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,
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Division of Developmental and Clinical Immunology, Departments of
Medicine and
Microbiology, and
§
Specialized Center for Caries Research, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The capacity of lymphocytes to generate a heterogeneous repertoire of Ag-binding proteins is the basis of their ability to recognize a broad range of specific Ags. The Ag receptors are encoded by families of variable (V), diversity (D), and joining (J) segments and constant (C) domains that undergo rearrangements exclusively in lymphoid cells (3). The Ag binding site of the B cell Ag receptor is created by the V domains of paired heavy (H)3 and light (L) chains. Each V domain contains three intervals of highly variable sequence (the complementarity-determining regions (CDRs)) that are separated from each other by four intervals of conserved sequence (frameworks (FRs)) (9, 10). The H and L chain CDR3s form the center of the Ag-binding site, with CDRs 1 and 2 forming the outside boundaries and the whole being supported by a scaffold formed by the FRs that ensures the juxtaposition of the H and L CDRs. In the preimmune repertoire, CDRs 1 and 2 are exclusively encoded by germline V gene segment sequence. LCDR3 is created by the joining of VL and JL gene segments and introduces a moderate amount of somatic diversity to the repertoire.
The third heavy (H) chain CDR (HCDR3) is the most diverse component of the Ab-binding site (11, 12) and typically plays a critical role in defining the specificity of the Ab (10, 13). Most HCDR3 intervals include a D gene segment that is typically flanked by runs of non-germline-encoded nucleotides (N regions), which are added by TdT (11). Nucleotides palindromic to the termini of the rearranging gene segments, termed P junctions, can also be added (14). Inclusion of a D gene segment, addition of N nucleotides, and gene segment extension by up to four sets of P junctions make HCDR3 the focus of somatic diversification of the preimmune repertoire (11, 12).
Regulation of the diversity of HCDR3 is one mechanism by which the range of Ag-binding sites available to the B cell repertoire can be controlled during ontogeny. In the mouse, although DH and JH utilization are similar in the neonate and the adult, neonatal HCDR3s lack N nucleotides (6, 7). This absence of N nucleotide addition results in neonatal H chains that contain similar germline-encoded HCDR3 sequences of restricted hydropathicity (15). The observation that neonatal HCDR3 intervals are, on average, shorter than those found in the adult has been attributed to this lack of N addition. Limitation or prevention of the production of longer Ag receptors that are a normal component of the adult repertoire has thus been ascribed to a passive process of repertoire development that is regulated at the genomic level.
In the human, the length of the HCDR3 sequences in Abs obtained from second trimester fetuses are also considerably shorter than those obtained from the blood of adults (8, 16, 17). Unlike the mouse, the patterns of DH and JH utilization differ between the fetus and the adult; and N nucleotides can be found in VDJ transcripts cloned from 8 wk gestation and through maturation to adulthood (8, 16). However, in spite of these apparent differences in the mechanisms of recombination and rearrangement that create the human HCDR3 repertoire, the average length of human fetal HCDR3 intervals is very similar to the average length of mouse neonatal HCDR3 intervals. The similarities in the final Ig products of B cell development in the fetal human and neonatal mouse, in spite of the differences in the specific mechanisms that contribute to their creation, argue that constraints on the length of the HCDR3 interval in early life may be functional significance and may not represent a simple passive case of ontogeny recapitulating phylogeny.
To determine when the HCDR3 repertoire matures in the human and to gain insight into the mechanisms that regulate its development, we have surveyed the distribution of HCDR3 lengths in first and second trimester fetuses, in neonates and young infants, and in adults; and we have compared the composition of HCDR3 in fetal and adult VDJ transcripts, which can be selected by Ag, to that of DJ transcripts, which cannot. We find that the range of HCDR3 structures is highly constrained in the fetus, is still restricted at the time of birth, and reaches a steady state by 2 mo of age. This restriction in HCDR3 length reflects both genetic regulation of VDJ rearrangement and a process of somatic selection that actively limits the range of Ag-binding structures available in the periphery to the fetus and the neonate and appears to influence the representation of JH gene segments and the contribution of N addition to the expressed repertoire. Moreover, a reevaluation of previously published neonatal and adult sequences from mouse (7) indicates that active selection for length is not unique to the human, but may play a major role in distribution of HCDR3 lengths during ontogeny in the mouse as well.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Liver and spleen from fetuses of 7–28 wk gestation were obtained from the Laboratory of Embryology of the University of Washington over a 10-yr period. Cord blood was obtained from 10 normal term infants. Peripheral blood was obtained from 62 infants ranging in age from 2 wk to 18 mo and from 5 patients with X-linked agammaglobulinemia (XLA) ranging in age from 5 to 32 yr of age. Fetal bone marrow (FBM) cells were flushed from the long bone specimens of a 19 and a 22 wk gestation fetus (determined by fetal foot length), adult bone marrow (ABM) was derived from the resected ribs of two kidney donors (30 and 48 yr old), and XLA bone marrow was obtained from the superior iliac crest of a 5-yr-old patient with XLA. All six patients with XLA have been shown to have mutations that abrogate function of the btk gene (18). All tissue collections and distributions were performed in accordance with policies established by the Human Use Institutional Review Board of the University of Alabama (Birmingham, AL).
