell receptor (pBCR) on the surface (3). When pre-B cells undergo successful light chain rearrangement, they develop into immature B cells and express the mature BCR (4). Immature B cells produced in the bone marrow migrate into the periphery to complete their maturation process (5).
Expression of the pBCR and BCR affords the possibility that the primary
B cell repertoire is shaped and directed by continuous clonal selection
beginning at the pre-B cell stage of differentiation (6).
Analysis of VH and V
gene expression in
different stages of development, in both humans and mice, has suggested
developmentally controlled nonrandom use of V(D)J genes and unique
patterns of junctional diversity (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). With better
understanding of BCR signaling, recent studies have provided direct
evidence that bias in the Ab repertoire is a result of selection
mediated through pBCR and BCR (18, 19, 20). It has also been
demonstrated that the migration of immature B cells into the periphery
and competition for entry into the peripheral immune system are active
processes mediated by signals through the BCR. The net result of these
events is a marked narrowing of the Ig repertoire (3, 21).
However, the ligands that drive and select B cells during these stages
of differentiation are still not known. It has been suggested that
certain levels of BCR signaling by interaction with self Ag might be
essential for survival and maturation of B cells (6, 22).
The fetal microenvironment is different from that of the adult, because it is not exposed to external Ags, but presumably abundantly exposed to self Ags. Therefore, the repertoire that develops during fetal life, the preimmune repertoire, may be considerably different from that found in adults. Mechanisms that restrict and shape the human adult primary Ig repertoire have been studied intensively. However, molecular and selective mechanisms involved in shaping the human preimmune repertoire have not been examined in detail. Analysis of the fetal B cell repertoire could, therefore, lead to a better understanding of the impact of the different influences that shape the expressed Ig repertoire.
In the current study, detailed Ig gene repertoire analysis was
conducted using genomic DNA obtained from individual
IgM+ B cells from fetal spleen. The goal was to
analyze the preimmune repertoire in humans to understand in greater
detail the impact of the molecular mechanisms and selective influences
that shape it, and to compare this with the adult human V
repertoire. The technique allowed us to analyze both productive and
nonproductive rearrangements without introducing an activation-related
bias. Using this methodology, molecular mechanisms and selective
influences that shape the human gene repertoire in peripheral
IgM+ B cells from normal adult donors have
recently been assessed (23). This adult V database was
used for comparison with the fetal preimmune repertoire. The results
indicate that there was a nonrandom rearrangement pattern in the
gene repertoire at the fetal stage of development. Positive selection
of individual V genes and expansion of B cells expressing particular
V rearrangements with homology-mediated joining independent of heavy
chain expression strongly influenced the fetal repertoire, resulting in
predominance of particular VJ junctions in the productive
repertoire. Evidence for more active peripheral receptor editing in the
fetal, compared with the adult V repertoire was also found. These
events occur before exposure of the fetus to exogeneous Ag and,
therefore, must be mediated by intrinsic developmental signals and/or
self Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Single cell preparations were made from three fetal spleens at 18 wk of gestation by mechanical disruption of tissue fragments, followed by filtration through nylon mesh. Fetal bone marrow cells were flushed from long bone specimens of an 18-wk fetus. All tissue collections and processing were done in accordance with policies established by the institutional review board for human experimentation at the University of Texas Southwestern Medical Center (Dallas, TX). Mononuclear cells were enriched by Ficoll-Hypaque density-gradient centrifugation, as described (24). The cells were then stained with a PE-labeled anti-CD19 mAb (Becton Dickinson, Mountain View, CA) and a FITC-labeled anti-human IgM mAb (Caltag, Burlingame, CA). The CD19+/IgM+ B cells were sorted using a FACStarPlus flow cytometer (Becton Dickinson, San Jose, CA) outfitted with an automated single cell deposition unit, and one cell was deposited into each well of a 96-well PCR plate assembled on a microAmp base, as described previously (24, 25). Each well contained 10 µl of PCR buffer (50 mMKCl, 10 mM Tris-HCl, pH 9, 0.1% Triton X-100).
PCR amplification
PCR amplification included an initial primer extension
preamplification (24) and subsequent nested PCR steps
(23). Single cells in 10 µl of PCR buffer were incubated
with 0.4 mg/ml proteinase K (Sigma, St. Louis, MO) for 1 h at
55°C, and the enzyme was inactivated by heating at 95°C for 10 min.
