Skip to main content

Main menu

  • Home
  • Articles
    • Current Issue
    • Next in The JI
    • Archive
    • Brief Reviews
      • Neuroimmunology: To Sense and Protect
    • Pillars of Immunology
    • Translating Immunology
    • Most Read
    • Top Downloads
    • Annual Meeting Abstracts
  • COVID-19/SARS/MERS Articles
  • Info
    • About the Journal
    • For Authors
    • Journal Policies
    • Influence Statement
    • For Advertisers
  • Editors
  • Submit
    • Submit a Manuscript
    • Instructions for Authors
    • Journal Policies
  • Subscribe
    • Journal Subscriptions
    • Email Alerts
    • RSS Feeds
    • ImmunoCasts
  • More
    • Most Read
    • Most Cited
    • ImmunoCasts
    • AAI Disclaimer
    • Feedback
    • Help
    • Accessibility Statement
  • Other Publications
    • American Association of Immunologists
    • ImmunoHorizons

User menu

  • Subscribe
  • My alerts
  • Log in
  • Log out

Search

  • Advanced search
The Journal of Immunology
  • Other Publications
    • American Association of Immunologists
    • ImmunoHorizons
  • Subscribe
  • My alerts
  • Log in
  • Log out
The Journal of Immunology

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Next in The JI
    • Archive
    • Brief Reviews
    • Pillars of Immunology
    • Translating Immunology
    • Most Read
    • Top Downloads
    • Annual Meeting Abstracts
  • COVID-19/SARS/MERS Articles
  • Info
    • About the Journal
    • For Authors
    • Journal Policies
    • Influence Statement
    • For Advertisers
  • Editors
  • Submit
    • Submit a Manuscript
    • Instructions for Authors
    • Journal Policies
  • Subscribe
    • Journal Subscriptions
    • Email Alerts
    • RSS Feeds
    • ImmunoCasts
  • More
    • Most Read
    • Most Cited
    • ImmunoCasts
    • AAI Disclaimer
    • Feedback
    • Help
    • Accessibility Statement
  • Follow The Journal of Immunology on Twitter
  • Follow The Journal of Immunology on RSS

IgM Heavy Chain Complementarity-Determining Region 3 Diversity Is Constrained by Genetic and Somatic Mechanisms Until Two Months After Birth

Satoshi Shiokawa, Frank Mortari, Jose O. Lima, César Nuñez, Fred E. Bertrand III, Perry M. Kirkham, Shigui Zhu, Ananda P. Dasanayake and Harry W. Schroeder Jr.
J Immunol May 15, 1999, 162 (10) 6060-6070;
Satoshi Shiokawa
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Frank Mortari
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jose O. Lima
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
César Nuñez
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Fred E. Bertrand III
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Perry M. Kirkham
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shigui Zhu
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ananda P. Dasanayake
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Harry W. Schroeder Jr.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Due to the greater range of lengths available to the third complementarity determining region of the heavy chain (HCDR3), the Ab repertoire of normal adults includes larger Ag binding site structures than those seen in first and second trimester fetal tissues. Transition to a steady state range of HCDR3 lengths is not complete until the infant reaches 2 mo of age. Fetal constraints on length begin with a genetic predilection for use of short DH (D7-27 or DQ52) gene segments and against use of long DH (e.g., D3 or DXP) and JH (JH6) gene segments in both fetal liver and fetal bone marrow. Further control of length is achieved through DH-specific limitations in N addition, with D7-27 DJ joins including extensive N addition and D3-containing DJ joins showing a paucity of N addition. DH-specific constraints on N addition are no longer apparent in adult bone marrow. Superimposed upon these genetic mechanisms to control length is a process of somatic selection that appears to ensure expression of a restricted range of HCDR3 lengths in both fetus and adult. B cells that express Abs of an “inappropriate” length appear to be eliminated when they first display IgM on their cell surface. Control of N addition appears aberrant in X-linked agammaglobulinemia, which may exacerbate the block in B cell development seen in this disease. Restriction of the fetal repertoire appears to be an active process, forcing limits on the diversity, and hence range of Ab specificities, available to the young.