Due to sample size, fetal tissue was directly used for RNA extraction
in samples of 
11 wk gestation. In fetal samples of age >12 wk,
mononuclear cells were purified by Ficoll-Hypaque gradient
centrifugation. Liver and splenic tissue samples were minced and bone
marrow cells were flushed from the ribs and the long bones. Buffy coat
samples were obtained from cord blood and peripheral blood samples of
infants and young children. In selected cases, FACS was used to isolate
B cell progenitors from the bone marrow lymphoid cells (see below).
FACS and separation of B lymphocyte progenitor subpopulations
Purified bone marrow mononuclear cells were divided into two
aliquots, one of which was stained with 4G7 anti-CD19
(
1
), (Becton Dickinson, Mountain View, CA) and 8G12
anti-CD34 (1), (Becton Dickinson). The other
aliquot was stained with the anti-CD19 and anti-secretory
(s)IgM (Southern Biotechnology Associates, Birmingham AL). In initial
experiments, cells were sorted into
CD19+,sIgM- and
CD19+,sIgM+ subpopulations on a
FACStarPlus instrument (Becton Dickinson). In later
studies, the following subpopulations were sorted:
CD34+/CD19+,
CD19high/sIgM-,
CD19+/sIgMlow, and
CD19+/sIgM+ (19). Postsort analysis (FACScan
instrument) revealed >97% purity.
RNA isolation and cDNA synthesis
Total RNA was isolated using Tri-Reagent (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s protocol (20). Using standard protocols (21), one-third of each RNA sample was used to synthesize first strand cDNA with avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim, Indianapolis, IN). For each cDNA preparation, a control synthesis reaction was performed without reverse transcriptase to rule out genomic DNA contamination of the RNA.
PCR amplification for IgH transcripts
Rearranged D7-JCµ, D3-JCµ, and VDJCµ transcripts were amplified from 1 µl of the cDNA reaction product from each sample. PCR was performed using a Perkin-Elmer (Norwalk, CT) Cetus model 9600 thermal cycler. Each PCR amplification was performed under the following reaction conditions: 31.7 µl dH2O, 5 µl 10x PCR buffer with 15 mM MgCl2 (Boehringer Mannheim), 1.5 µl 50 mM MgCl2, 8 µl 1.25 mM dNTPs, 2 µl 20 µM each primer, and 1.25 units Pwo DNA polymerase (Boehringer Mannheim). Each reaction underwent one initial cycle of denaturation at 95°C for 1 min, followed by 30 cycles of amplification with denaturation at 95°C for 30 s, primer annealing for 1 min, and extension at 72°C for 1.5 min. A final extension was performed at 72°C for 5 min. Primer annealing temperatures were 55°C for D7-JCµ, 60°C for D3-JCµ, and 56°C for VDJCµ amplifications. Primer combinations used to amplify cDNA, in separate reactions, were: D7-JCµ (5'-GAG CTG AGA ACC ACT GTG-3' and 5'-CCC GGG TGC TGC TGA TGT CAG-3') (22, 23), D3-JCµ (5'-GAG GTC TGT GTC ACT GTG-3' and 5'-GGA GAA AGT GAT GGA GTC GGG-3' (24) or 5'-GGA CAT CCC (AG)GG TTT CCC CAG-3' and 5'CCC GGG TGC TGC TGA TGT CAG-3') (25), and VD-JCµ (5'-CGC AAG CTT ACA C(TGA)G C(CT)G-3' and 5'-CCC GGG TGC TGC TGA TGT CAG-3') (26). (The first set of D3 primers amplified members of the D2, D3, and D6 families. The second set was specific for D3.) Actin mRNA was analyzed as a control for the reliability of the cDNA synthesis, using the primers 5'-GTG GAC TTG GGA GGA GGA CTC TGG G-3' and 5'-GCGGGA AAT CGT GCG TGA CAT T-3' (27).
Assessment of HCDR3 length by spectrotype
A 1-µl aliquot of first-round VDJCµ product underwent a
second round of PCR amplification using the original Framework 3
consensus region V primer (5'-CGC AAG CTT ACA C(TGA)G C(CT)G-3') in
association with an internal, nested Cµ primer (5'-AAT TCT CAC AGG
AGA CGA G-3'). This latter primer was [-32P]ATP
end-labeled with polynucleotide kinase (Boehringer Mannheim), as per
the manufacturer’s recommendations. The reaction conditions were the
same as the first round, with the exception that amplification
proceeded for only five cycles. A 10-µl aliquot of second round PCR
product was mixed with 2 µl of loading dye (21), boiled for 2 min in
a water bath, quick cooled, and a 6-µl aliquot of the mix was loaded
on 7.5% polyacrylamide sequencing gel. Each gel included a set of
lanes with sequenced M13 mp18, and one lane containing an amplified
HCDR3 interval from a VDJCµ clone of known sequence as controls for
length and amplification. After electrophoresis, the gel was exposed to
x-ray film (Kodak, Rochester, NY) for an average of 4 h, and to an
imaging plate (Fuji Photo Film, Japan) for an average of 30 min. The
image plate was scanned with a Fujix Bas 1000 Phosphoimager, and the
generated images were analyzed using MacBass V2X software (Fuji). After
subtraction for background, the intensity of each section of a given
lane corresponding to a three-nucleotide interval was quantified and
divided by the total intensity of the lane that corresponds to lengths
of 6–30 codons. This percentage contribution was multiplied by the
corresponding HCDR3 length in codons. Finally, the contribution of each
codon was added to determine the average length of the HCDR3 intervals
in each lane.