Primer extension preamplification employing random 15 mers and 60
rounds of amplification with Taq polymerase (Promega,
Madison, WI) was performed to produce sufficient DNA for multiple
subsequent DNA amplifications. Rearranged VJ and
VHDJH genes were then
amplified, as described previously (23, 24).
Sequence analysis
PCR products were separated by electrophoresis on a 1.5% Seakem
agarose gel (FMC Bioproducts, Rockland, ME) and purified using GenElute
agarose spin columns (Supelco, Bellefonte, PA). Purified products were
directly sequenced using the ABI Prism Dye Termination Cycle Sequencing
kit (Perkin-Elmer, Foster City, CA) and analyzed with an automated
sequencer (ABI Prism 377; Applied Biosystems, Foster City, CA). For
identification of the germline V and VH gene
segments, the V BASE Sequence Directory (26) was used in
conjunction with the software programs Sequencher (Gene Codes, Ann
Arbor, MI) and Gene Works (release 2.45; IntelliGenetics, Mountain
View, CA). V, J, VH, D,
JH gene nomenclature was adopted according to the
V BASE Sequence Directory.
A rearrangement was considered productive if the VJ junction
maintained the reading frame into the J segment. Rearrangements that
failed to maintain the reading frame into the J gene segment
(n = 66), and those rearrangements that introduced a
stop codon at the junction during the rearrangement process
(n = 4) were considered as nonproductive.
Rearrangements that involved pseudogenes were always considered
nonproductive (n = 9). At the junctions, sequences were
considered to be germline if at least two contiguous nucleotides
matched the germline sequence. Junctional additions were scored as
either inverted repeats at full-length coding ends (P nucleotides; P),
inverted repeats at nucleolytically processed coding ends
(Pr nucleotides; Pr), or
nontemplated junctional additions (N nucleotides; N). In cases in which
a nucleotide could be scored as either a P or a
Pr, it was scored as a P. When junctions without
N additions contained repeated nucleotides that could not be
unequivocally assigned to either coding end, these were assigned as
junctional microhomologies (H).
A total of 184 fetal sequences was obtained from three fetal spleens all at 18 wk of gestational age. For comparison, 227 adult sequences that had been published previously (23) were used. In brief, adult sequences were obtained from two healthy normal male donors (26 and 45 years old) and included two populations, CD19+/IgM+/CD5+ or CD5- B cells. In the analysis, clonally expanded rearrangements in the productive repertoire were considered as the same sequence. A total of 154 fetal sequences, including 84 nonproductive and 70 productive rearrangements, and a total of 201 adult sequences, including 146 productive and 55 nonproductive rearrangements, were analyzed. The numbers derived from the adult sequence analysis were calculated after clones were removed, and hence are different from what was previously published (23).
Estimation of Taq-polymerase error rate
The maximal PCR error rate for this analysis has been documented to be 1 x 10-4 (23). Thus, few, if any, of the nucleotide changes encountered in this analysis can be ascribed to PCR amplification errors.
Statistical methods
2 tests were used to compare the gene usage and
junctional nucleotide differences found in the productive and
nonproductive repertoire as well as between fetal and adult
repertoires. The goodness-of-fit 2 test was used to
assess differences between the observed frequencies of V genes as
would be expected by random usage based on the number of V genes
known to be in the genome. Values of p 
0.05 were
considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the fetal bone marrow, CD19+
IgM- pro/pre-B cells formed the major
population, comprising 52% of the B cell population, whereas in fetal
spleen, CD19+ IgM+ B cells
constituted the major B cell population, comprising 95% of the total B
cell population (Fig. 1
). The ratio of
IgM- pro/pre-B cells to
IgM+ B cells was 1.1:1 in the fetal bone marrow
compared with 1:19 in the spleen. For the current study, the
IgM+ B cell population from fetal spleen was
sorted for Ig repertoire analysis.
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rearrangements in the fetal repertoire
Because nonproductive rearrangements fail to give rise to
functional proteins, the frequency of gene segments observed in the
nonproductive repertoire should reflect the relative frequency of
rearrangement of each individual gene segment in the absence of any
putative selection. To determine whether the rearrangement process
itself is different between fetus and adult, the distribution of
nonproductive VJ rearrangements in fetal splenic B cells was
compared with that in adult donors (Fig. 2, upper panel). Although the
usage of individual V gene segments in the fetus and the adults
showed some differences, the general rearrangement pattern of
nonproductive V gene segments was very similar between the fetus and
the adult. Of a total of 51 functional V genes and pseudogenes, 19
were detected in the fetal repertoire compared with 18 in the adult
repertoire. These included 2 pseudogenes in the fetal repertoire (2A1,
5A) and 4 pseudogenes (2A1, 3A2, 7C, and 5A) in the adult repertoire.