In the human, the ability to mount an effective humoral response to several Ags, including specific microorganisms and vaccines, is delayed until well after infancy (1). This sequential acquisition of the ability to respond to particular Ags has been shown to be a characteristic feature of the developing immune system (2). These observations represent a paradox in that they suggest an element of order in the development of certain components of the Ab repertoire even though the mechanisms that underlie the generation of the Ab are stochastic (3). This paradox has been in part resolved by the observation that, in contrast to the adult, the fetus does not make full use of all the mechanisms that are available for the generation of Ab diversity (4, 5, 6, 7, 8).

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.

Materials and Methods

Sample collection

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 (γ1κ), (Becton Dickinson, Mountain View, CA) and 8G12 anti-CD34 (γ1κ), (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 10× 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 [γ-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 10× 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 κ isotype and its use of a VH gene segment from the same evolutionary clan as human V3-23. The mouse κ L chain variable domain was first altered to form a humkv325-Jκ1. 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.

Results

During ontogeny there is an inverse relationship between DH and JH utilization and chromosomal position relative to the boundary between DH and JH

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, χ2). 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, χ2).

FIGURE 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 1.

HCDR3 diversity during human ontogeny. Top row, First trimester fetal liver VDJCμ+ transcripts. Middle row, Second trimester fetal liver and bone marrow VDJCμ+ transcripts. Bottom row, Adult blood VDJCμ+ transcripts from the published work of Yamada et al. (17). Left column, Percentage of transcripts that utilize members of the designated DH families. A “?” designates transcripts where the DH family cannot be determined. The D7 (DQ52) gene segment contributed to 19 of 37 first trimester, 40 of 117 second trimester, and 2 of 99 adult VDJCμ transcripts; whereas D3 (DXP) gene segments contributed to 2 of 37, 13 of 117, and 29 of 99 transcripts, respectively (p < 0.0001 χ2). Middle column, Distribution of the lengths of the CDR 3 intervals of the transcripts (residues 93–102 (8;9;12)) divided into 3 residue intervals (e.g., <9, 10–12, 13–15, 16–18, 19–21, 22–24, and >24 codons). Right column, Percentage of transcripts that utilize the designated JH gene segment. For each developmental stage, the number of transcripts analyzed is indicated by the value of n. The arrows are intended to emphasize the major differences between fetal and adult transcripts.

The distribution of HCDR3 lengths is restricted in the fetus and neonate

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, χ2). 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, χ2).

FIGURE 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 2.

HCDR3 length distribution during human ontogeny. Shown are average HCDR3 lengths in VDJCμ transcripts from: A, 34 fetal liver samples ranging in age from 8 to 28 wk gestation, B, 35 fetal spleen samples of which 17 derive from the first trimester of gestation and 16 from the second trimester, and C, 72 samples of cord blood and peripheral blood from infants ranging in age from birth to 18 mo of age. The dotted lines denote 5% and 95% confidence limits for the distribution of lengths over time.

D7- and D3-containing sequences were analyzed to elucidate the contribution of individual gene segments to HCDR3 length

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.

FIGURE 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 3.

A dissection of the components contributing to HCDR3 length in D3- and D7-containing H chain variable domains. The potential contribution of the germline sequence of VH gene segments, P junctions, N region addition, DH gene segments, and JH gene segments to HCDR3 length is illustrated. The actual contribution of these components to 28 adult D3-containing HCDR3 intervals, 12 fetal D3-containing intervals, and 59 fetal D7-containing intervals is shown below. Also shown beside the DH gene segment is the average 5′ and 3′ loss of sequence for D3 and D7 gene segments. All components are shown to scale.