Sequence analysis of DJ and VDJ transcripts
A total of 5 µl of the initial PCR products were reamplified using: D7 (5'-TTT AAG CTT GAG CTG AGA ACC ACT GTG-3'), D3 (5'-CGG AAG CTT GAG GTC TGT GTC ACT GTG-3'), or V (5'-CGC AAG CTT ACA C(TGA)G C(CT)G-3') primers in association with a nested Cµ primer (5'-GGA GAA AGT GAT GGA GTC GGG-3'). There is an internal EcoRI restriction site in Cµ just upstream of this latter primer. Reamplification was performed under the following conditions: 60.5 µl dH20, 10 µl 10x buffer with 15 mM MgCl2 (Boehringer Mannheim), 16 µl 1.25 mM dNTPs, 4 µl 20 µM primers, 1.5 U Pwo DNA Polymerase (Boehringer Mannheim), and 5 µl from the first round. Second-round PCR products were digested with EcoRI and HindIII, cloned into either pBluescript SK (Stratagene, San Diego, CA) or pZero (Invitrogen, Carlsbad, CA), and screened with a Cµ probe (AAT TCT CAC AGG AGA CGA G). DNA sequencing was performed by the dideoxy termination technique (28).
HCDR3 is defined as the interval between the conserved cysteine (amino acid 92) at the carboxy terminus of Framework 3 and the conserved tryptophan (amino acid 103) at the amino terminus of Framework 4 (9, 12). To classify the DH germline sequence (25) within the HCDR3 interval of each of the 253 sequences analyzed, the criteria of a minimum of either seven nucleotides of uninterrupted identity with germline sequence, including P junctions, or eight nucleotides with one mismatch with at least two nucleotides of identity at both the 3' and 5' end of the candidate DH gene segment was used.
A total of 37 HCDR3 intervals, of which 28 were in-frame, were cloned from 15 different fetal liver samples of 7–10 wk gestation. This compilation includes 13 previously reported intervals (8). Fetal liver sample size precluded sorting cells into subpopulations; thus, these intervals derive from a mixture of B cells and B cell progenitors. A total of 32 second trimester fetal liver HCDR3 intervals, 26 in-frame, were also analyzed. Of these, 26 HCDR3 intervals from 2 fetal liver cDNA libraries of 15 and 19 wk gestation (23, 26) have been previously reported. Six additional cDNAs were isolated from the same 15-wk cDNA library (23). Mononuclear cells from 19-wk FBM were sorted on the basis of surface expression of CD19 and IgM. Twenty-two HCDR3 intervals from CD19+,sIgM- pro and pre-B cells, 20 in-frame, and 63 HCDR3 intervals from CD19+,sIgM+ B cells, all in-frame, were obtained. These sequences were compared with a previously published set of 99 HCDR3 sequences cloned from the blood of 6 normal adults (17).
A total of 55 fetal and 33 adult D3-J and 55 fetal and 22 adult D7-J transcripts were analyzed. A preliminary evaluation of a subset of these DJ transcripts has been previously reported (15). The 8-wk fetal liver samples yielded 14 D3-J transcripts and 14 D7-J transcripts. A 12-wk fetal liver sample yielded 16 D3-J and 22 D7-J transcripts. CD19+,sIgM- B cell progenitors from a 19-wk FBM sample yielded 25 different D3-J and 19 D7-J transcripts. CD19+,sIgM- B cell progenitors from 2 different ABM samples yielded 17 D3-J and 10 D7-J transcripts. An additional 16 D3-J and 12 D7-J transcripts were obtained from unsorted bone marrow of a third adult donor. Lastly, 31 D3-J transcripts were obtained from the peripheral blood and bone marrow of 6 different patients with XLA. Due to message abundance, only two D7-J transcripts were obtained from the same samples. Each of these reported sequences are unique; repeat sequences were not included in this analysis.
Statistical analysis
Statistical analysis was performed using the program JMP (SAS Institute, Cary, NC). Average values are reported with the SEM.
Molecular modeling
All molecular modeling was performed on a Silicon Graphics
IRIS/4D220GTX computer system using the QUANTA modeling software
(Polygen, Waltham, MA). The crystallographic structure of mouse J539
Fab (pdb file 2FBJ (29); obtained from the Brookhaven National Protein
Database (30, 31)) was chosen for homology-based modeling because of
its use of the isotype and its use of a VH gene segment
from the same evolutionary clan as human V3-23. The mouse L chain
variable domain was first altered to form a humkv325-J1. This
mutation was followed by 100 cycles of steepest descents minimization
to clear the structure of any bad amino acid contacts caused by
mutation. The J539 VH segment was then mutated to the
appropriate sequence. The complete structure was subjected to extensive
minimization, which included 100 cycles of steepest descents
minimization followed by at least 500 cycles of conjugate gradient
minimization with the objective of gaining an rms force applied under
0.6 for 10 consecutive cycles.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The single member of the D7 (DQ52) family, D7-27, contributed to
one-half of first trimester, one-third of second trimester, but only
one-fiftieth of the adult HCDR3 intervals examined (Fig. 1
). This gene segment is located
immediately adjacent to the JH locus. The five members of
the D3 (DXP) family (D3-3, D3-9, D3-10, D3-16, and D3-22) are scattered
20–50 kb upstream of JH (25). These gene segments
contributed to one-twentieth of first trimester, one-tenth of second
trimester, and nearly one-third of adult sequences
(p < 0.0001, 
2). Among VDJCµ
transcripts, JH4 was the most commonly used JH
gene segment at all stages of ontogeny. However, JH2 was
the second most commonly utilized JH gene segment among
first trimester transcripts, JH3 was the second most common
among second trimester transcripts, and JH6 was the second
most common among adult transcripts (p <
0.0001, 2).