Of the functional V genes detected, 2A2, 2B2, and 1G were
significantly overrepresented in the nonproductive repertoire, in both
the fetus and the adult, compared with the expected frequency based on
random usage. However, 4B, which was overrepresented in the
adult, was not detected in the fetus, and 6A, which was not
overrepresented in the adult, was overrepresented in fetus. When
particular V gene segment usage was compared, only one gene, 4B,
which is the most distal V gene from the J-C region, was
rearranged significantly less frequently in the fetus compared with the
adult (p = 0.005). J segment use in the
nonproductive repertoire showed similar patterns in both fetus and
adult (Fig. 3). J7 was used most often
(61.4% vs 60%), followed by J2/3 (35.7% vs 34.5%), and J1
(2.9% vs 5.5%). These results demonstrate that a similar repertoire
bias is introduced in VJ rearrangements in both the fetus and the
adult, and nonrandom use of V and J gene segments, as shown in
the adult, is already present at the fetal stage of development.
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gene rearrangement during the
fetal stage of development
To investigate whether chromosomal position can affect the
rearrangement of V genes during the different stages of development,
we analyzed V gene usage in nonproductive rearrangements according
to V gene clusters. The V genes are divided into three clusters,
clusters A, B, and C, with cluster A being most J proximal and
cluster C being most J distal (27). Frequencies of
cluster A, B, and C V genes in the nonproductive repertoire of fetus
were comparable with the adult (Fig. 3
). When individual V gene
segment usage was analyzed according to V gene clusters,
rearrangement of V genes in the fetus occurred throughout the entire
V locus, as shown in adult, except for the two most distal genes in
cluster C, 8A and 4B (Fig. 2, upper panel). Observations
that the two most distal V genes in cluster C were less frequently
rearranged in the fetus suggest that some positional effects influence
V gene rearrangement during fetal stages of development.
The expressed fetal gene repertoire is restricted and shaped by
positive selection
The distribution of productive (and thus potentially selectable)
VJ rearrangements was analyzed to define differences in the
expressed repertoires between the fetus and the adult (Fig. 2). The
distribution of V gene segments in the productive repertoire was
found to differ considerably between the fetus and the adult. Relative
restriction of V gene segment usage was noted in the fetal
repertoire. Of the functional V genes expressed, five V genes,
2E, 2A2, 1C, 1G, and 3L, were found more often than expected in the
fetal productive repertoire, whereas seven genes, 3R, 2A2, 3H, 2B2, 1C,
1G, and 1B, were found more often than expected in the adult. A number
of V genes were significantly less frequent in the fetal productive
repertoire compared with the adult, including 3R and 1B, whereas 2E and
3L were more frequent in the fetus. Certain V gene segments within
each V family were noted most frequently in both fetus and adult.
However, 1G was the most frequent gene in the V1 family, 2E in the
V2 family, 3L in the V3 family, and 4A in the V4 family in the
fetus, compared with 1B, 2A2, 3H, and 4B, respectively, in adult.
Overall, a few V genes were overexpressed by a large number of cells
in the productive repertoire of the adult, and this tendency was more
marked in the fetus.
Each V gene was analyzed for differences in its distribution between
the productive and nonproductive repertoires to determine whether they
were influenced by selection (Fig. 2). In the fetus, no functional gene
was found at a significantly lower frequency in the productive than
nonproductive repertoire. Thus, no V gene was negatively selected in
the fetal repertoire, whereas 4C was negatively selected in adult
(p = 0.032). In the adult, no positive
selection was observed. However, in the fetus, 2E was found at a
significantly higher frequency in the productive than nonproductive
repertoire (p = 0.025), suggesting that it was
positively selected.
Junctional diversity in the fetal repertoire is influenced by positive selection
There were no significant differences in the complexity of the
VJ junctions assessed by the frequency of nucleotytic processing
at the coding ends, P nucleotide addition, and N nucleotide addition
between the fetal and the adult nonproductive repertoires (Fig. 4). When productive and nonproductive
junctions in the fetal repertoire were compared (Fig. 4), evidence of
selection based on combinatorial diversity was obtained. Junctions with
untrimmed ends, P nucleotides, Pr nucleotides,
and N nucleotides, were all comparable in the fetal productive and
nonproductive repertoires. However, in junctions without N additions,
microhomology-mediated joining (H joining) was observed significantly
more frequently in the productive rearrangements in fetus compared with
nonproductive rearrangements of the fetus (77.5% vs 28.6%;
p = 0.00002), suggesting positive selection. No similar
difference was observed between productive and nonproductive
rearrangements in the adult (55.7% vs 47.3%).