Given the difference in lengths, we expected that fetal HCDR3 intervals containing D3 gene segments would be longer than those containing D7. However, by sequence analysis, D3-HCDR3 and D7-HCDR3 intervals exhibited nearly identical average lengths of 12.8 ± 0.7 and 12.7 ± 0.3 codons, respectively (Fig. 3⇑).

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 × 10−6, χ2).

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, χ2). 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, χ2). 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, χ2). In the adult, differences in the prevalence of JH6 in D3-J and D7-J transcripts did not reach statistical significance (p = 0.24, χ2).

FIGURE 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 4.

JH utilization among DJ transcripts during human ontogeny. Depicted are the percentage of transcripts that use the designated JH gene segment. Left column, D3-J transcripts. Right column, D7-J transcripts. Shown by row from top to bottom are transcripts from 8-wk fetal liver, 12-wk fetal liver, 19-wk FBM, ABM, and the blood and bone marrow of patients with XLA, respectively. For each category, the number of transcripts analyzed is indicated by the value of n. The arrows are intended to emphasize the major differences between fetal and adult transcripts.

The site of rearrangement in JH also differed by D gene segment. Due to greater abundance, this was best demonstrated among those transcripts that contained JH4. On average, JH4 lost one more nucleotide at the 5′ terminus in D7-JH4 transcripts vs D3-JH4 transcripts (5.4 ± 0.4 vs 4.2 ± 0.4 nucleotides, respectively; p < 0.02, Student’s t test). The presence or absence of N nucleotides had no effect on the site of JH rearrangement. The greater loss of JH sequence in D7-JH4 rearrangements was unexpected, given that on average, D7-HCDR3 intervals contained more nucleotides derived from JH sequence than D3-HCDR3 intervals (Fig. 3⇑).

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.

FIGURE 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 5.

Patterns of N addition during human ontogeny. Distribution of the extent of N addition in D7- and D3-containing DJ transcripts from normal fetal and adult B lineage cells, and from the blood and bone marrow of patients with XLA. Statistically, there was no difference in N addition between D7-J and D3-J transcripts in the adult (8.5 ± 1.2 vs 7.8 ± 1.0 nucleotides, respectively; p = 0.67, Student’s t test).

JH utilization appeared to vary between those transcripts that contain N addition vs those that did not. Of the 14 D3-J sequences from first trimester tissues, only one used JH6, and this sequence was the only one with N addition. One-third (4 of 13) of all the fetal D3-J sequences that contain N addition used JH6, whereas only one-tenth (4 of 41) of the sequences without N addition used JH6. Thus, control of N addition and JH utilization may be linked.

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, χ2), 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, χ2).

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⇓).

FIGURE 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 6.

Distribution of average HCDR3 lengths of VH3DJCμ transcripts from FBM and ABM as a function of B cell differentiation.

HCDR3 length is also regulated in the mouse

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).

FIGURE 7.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 7.

The distribution of HCDR3 lengths is controlled in mouse. The distribution of HCDR3 lengths in VDJCμ+ transcripts from neonatal liver, spleen, and adult spleen (7). Shown is the average length of sequences using the DFL16.1 and DQ52 gene segments, and members of the DSP family, as well as the average length of HCDR3 intervals for the population as a whole.

Even in the presence of DH gene segments of human length, HCDR3 length was preserved in the mouse. We evaluated the distribution of HCDR3 intervals from mice with a transgenic human IgH minilocus containing representatives of all of the human DH gene segments, as well as the complete JH locus (41). Among 32 in-frame transcripts, the average HCDR3 length was 12.1 ± 0.6 codons, which is similar to the average for fetal liver and bone marrow in human, but 5 codons less than that seen in the peripheral blood of children and adults (see above).

Discussion

By a combination of genetic and somatic mechanisms, humans are prevented from expressing Igs with long HCDR3 intervals during fetal life. In the third trimester of gestation, B cells expressing Abs with HCDR3 intervals of longer length begin to appear. However, constraints on length are still apparent at birth and do not disappear completely until the infant reaches the age of 2 mo (Fig. 2⇑).