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The average length of HCDR3 length distributions also changes
during ontogeny. By sequence analysis, although the average length was
the same in first and second trimester sequences, it was approximately
five codons longer in the blood of adults (12.2 ± 0.5, 12.5
± 0.2, and 17.7 ± 0.5 codons, respectively; p <
0. 0001, 2). The similarity in average HCDR3 length
between first and second trimester samples led us to assess the
relationship between average HCDR3 length and the maturation of the IgM
repertoire. By spectrotype analysis, the average HCDR3 length
distribution was found to remain relatively unchanged in fetal liver of
8- to 28-wk gestation, exhibiting a mean of 13.2 ± 0.2 codons
(Fig. 2). Fetal liver is a hematopoietic
organ, enriched for B cell progenitors, whereas spleen is a lymphoid
organ that contains a higher proportion of mature B cells as the fetus
develops (32). Unlike liver, where the HCDR3 length was similar in
first and second trimester tissues, by spectrotype analysis, HCDR3
intervals from first trimester spleen were smaller than those from
second trimester (12.6 ± 0.2 vs 13.3 ± 0.2 codons,
respectively; p < 0.02, Student’s t test)
(Fig. 2). In the cord blood, which contains primarily naive
sIgM+/sIgD+ B cells, the average HCDR3 was two
residues longer by spectrotype analysis than either second trimester
spleen or liver (15.4 ± 0.1 codons) (Fig. 2). By spectrotype
analysis, a steady state HCDR3 average of 17.3 ± 0.2 was reached
at 2 mo of age (p < 0.0001, 2).
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D7 and D3 gene segments were chosen because they best represent
the change in DH utilization between fetus and adult (Fig. 1
). Among our database of 253 VDJCµ sequences, 14 fetal D3-, 59 fetal
D7-, 28 adult D3-, and 2 adult D7-containing HCDR3 intervals were
identified. The adult D7-HCDR3 sequences were both out-of-frame and of
insufficient numbers for comparison. Two of the fetal D3 sequences that
had undergone rearrangement by inversion were also excluded from the
analysis. In the adult, D3-containing HCDR3 intervals had the same
length as the average for the population of HCDR3 intervals as a whole
(17.7 ± 0.7 codons vs 17.5 ± 0.5 codons, respectively, by
sequence analysis).
DH and JH gene segment lengths differ and might
be expected to influence the length of HCDR3 (Fig. 3). D7-27 is the shortest DH,
consisting of only 11 nucleotides. With a length of 31 nucleotides,
D3-3, -10, -11, and -22 are among the longest conventional
DH gene segments (25). JH4 is the shortest
human JH, contributing only 14 nucleotides to HCDR3;
whereas JH3 can contribute 16; JH5, 17;
JH1, 18; JH2, 19; and JH6 can
contribute up to 29 nucleotides, respectively. When limited to germline
sequence, D3-HCDR3 intervals can achieve lengths of 17
(JH4) to 22 (JH6) codons, whereas D7-containing
HCDR3 intervals can only achieve lengths of 11–16 codons,
respectively. To attain lengths longer than 16 or 22 codons,
respectively, P junctions or N nucleotides must be added.
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). Differences in fetal and adult HCDR3 length distributions were the product of D-specific preservation of DH sequence
Fetal and adult HCDR3 sequences containing D3 and D7 gene segments
were dissected to assess the role of DH length,
JH utilization, and N region addition in controlling HCDR3
length (Fig. 3). One very significant factor was the greater resistance
of D7 gene segments to the loss of terminal nucleotides. D7 gene
segments lost only an average of 0.2 ± 0.2 nucleotides at the 3'
terminus and 2.2 ± 0.2 at the 5' terminus, whereas D3 gene
segments lost 8.3 ± 0.9 and 11.5 ± 0.9 nucleotides at each
terminus, respectively (p < 0.0001, Student’s
t test) (Fig. 3). D7 elements also contained more P
junctions. There was an average of 0.5 P nucleotides added to the 3'
end of the D7 gene segments, whereas D3 elements had no P junctions
(p < 0.04, Student’s t test).
JH length and N addition appeared to play lesser roles.
(Fig. 3). The JH portion of HCDR3 and the extent of N
addition between D and J were slightly greater in D7- vs D3-HCDR3
intervals, but neither of these trends achieved statistical
significance (JH length: 11.5 ± 0.5 vs 9.9 ±
1.2 nucleotides, respectively; p = 0.08, Student’s
t test. N addition: 4.3 ± 0.5 vs 2.8 ± 1.0
nucleotides, respectively; p = 0.25, Student’s
t test). (Fig. 3).
In the adult, preservation of DH sequence was a major
factor contribution to the increase in the average length of HCDR3.
When compared with the fetus, adult D3-containing sequences retained
eight additional nucleotides at the 5' end (loss of 3.7 ± 0.9 vs
11.5 ± 0.9 nucleotides in adult and fetus, respectively;
p < 0.0001, Student’s t test) (Fig. 3).