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-light chains
When fetal and adult VJ junction sequences from productive
rearrangements were analyzed in detail, it was noteworthy that numerous
identical junctions in both the fetus and adult were observed (Figs. 5 and 6). In the fetus, 19%
of the total productive rearrangements were represented more than once.
It was particularly striking that nine identical versions of the
3L-J7 rearrangements, 5-3.3.1H, were detected. In the adult,
although the frequency was lower than in the fetus, 13.7% of the total
productive rearrangements were found more than once. Notably, however,
75% of the identical junctions in the adult were found within the
CD5+ population, whereas the frequency of
identical -light chain sequences was much lower in the
CD5- population. The frequent occurrence of
identical V sequences in the productive repertoire was in contrast
to their significantly lower frequency in the nonproductive repertoires
in both the fetus and the adult (5.7% in each) (Fig. 7A). Notably, 43.8% of the
productive rearrangements in the fetus with identical junctions used H
joining compared with 15% in adult (p = 0.018)
(Fig. 7B). This compared with 0/4 and 1/3 of the
nonproductive rearrangements in the fetus and adult, respectively, that
employed identical junctions with H joining. These findings indicate
that fetal B cells expressing identical productive V-chains are much
more likely to use H joining. To determine whether B cells with
identical -chains were truly clonal, rearranged heavy chain genes
were amplified from the same cells that exhibited identical -chains.
In eight of sixteen sets of fetal B cells with identical -light
chains, we were able to amplify productive
VHDJH rearrangements from
at least two members of the set. Although -light chains were
identical, heavy chain rearrangements were different in seven of eight.
The exception was 5-3.3.5D and 5-3.3.6G, which both employed
VH3-07/D7-27/JH3 along with
V3L/J7 (Fig. 8). However, five
additional B cells expressing an identical arrangement involving V3L
employed different VHDJH
rearrangements. These findings indicate that the fetal
IgM+ B cells with common V rearrangements are
not clonal with regard to heavy chain use.
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rearrangements
The average CDR3 length of rearrangements was similar in the
fetus and the adult. The mean (±SEM) length of the CDR3 of
productively rearranged VJ genes in the fetus was 31 ± 0.3
bp compared with 31.5 ± 0.2 bp in the adult. The CDR3 length
distribution also showed a similar Gaussian distribution pattern in
both the fetus and the adult productive rearrangements, with a range of
24–39 bp in both groups (Fig. 9, upper panel). In the nonproductive rearrangements, the
distribution of CDR3 length maintained a similar Gaussian distribution
pattern as was seen in the productive rearrangements, but they were
somewhat more broadly distributed, with a range of 14–45 bp in the
fetus and 23–45 bp in the adult (Fig. 7, lower panel). The
mean (±SEM) length of the CDR3 of nonproductively rearranged VJ
genes was 31.5 ± 0.6 bp in the fetus and 32.5 ± 0.6 bp in
the adult, not significantly different from that of the productive
rearrangements.
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To address whether receptor editing played a role in
shaping the fetal repertoire, the relationship between the usage of the
V cluster and J segments was analyzed. Receptor
editing/replacement would be expected to produce bias in the frequency
of association of the most 5' cluster C V genes to the most 3' J
gene, J7, more reflected in the nonproductive rearrangement compared
with the productive rearrangement. In the fetus, 90% of cluster C V
genes were associated with the J7 gene segment in the nonproductive
rearrangements and 80% in the productive rearrangement, whereas in the
adult, 66.7% of cluster C V genes associated with J7 in the
nonproductive rearrangements and 88.2% in the productive
rearrangements (Fig. 3). These data are consistent with the conclusion
that there is more extensive receptor editing/replacement in the fetus
compared with the adult.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene segments (12, 28).