Loss of these eight terminal nucleotides in fetal µ transcripts
reduces the likelihood of incorporating the only charged amino acid
encoded by the 5' end of D3 gene segments, aspartic acid. N addition
and the contribution of JH sequence to HCDR3 length was
enhanced in the adult, but these differences again failed to achieve
statistical significance (Fig. 3).
Fetal constraints on HCDR3 length are already present in non-Ag selectable DJ transcripts
The change in HCDR3 length distributions between fetus and adult could reflect differences in the interaction between the recombinase and the IgH locus, Ag receptor-influenced selection of the final products, or both. Human DJ joins, the intermediate products of H chain rearrangement, are transcribed but lack the conserved upstream translation start codon that allows translation to a Dµ protein product in the mouse (25, 33). Analysis of DJ transcripts allows assessment of the site of 3' DH rearrangement, N region addition between D and J, patterns of JH utilization, and the site of JH rearrangement without concern for selection for a protein product.
Within each DH family, processing of the 3' coding ends was
similar in fetus and adult. Between families, however, recombination
led to a greater loss of 3' D coding sequence in D3-J transcripts than
in D7-J transcripts (5.3 ± 3.9 vs 1.3 ± 3.2 nucleotides at
the 3' terminus, respectively; p < 0.0001, Student
t test). Fewer D3-J transcripts incorporated P junctions
than D7-J transcripts (1 of 88 vs 27 of 77 sequences with P junctions,
respectively; p < 1 x 10-6,
2).
JH utilization in fetal and adult DJ transcripts differed by both age and D gene segment
Mirroring the pattern seen in VDJ rearrangements, sequence
analysis of the DJ transcripts revealed that JH4 was the
predominant JH gene segment in both D3-J and D7-J
transcripts at all ages studied (Fig. 4).
Use of JH6 increased from one-seventh of fetal D3-J
transcripts to one-third of adult D3-J transcripts (8 of 55 vs 11 of
33, respectively; p < 0.02, 2). Among
D7-J transcripts, JH6 increased from none in the
fetus to one-seventh of the adult D7-J transcripts (0 of 54 vs 3 of 22,
respectively; p = 0.03, 2). Between D
gene segments, in the fetus, JH6 was used in none of the
D7-J transcripts and in one-seventh of the D3-J transcripts (0 of 54 vs
8 of 55; p < 0.001, 2). In the adult,
differences in the prevalence of JH6 in D3-J and D7-J
transcripts did not reach statistical significance
(p = 0.24, 2).
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). The extent of N addition differed by both age and D gene segment
By sequence analysis, the extent of N addition between D and J was
similar in adult D3-J and D7-J transcripts, but differed markedly
between fetal D3-J and D7-J transcripts and between fetal and adult D-J
transcripts in general (Fig. 5). Fetal
D7-J transcripts contained five times more N addition than fetal D3-J
transcripts (5.0 ± 0.6 vs 0.9 ± 0.3 nucleotides,
respectively; p < 0.0001, Student’s t
test) (Fig. 5). Between fetus and adult, N region addition in D7-J
transcripts increased by 70% (5.0 ± 0.6 vs 8.5 ± 1.2
nucleotides, respectively; p < 0.01, Student’s
t test), whereas there was an 800% increase of N addition
in D3-J transcripts (0.9 ± 0.3 vs 7.8 ± 1.0 nucleotides,
respectively; p < 0.0001, Student’s t
test) (Fig. 5). Although the proportion of sequences containing N
addition increased from 7% at 8 wk to 25% at 19 wk, in the adult 6%
(2 of 33) of the D3-J transcripts still lacked N addition. (Fig. 5).
Thus, even in the adult, TdT did not add N nucleotides to all
rearranging gene segments.
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Absence of N addition had no apparent effect on DH reading frame preference
Unlike the mouse, where preferential rearrangement at sites of sequence identity between D and J helps dictate use of DH reading frame 1 (rf1) (6, 7, 34), absence of N addition had a minimal effect on reading frame preference. One-half of the first trimester D3-J transcripts, one-fourth of the second trimester D3-J transcripts, and one-twentieth of the adult D3-J transcripts had D-J rearrangements that occurred at sites of identity between D and J (14 of 30, 9 of 33, and 2 of 33, respectively). Following the pattern seen in mouse DFL and DSP sequences, rf1 in human D3 gene segments is enriched for tyrosine, serine, and glycine, rf2 encodes hydrophobic amino acids, and rf3 contains many termination codons (8, 25). Among the 25 transcripts with overlapping sequence, reading frame utilization appeared random with nine using rf1, nine using rf2, and seven using rf3. Among the transcripts that contained N addition, one-half used rf1, one-fourth used rf2, and one-sixth used rf3 (a ratio of 36:16:11; p < 0.0001, binomial distribution). Among all of the D3-J transcripts, one-half used rf1, one-fourth used rf2, and the remaining one-fifth used rf3 (a ratio of 45:25:15; p < 0.0003, binomial distribution). There was no significant difference in reading frame preference between fetal and adult D3-J transcripts, or in fetal and adult D7-J transcripts. A reading frame preference was also apparent among D7-J transcripts, with one-third using rf1, one-fourth using rf2, and one-half using rf3 (a ratio of 23:20:34; p = 0.02, binomial distribution). A preference for rf1 in D3-HCDR3 and for rf3 in D7-HCDR3 intervals has been previously reported (25). Similarity in reading frame preference by both precursor DJ and mature VDJ transcripts suggests that the pattern of reading frame utilization is established early in B cell development.