Preferential gene use reflected the proximity of gene segments in
chromosomal position (8, 9, 12). Fetal diversity was also
reported to reflect limited diversification of the CDR3 region during
early ontogeny. This was related to a relative paucity of N region
additions (7, 29), a high frequency of homology-directed
recombination (17), and generation of shorter CDR3 domains
during the fetal life (30). It has been suggested that the
restricted diversity of the fetal Ig repertoire predisposes to the
generation of multireactive, low affinity self-reactive Abs at this
stage of development (31). The physiologic impact of
self-reactive, germline-encoded Abs remains obscure, but it has been
suggested that self-reactivity of these Abs is an essential component
for B cell survival and maturation (6), and mediates
positive selection (22). In addition, cross-reactivity
with exogenous microbes may play a role in host defense to the small
number of pathogens that may threaten the survival of an individual
during the perinatal period (32).
Most of the information on the fetal Ig repertoire derives from the
analysis of the murine repertoire. Limited analyses of human
VH and V repertoires suggested that there is
also restricted Ag specificity and a high degree of self-reactivity,
although some of the mechanisms leading to these features may differ
from those observed in the mouse. However, no information is available
regarding the development and selection of the VJ repertoire in
the human fetus. The current data provide a sufficiently detailed
analysis of the VJ rearrangement in fetal spleen to assess the
characteristics of the repertoire and delineate some of the influences
that shape it.
By comparing the distribution of B cells in the bone marrow and the spleen of an 18-wk fetus, we were able to define that during the second trimester of gestation, the pro/pre-B cell population was mostly found in the bone marrow, whereas less than 5% of splenic B cells were this B cell precursor population. Most of the B cells found in the fetal spleen at this stage were surface IgM+ B cells. Our data confirm that very little, if any, B cell generation takes place in the second trimester fetal spleen, but rather, B cells generated in the bone marrow migrate to this organ to complete their maturation process (33, 34). By the second trimester of fetal development, the spleen functions mainly as a peripheral lymphoid organ as in postnatal life. Fetal spleen, therefore, serves as a good source for sampling the fetal B cell repertoire.
By analyzing nonproductive rearrangements from
IgM+ B cells from fetal spleen and comparing them
with the adult, we were able to assess possible differences in the
rearrangement process involved in the development of the fetal VJ
repertoire compared with the adult in a relatively unbiased way. One of
the recombination mechanisms contributing to the generation of the
rearranged VJ repertoire is preferential rearrangement of V
and J gene segments. Analysis of the fetal nonproductive repertoire
revealed that the nonrandom ultilization of the V and J segments
in the fetal rearrangements was similar to that noted in the adult
(23). Overrepresentation of 2A2, 2B2, 1G V genes in the
adult nonproductive repertoire was also observed in the fetal
repertoire. J7 was most frequently used of the four functional J
segments in both repertoires, followed by J2/3 and J1. However,
notable differences were observed when individual V genes were
analyzed. Although the rearrangement of V genes in the fetus
occurred throughout the entire V locus, we found that V genes, 4B
and 8A, were not rearranged in the nonproductive repertoire of the
fetus. In the adult nonproductive repertoire, the 4B gene was not only
detected, but it was the second most frequently rearranged gene.
Cluster C genes are the most J distal, located more than 600 kb
upstream of the J1 locus, and 8A and 4B are located at the most
distal region of this cluster, about 800 kb upstream of J1 locus
(27, 35). The finding that the two most distal V genes
were not detected in the fetal nonproductive repertoire suggests that,
as in the mouse, proximity of gene segments in the chromosome position
plays a role in preferential VJ rearrangements early in human
ontogeny. However, it is different in that this positional effect is
not as profound as in mouse (8, 9, 36), because
rearrangement of V genes in the human fetus occurred throughout the
entire V locus, except the most distal two genes. These findings
indicate that nonrandom gene segment use largely reflects a regulatory
process that is intrinsic to the specific gene element and is
independent of the stage of B cell maturation. Imposed upon this is a
modest maturation-dependent influence of chromosomal location.
Presumably, because of immaturity in the accessibility or promoter
influence of V genes, rearrangement to the most J distal V
genes was restricted in the fetus, contributing to a modest
developmental bias in the distribution of V genes in the fetal
nonproductive repertoire.
During recombination, diversity can be introduced at the VJ
junctions by addition and deletion of a number of nucleotides
(37). Analysis of the VJ junctions in the
nonproductive rearrangements revealed no significant differences in the
complexity of the VJ junctions between the fetal and adult
repertoires. However, a trend toward restriction of the complexity of
the VJ junctions was observed in the fetal repertoire. In the
fetal nonproductive VJ junctions, biochemical events that occur
at coding ends during recombination resulted in use of more
germline-encoded sequences, in that there was less nucleotide
processing at both the 5' and 3' coding ends, more use of
germline-templated P nucleotides, and less use of nongermline-templated
N regions. Although VJ rearrangements containing N segments were
less frequent in the fetus compared with adult, 50% of fetal
rearrangements contained N segments.