In the adult, individual D3 gene segment utilization was similar
between DJ transcripts and VDJ transcripts (p =
0.23, 2), with D3-22 most common, followed by D3-3,
D3-9, and D3-10 in that order. In the fetus, D3-3 rather than D3-22 was
the most commonly used DH in DJ transcripts
(p = 0.03, 2).
Absence of btk expression can influence the extent of N addition
Examination of DJ transcripts from patients with XLA confirmed
that enhancement of N addition could be associated with prolonged
exposure to TdT in vivo. Patients with XLA have loss of function
mutations in the cytoplasmic tyrosine kinase btk (35, 36).
These mutations lead to a block in B cell maturation at the pro-B/pre-B
cell stage. In normal individuals, TdT is detected in <1% of
cytoplasmic Cµ+ cells. In XLA, however, TdT expression is
prolonged with up to one-third of cytoplasmic Cµ+ cells
still expressing this enzyme (37, 38). By sequence analysis, XLA D3-J
transcripts were found on average to contain almost twice as many N
nucleotides as D3-J transcripts from normal ABM (13.1 ± 1.9 vs
7.8 ± 0.9 nucleotides, respectively; p = 0.01,
Student’s t test) (Fig. 5). The HCDR3 lengths of VDJCµ
transcripts in the peripheral blood of some of the XLA donors were then
evaluated by spectrotype analysis to see if long DJ rearrangements
dictated the presence of long HCDR3s. Of the four patients evaluated,
VDJCµ transcripts could not be detected in one, a second patient had
a single band likely representing only one productive rearrangement, a
third patient exhibited three such bands, and the fourth had eight.
Presuming each of these 12 bands represented a unique transcript, an
average HCDR3 length was calculated and found to be similar to the
average for cord blood (14.8 ± 0.9 vs 15.4 ± 0.1 codons,
respectively).
Convergence of fetal D3- and D7-HCDR3 lengths is likely the result of selection
In the fetus, JH utilization and the extent of N
addition in VDJ transcripts was not representative of that observed
among DJ progenitors. Among fetal D3-J transcripts, the extent of N
addition at the DJ junction increased 3-fold between DJ and VDJ
(0.9 ± 0.3 vs 2.8 ± 1.0 N nucleotides, respectively;
p = 0.01, Student’s t test) (Figs. 3 and 5). In the adult, the differences in N addition were not significant
(7.8 ± 0.9 vs 6.5 ± 1.3 N nucleotides, respectively;
p = 0.4, Student’s t test) (Figs. 3 and 5).
Similarly, among fetal sequences using the D7 gene segment, there was
little difference between DJ transcripts and VDJ transcripts (5.0
± 0.6 vs 4.3 ± 0.5 N nucleotides, respectively;
p = 0.3, Student’s t test) (Figs. 3 and 5).
Note that for those sequences using the short D7 gene segments, N
addition between DJ and VDJ transcripts did not enhance length; and
that for sequences using the longer D3 gene segments, N addition was
more likely to increase length (Fig. 3).
Trimming of DH and use of alternative JH gene segments appear to be the two most important factors in allowing the length distribution of D3- and D7-HCDR3 intervals to converge. When compared with DJ transcripts, fetal D3 gene segments in VDJ transcripts lost twice as many nucleotides at the 3' terminus (4.7 ± 0.5 vs 8.3 ± 0.9 nucleotides, respectively; p < 0.006, Student’s t test), trimming the average length by one codon. There was no significant difference in the loss of 3' nucleotides between adult DJ and VDJ transcripts. In contrast, D7-HCDR3 intervals preserved 3' DH sequence (loss 1.3 ± 0.4 nucleotides in DJ transcripts vs 0.2 ± 0.2 nucleotides in VDJ transcripts; p = 0.02, Student’s t test).
A shift in JH utilization between DJ and VDJ helped
lengthen D7-HCDR3 intervals in the fetus, but no such shift was
detected in adult. Among 19 first trimester D7-VDJ transcripts, 5
contained JH2, whereas none of 14 D7-J transcripts
contained JH2 (p < 0.05, Fisher’s
exact test) (Fig. 4). On average, D7-HCDR3 intervals with
JH2 were longer by six codons than those using
JH4 (15.6 ± 0.8 vs 9.0 ± 0.6 nucleotides,
respectively; p < 0.001, Student’s t
test). The number of adult D7-HCDR3 intervals and fetal D3-HCDR3
intervals was not sufficient for statistical evaluation of
JH utilization. Among adult D3-HCDR3 intervals,
JH utilization mirrored that seen in DJ transcripts (Fig. 4).