These observations indicate that the basic molecular mechanism
governing the recombination of the -light chain genes is not
significantly different between the fetus and the adult. Although there
was some degree of restriction in diversity introduced during the
VJ recombination in the fetus, this bias was very modest,
implying that recombination mechanisms governing the adult V(D)J
recombination is already established at the second trimester of
fetal life.
The productive rearrangements, which encode functional protein, can
potentially be influenced by selection. Comparison of the productive
repertoire to the nonproductive repertoire allows for a determination
of positive and negative selective influences that shape the Ig
repertoire. Because of differences in the environment between the
prenatal and the postnatal period, it is possible that different
selection mechanisms are operative at these stages of life. It has been
clearly demonstrated that negative selection events shape the primary
repertoire of Ag-reactive B cells (38, 39, 40). Negative
selection occurs upon encounter with self Ag and ensures elimination of
autoreactive B cells that might have emerged during recombination.
However, B cells with self-reactivity do occur in the primary Ab
repertoire, although such processes seem to be restricted to early
ontogeny and perhaps to a particular B cell subset (B-1 cells)
(41, 42). Based on these findings, it has been suggested
that B cells can be subjected to positive selection and maintained on
the basis of their autoreactivity (22, 29, 42), but the
role of positive selection in shaping the primary Ig repertoire is
still unclear. During prenatal life, autoreactive Abs are abundantly
generated, and this reactivity may serve as the basis of positive
selection rather than negative selection. The current data demonstrate
that positive selection is likely to contribute to shaping the fetal
V repertoire, whereas negative selection mainly shapes the adult
V repertoire.
In the adult, seven functional V genes were overrepresented in the
productive repertoire, but positive selection was not evident. Rather,
negative selection of V gene 4C was observed. These findings implied
that this particular gene may have a propensity for autoreactivity and,
thus, be deleted from the expressed repertoire (23).
However, in the fetus, a smaller number of V genes were
overrepresented compared with the adult, but positive selection was
apparent. The V gene, 4C, which was negatively selected in the
adult, was not apparently influenced by negative selection in the
fetus, and the 2E gene, which was not overrepresented in the productive
repertoire of the adult, was significantly overrepresented and also
positively selected in the fetus. Because restriction of the repertoire
was not found in the nonproductive repertoire, relative restriction of
the fetal repertoire appears to result from selection. Thus, it is
possible that restricted V gene usage and selective use of certain
V genes in the fetus predispose to the generation of multireactive,
low affinity, self-reactive Abs required for immunologic needs at this
stage of development, and through positive selection, these
autoreactive Abs may be maintained.
Junctional diversity of the fetal repertoire was also influenced by
selection. In the nonproductive junctions, no significant differences
in the complexity of VJ junctions were observed between fetus and
adult. However, there was a trend toward use of more germline-encoded
sequences at the junctions in the fetal repertoire. In the fetal
productive repertoire, junctions showed a greater tendency toward
diversification. The 5' nucleotide processing was more frequent, P
nucleotides were less frequent, and N nucleotide addition was more
frequent in the productive repertoire compared with the nonproductive
repertoire of the fetus. A striking finding was that junctions
employing microhomology-mediated joining were significantly more
frequent in the productive rearrangements of the fetus compared with
fetal nonproductive repertoire, indicating that these junctions were
positively selected. This selection was not observed in the adult.
These results imply that the expressed fetal repertoire is both
positively and negatively selected based upon junctional diversity.
Selection of rearrangements with H joining causes major constraints on
the junctional diversity mechanism observed in the productive
repertoire, which appears to be the major outcome of selection in the
fetus (17, 43).