Selection of HCDR3 length is associated with the surface expression of µ H chain
Trimming of DH and the shift in JH
utilization supports the view that HCDR3 intervals could be actively
selected for length. Studies showing a decrease in HCDR3 length
distribution in B cell progenitors compared with B cells in ABM (39),
enhanced length in nonfunctional vs functional rearrangements (40), and
the decrease in length distribution between first trimester fetal liver
and spleen in this work suggest that selection in the fetus could occur
during the process of differentiation from a pre-B cell to B cell. We
had previously amplified VH3-containing VDJCµ transcripts
from the bone marrow of a 22-wk fetus and a 47-yr-old adult. We
reamplified these products and analyzed the distribution of HCDR3
lengths by spectrotype (Fig. 6). In both
fetus and adult, the average HCDR3 length decreased during B cell
differentiation. (Fig. 6). The most dramatic alteration of lengths was
observed between those cells expressing only cytoplasmic µ H chain
and those expressing a low level of surface IgM (Fig. 6).
|
Barring additional experiments of nature such as XLA, analysis of
the mechanism by which HCDR3 length is selected remains a primarily
descriptive endeavor. To determine whether a similar process occurs in
a species more amenable to experimental manipulation, we evaluated the
HCDR3 length distributions of previously reported VDJCµ transcripts
from newborn liver, newborn spleen, and adult spleen of mouse (7) (Fig. 7). With a length of 11 nucleotides,
mouse DQ52 is 2 codons shorter than the 17-nucleotide long members of
the DSP family. In turn, 23-nucleotide long DFL16.1 contains 2 more
codons than DSP gene segments. On average, neonatal liver DQ52 HCDR3
intervals were three codons shorter than DSP HCDR3s; and DSP HCDR3s
were two codons smaller than DFL16.1 HCDR3s (7.2 ± 0.8, 10.9
± 0.6, and 13.0 ± 0.5 codons, respectively; p <
0.0002, one way ANOVA) (Fig. 7). In spleen, HCDR3 lengths appear to
have been adjusted by selection. Although still exhibiting unique
length distributions, DQ52 HCDR3 intervals were lengthened, DSP
intervals retained a similar length, and DFL16.1 intervals were
shortened when compared with liver (8.0 ± 0.5, 9.7 ± 0.3,
and 11.1 ± 0.6 codons, respectively; p < 0.0002,
one way ANOVA) (Fig. 7). The difference between an average length for
neonatal liver and spleen DFL16.1-containing HCDR3 intervals was
significant at p < 0.05 (Student’s t
test). In adult spleen, DQ52, DSP, and DFL16.1 HCDR3 intervals
converged to the same average length (10.6 ± 0.6, 11.1 ±
0.5, and 11.8 ± 0.6; DSP codons, respectively; p
= 0.4, one way ANOVA) (Fig. 7). Although DFL16.1 lengths remained the
same between neonatal and adult spleen, the increase in length for
splenic DQ52- and DSP-containing HCDR3 intervals was significant at
p < 0.05 (Student’s t test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
One of several genetic mechanisms controlling HCDR3 length is
regulation of DH gene segment rearrangement. In previous
studies, we have shown that D7-J rearrangements, which are more likely
to generate short HCDR3 intervals, occur in fetal
CD34+/CD19- B cell progenitors, but are not
detected in their adult counterparts (19). The present study documents
that the differences between D3- and D7-containing Abs reflect more
than just timing of rearrangement. D7 gene segments exhibit different
patterns of JH utilization, preserve more germline
sequence, and undergo addition of more N nucleotides (Figs. 3-5).
Together, these patterns point to a fundamental difference in the
nature of the interaction between the recombinase complex and
individual DH and JH gene segments in fetal vs
adult cells.
In part, differences in the way these gene segments are processed
appear inherent to the sequences of the DH and
JH gene segments themselves. For example, the 3' sequence
of the D7-27 gene is GC-rich (5'-CTAACTGGGGA-3'), whereas the 3'
termini of D3 sequences are AT-rich (5'-... TGGTTATTATAAC-3').
Increased preservation of 3' D7 sequence, including the addition of P
junctions, may reflect greater stability of a GC-rich 3' terminus after
recombinase cleavage, endowing D7-27 gene segments with resistance to
exonuclease activity (42, 43). Recombination signal sequences (RSS) can
also influence rearrangement frequency. Each JH has a
unique RSS (9). The JH4 RSS adheres most closely to the
consensus established by Wu and colleagues (44), followed by the RSS of
JH6, JH5, JH3, JH2, and
JH1, in that order. The "quality" of the JH
RSS parallels the pattern of JH utilization in adult
tissues and may explain why JH4 is the preferred
recombination partner for both D3 and D7 gene segments (45). A
preference for the substrate formed by the pairing of the D3 and
JH6 RSS over the RSS of D7 and JH6 could
explain why the rearrangement frequency of D3
JH6 and
D7JH6 differ.
There must be more to control of JH utilization, however,
than just a preference for different recombination partners. Use of
both D3 and JH6 is enhanced in adults. One potential
mechanism is secondary rearrangement. For example, an initial
preference for JH-proximal D7-JH4 rearrangement
at all ages might be followed in the adult by secondary rearrangement
of an upstream D3 to a downstream JH6. However, in the
adult, use of downstream JH6 gene segments increases in
D7-J transcripts as well. By nature of their chromosomal position,
these D7-JH6 transcripts cannot be the products of a
secondary DJ rearrangement. The implication is that activation
or enhancement of the accessibility of JH6 for
recombination is another mechanism by which HCDR3 composition can be
controlled.
As in the thymus, N addition in fetal B cells is not a simple all or
none phenomenon (46). Control of the extent of N addition appears to be
a third mechanism by which the length and the hydropathicity of HCDR3
can be regulated (15). The extent of N addition has been shown to
differ by stage of development (e.g., neonatal vs adult (6, 7)) and by
stage of differentiation (e.g. VDJ vs DJ (6, 47)). These
quantitative as well as qualitative differences in the number of N
nucleotides added have been attributed to differences in the levels of
TdT expression, chromatin structure, the accessibility of TdT to
chromatin, or the composition of the recombinase complex itself (6, 46, 47).