One of the remarkable findings of this study was the identification of
numerous identical VJ junctions in both fetus and adult. In the
fetus, 19% of the total VJ productive rearrangements were
represented more than once. Surprisingly, however, when rearranged
heavy chain genes were analyzed from the same fetal B cells that
expressed identical VJ junctions, most were disparate. Therefore,
these fetal B cells were clonal at the -light chain locus, but
obviously not typical clones when the heavy chain rearrangements were
considered. A similar finding was made when the adult productive
repertoire was analyzed with B cells identified that expressed
identical -light chains but disparate heavy chain rearrangements
(data not shown). These -light chain clones were significantly more
frequent in the productive compared with nonproductive repertoire in
the fetus and the adult, and significantly more frequent in fetal
compared with adult productive repertoires, especially when only
CD5- adult B cells were considered. These
results imply that selection rather than bias in recombination played a
major role in the appearance of the expanded -light chain clones,
and this selection bias was more apparent in the fetus. These data
imply that B cells expressing specific V-light chain rearrangements
are positively selected in fetal B cells and adult
CD5+ B cells, regardless of heavy chain
expression. These results suggest a unique V-specific mode of B cell
selection that expands B cells presumably by reacting with autoantigen.
Precedence for such a mechanism of B cell selections derives from an
analysis of avian B cells that exhibit extreme restriction of
junctional diversity with predominance of one
VL-JL joint as a result of
developmental selection that takes place in the embryonic spleen
(20). This embryonic selection process is not restricted
to avian B cells, because similar junctional restriction was shown in
Ig genes of neonatal murine B cells (7, 17). Based on the
current data, developmental selection and expansion of certain VJ
junctions also occur in the human fetus, and strikingly appear to be
strictly based on sequences in the CDR3 domain of the -light chain,
irrespective of the heavy chain. Because this occurs in the fetal
spleen, the current data indicate that B cells undergo selective
expansion based on the -light chain CDR3 independent of external Ag,
and probably, therefore, mediated by self Ag (22, 44).
Selective B cell expansion based on only the -light chain in the
fetus may reflect the nature of unique Ag-Ab interactions in the fetal
life, potentially reflecting self Ag-mediated interactions essential
for survival and maintenance of B cells and the need for creation of
primitive immune response in the immediate postnatal period. In the
adult, although the frequency was lower than in the fetus, expansion of
B cells expressing specific -light chains was found in 13.7% of the
total productive rearrangements. However, most of these cells were
found within the CD5+ population in the adult.
Previously, in adult mice, B cells with canonical receptors containing
particular
VH-D-JH/V-J junctions
were reported in association with the B1 phenotype (22, 45, 46). In addition, selection of certain
VH-J-bearing clones into the mature long-lived
B cell pool (46), and BCR-directed selection of
VH81x-V1C and
VHS107-V24 clones into the splenic marginal
zone were recently reported (47). It is possible that
-light chain clones found in IgM+ B cells in
the fetus may persist to the adult stage as a long-lived B cell pool,
and mainly remain restricted to the special subset of B cells
expressing CD5 in adult life.
There were some differences between the expanded B cell populations
with identical -light chains in the fetus and the adult. The first
was the greater use of H joining in the fetus. H joining was found to
be used significantly more frequently in the B cells with identical
VJ rearrangements in the fetus compared with that of the adult.
When the amino acid introduced at the junctions was analyzed (data not
shown), it was noteworthy that two of the fetal B cells with identical
VJ junctions introduced proline at the junctions, which was not
found in the adult B cells with the same VJ junctions. Notably,
the two fetal junctions that introduced proline were the result of
joining by microhomology. By contrast, in the adult, three of the
identical VJ rearrangements introduced glycine into the CDR3,
which was not found in the fetal rearrangements. Introduction of
proline at a junction causes constraints on ligand binding by tightly
limiting the rotational degree of freedom at the Ag binding site,
whereas glycine allows free rotations at the Ag binding site. Selective
expansion of -light chains using H joining in the fetus may suggest
more tightly regulated Ag reactivity during the fetal period of the
development. This restriction of Ag reactivity in early life was shown
to be essential for the acquisition of complete functional adult
repertoire (48).
Except for the fetal B cells with identical rearrangements, CDR3 length
of the -light chain was not selected. The average length and also
the length distribution of the CDR3 region in both the productive and
nonproductive repertoire remained unchanged during the ontogeny.
Molecular mechanisms appear to impose strict limits on the CDR3 length
of the -chain by tightly regulating the mechanisms governing the
junctional diversity. It was shown in the human heavy chain that the
length of the CDR3 region was considerably shorter in fetal B cells
compared with those in adult (31) and a shorter CDR3
length was selected to restrict diversity in the fetal repertoire.
However, this restriction in fetal CDR3 length was not profound in
human -light chain (28) and clearly is not a feature of
the -light chain repertoire. The difference found between the CDR3
restriction in the heavy chain and the light chains may be related to
the greater range of lengths available to the CDR3 region in the heavy
chain, because heavy chain rearrangement involves D segments in
addition to V and J segments, and the very tight molecular regulation
of CDR3 length seen in - and -light chains evident in the
nonproductive rearrangements (23, 49).
During development, self-reactive B cells can be rescued from negative
selection by up-regulating RAG-1 and RAG-2 and replacement of
autoreactive BCR by secondary Ig gene rearrangements, i.e., receptor
editing/replacement (50, 51, 52, 53). To maintain a B cell pool in
the face of a changing environment continuously challenged with
autoantigen as in fetal life, it is possible that receptor editing is
actively revising the Ag receptor specificity to escape negative
selection (54, 55). Evidence of receptor editing can be
reflected in the Ig repertoire in the following two ways. First,
because receptor editing is achieved by rearrangements of V genes 5' to
the initially rearranged productive VJ rearrangements to 3' J elements
(50, 52), bias in the association of the most 5' V gene to
most 3' J gene would be evidence of secondary rearrangements. Because
only about one-third of the secondary rearrangements are expected to be
successful in producing productive joints, this bias would be reflected
both in yielding and nonproductive repertoire, with more bias in the
nonproductive repertoire if receptor editing were actively occurring.
We found bias in the association of the most J distal cluster C V
genes with J7 in both the productive and nonproductive repertoires
in the fetus, with more apparent bias in the nonproductive repertoire.
Ninety percent of cluster C V genes were associated with J7 in
nonproductive rearrangements, and 80% in the productive repertoire of
the fetus, reflecting evidence of receptor editing in the fetus.
However, this bias was not as apparent in the adult, in which 66.7% of
cluster C V gene associated with J7 in nonproductive
rearrangements and 88.2% in productive rearrangements. Appearance of
secondary nonproductive rearrangements would depend on another
secondary rearrangement that would introduce a productive light chain,
usually a rearrangement. It is possible that secondary
rearrangement is less efficient in the adult, so that bias in cluster C
V gene to J7 association was seen mostly in the productive
repertoire of the adult, whereas it was observed in both productive and
nonproductive rearrangements in the fetus.
Second, evidence of receptor editing can be reflected in the productive
to nonproductive ratio. Theoretically, if both alleles undergo ordered
rearrangement, the productive to nonproductive ratio in the detected
sequences should be close to 2.5:1. Because rearrangements are usually
one-third productive and two-thirds nonproductive, any additional light
chain rearrangement in which the nonproductive rearrangements can be
retained will bring the productive to nonproductive ratio below the
expected 2.5:1 ratio. We found that the productive to nonproductive
ratio in the adult was close to the expected ratio (2.6:1),
whereas the ratio in the fetus was 1:1, significantly lower than the
expected ratio. This implies that receptor editing was very active in
the fetus, leading to the introduction of additional nonproductive
rearrangements that appeared in the repertoire presumably because of
additional secondary productive rearrangement of the locus. This
finding implies that receptor editing is more actively revising the Ag
receptor specificity in the fetus compared with the adult to escape
negative selection and maintain a functional B cell repertoire.
The current data demonstrate that three mechanisms strongly
influence the shaping of the fetal -chain repertoire in human:
receptor editing, positive selection, and expansion of B cells
expressing specific -light chains. These events occur before
exposure of the fetus to exogeneous Ag and suggest, therefore, that the
fetal repertoire is strongly influenced by exposure to
autoantigens.
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Acknowledgments |
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Footnotes |
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2 Current address: Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892.
3 Address correspondence and reprint requests to Dr. Peter E. Lipsky, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 10 Center Drive, MSC 1820, Bethesda, MD 20892-1820.
4 Abbreviations used in this paper: BCR, B cell Ag receptor; CDR, complementarity-determining region; pBCR, pre-BCR.
Received for publication March 29, 2000. Accepted for publication August 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J repertoire. J. Immunol. 162:2137. light-chain locus on chromosome 22q11.2. Hum. Mol. Genet. 4:983.III gene usage is nonstochastic in both adult and newborn peripheral B cells and that peripheral CD5+ adult B cells are oligoclonal. J. Clin. Invest. 93:2093.
gene locus. Genome Res. 5:125. light chains is encoded by a single germline gene. Nucleic Acids Res. 13:6515. gene repertoire of IgM+ B cells. J. Clin. Invest. 99:1614.[Medline]
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