In this work, we show that at the same stage of maturation and development, N addition is being modulated in a gene-specific fashion, raising the possibility that D3 and D7 rearrangements are the products of two different types of B cell progenitors in the fetus. One progenitor population would express near adult levels of TdT activity and preferentially rearrange DQ52 in the context of a relatively inaccessible JH6 gene segment. The other population would express minimal levels of TdT activity and contain a more wide-open DH and JH locus, enabling rearrangement of upstream DH elements and downstream JH gene segments, including JH6.
Support for this latter hypothesis comes from the analysis of HCDR3
intervals from B precursor acute lymphocytic leukemias (B cell ALL).
HCDR3s of children 3 yr of age or younger have a paucity of N addition
between D and J when compared with intervals from similar leukemias
derived from older children (48, 49). These data have been interpreted
as compatible with the hypothesis that the leukemic event in the
younger children took place in fetal life. However, use of DQ52 in the
HCDR3 intervals from these younger children is rare when compared with
DQ52 utilization in fetal tissues as a whole (2 of 34 vs 59 of 154,
respectively; p < 0.001, 2). If there
are two progenitor populations, then it is possible that
DQ52-rearranging cells are more resistant to leukemic transformation.
Superimposed upon genetic mechanisms that constrain HCDR3 length is a process of somatic selection that not only prevents the expression of rare fetal Abs with long HCDR3s, but also regulates length in the adult. This process influences patterns of gene segment utilization. For example, the apparent preference for DH-proximal JH2 gene segments in first trimester tissues appears due to selection rather than preferential rearrangement.
In both fetus and adult, selection for length appears to occur during the progression from a cytoplasmic µ+ pre-B cell to a CD19+/sIgMlow cell. This latter population is largely comprised of cells expressing µ H chain in association with surrogate L chain and V pre-B (50, 51). In the mouse it has been shown that the ability of an H chain to form a pre-B cell receptor has a direct effect on the survival of the cell (52). Successful assembly of the pre-B receptor appears to inhibit B cell differentiation in the fetus, but enhance survival and differentiation in the adult (53). In the present study, the final, "preferred" length of the HCDR3 interval differs between fetus (12 codons) and adult (17 codons), suggesting that fetal and adult pre-B cell receptors are subject to different selective pressures in the human. By the time of birth, the average length of HCDR3 has already increased by two codons when compared with both fetal liver and spleen at 28 wk gestation. If a ligand or ligands play a role in the selection of HCDR3 intervals and the maturation of the repertoire, these ligands must thus be either endogenously produced or transmitted to the infant across the placental barrier (54), raising the possibility that the development of the neonatal repertoire could be modulated by challenging the mother with Ag.
Somatic selection for length appears to be intact in patients with XLA, and, thus, is not abrogated by the lack of btk function. If cells that express long HCDR3 domains are being eliminated, it is possible that this process of selection may contribute to the block in B cell maturation that characterizes XLA. In normal individuals, IL-7 down-regulates the expression of TdT (55). However, when exposed to IL-7, pro-B cells from XLA patients continue to express TdT at high levels (55). These observations suggest that the IL-7R signal transduction pathway can be used to modulate the extent of N addition.
Among the infants examined, there was individual variation in the average HCDR3 length expressed. A 2-mo-old infant with a restricted HCDR3 length distribution that averaged 15.2 codons still expressed small HCDR3 intervals exhibited an average of 15.5 codons at 4 mo of age. The maturation of HCDR3 length distributions may be delayed in some individuals. That such a delay may affect the ability of the individual to respond to Ag remains an intriguing possibility.
Conceptually, the human repertoire appears to include a third type of
structure, a "knob," in addition to the two general types of
Ag-binding surface, the "groove" and the "cavity," that have
been previously described in the mouse (56, 57). To better visualize
the consequences of lengthening HCDR3, we modeled three Fv domains with
HCDR3 intervals of 12, 18, and 24 codons (12) (Fig. 8). These models illustrate the
protrusion of the Ag-binding surface in longer HCDR3 intervals (57).
|
At least some of these developmentally regulated Ag specificities are likely to require V domains with long HCDR3s. Lengthening HCDR3 enlarges the Ag-binding surface of the Ab, increasing the potential for multireactivity, including self-reactivity. Analysis of a set of multispecific, self-reactive Abs from human B1-a cells revealed that most of these sequences, which were obtained from adults, contained long HCDR3 domains not representative of the fetal repertoire (58). It remains unclear whether expression of long HCDR3 domains is a detriment to fetuses or an advantage to the adult, or both. In either case, changes in the potential for self-reactivity could be a major factor.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Harry W. Schroeder, Jr., Division of Developmental and Clinical Immunology, Wallace Tumor Institute 378, UAB Station, University of Alabama, Birmingham, AL 35294. E-mail address:
3 Abbreviations used in this paper: H, heavy; L, light; CDR, complementarity-determining region; XLA, X-linked agammaglobulinemia; FBM, fetal bone marrow; ABM, adult bone marrow; s, secretory.
Received for publication December 18, 1998. Accepted for publication March 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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genes: implications for T cell lineages and for a novel intermediate of V-(D)-J joining. Cell 59:859.[Medline]
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