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 D7→JH6 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 D→J 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. V→DJ vs D→J (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, χ2). 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).

FIGURE 8.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 8.

Three dimensional molecular models of human Fv domains with emphasis on the consequences of lengthening HCDR3. A, Fv structures from the side. The Ag-binding site is at the top with Vκ on the left and VH on the right. B, Fv structures from the top, looking down into the Ag-binding site. The color scheme for both is as follows: (numbers are according to Kabat (9)). Blue = VH, violet = Vκ (Humkv325 with a 9-amino acid CDR3 and Jκ1), white = HCDR1 (31 to 35); HCDR2 (50 to 65), Vκ LCDR1 (24 to 34); LCDR2 (50 to 56); LCDR3 (89 to 97). Gray = HCDR3 (93–102). Top, V3-23 with D7-27 and JH3 and a 12-amino acid HCDR3. Bottom left, V3-23 with JH5 and an HCDR3 of 18 amino acids (clone 18/2 (59)). Bottom right, V3-23 with JH6 and a 24-amino acid HCDR3. The 3-dimensional raster representation includes side chains on all residues.

Structurally, lengthening the HCDR3 interval creates Ab-binding sites with the potential to bind a different range of epitopes than Abs with short HCDR3s, e.g., gaining the potential to insert a portion of the Ag-binding site into a cavity within an Ag. In complementary studies, Zimmer et al. (unpublished data) have analyzed the reactivity of plasma Abs from this same population of infants and children. Their studies indicate that IgM Abs against a range of bacterial and endogenous Ags first appear between the age of 2 and 6 mo. Given that the composition, as well as the length, of HCDR3 intervals diversifies with age, the change in Ab specificities cannot be attributed solely to the advent of long HCDR3 intervals. However, the emergence of these Abs appears to be a measure of the maturation of the repertoire.

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.

Acknowledgments

We thank Sarah Robinson and Liming Zhang for excellent technical assistance, Dr. Ann Feeney for reviewing our analysis of her published sequences, Dr. Nicholas Chiorazzi for his help in establishing the HCDR3 length distribution assay, the members of the Specialized Center for Caries Research for helping us obtain blood from normal young infants, and the Laboratory of Embryology of the University of Washington for their long term help in providing precious fetal samples.

Footnotes

  • ↵1 This work was supported, in part, by Grants AI33621, DE11147, RR00032, and HD36292.

  • ↵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: Harry.Schroeder{at}ccc.uab.edu

  • ↵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 December 18, 1998.
  • Accepted March 1, 1999.
  • Copyright © 1999 by The American Association of Immunologists

References

  1. ↵
    Stein, K. E.. 1992. Thymus-independent and thymus-dependent responses to polysaccharide antigens. J. Infect. Dis. 165: S49
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Silverstein, A. M.. 1977. Ontogeny of the immune response: a perspective. M. D. Cooper, ed. Development of Host Defense 1st Ed.1 Raven Press, New York.
  3. ↵
    Yancopoulos, G. D., F. W. Alt. 1986. Regulation of the assembly and expression of variable-region genes. Annu. Rev. Immunol. 4: 339
    OpenUrlCrossRefPubMed
  4. ↵
    Yancopoulos, G. D., S. V. Desiderio, M. Paskind, J. F. Kearney, D. Baltimore, F. W. Alt. 1984. Preferential utilization of the most JH-proximal VH gene segments in pre-B cell lines. Nature 311: 727
    OpenUrlCrossRefPubMed
  5. ↵
    Perlmutter, R. M., J. F. Kearney, S. P. Chang, L. E. Hood. 1985. Developmentally controlled expression of immunoglobulin VH genes. Science 227: 1597
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Gu, H., I. Forster, K. Rajewsky. 1990. Sequence homologies, N sequence insertion and JH gene utilization in VH-D-JH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation. EMBO J. 9: 2133
    OpenUrlPubMed
  7. ↵
    Feeney, A. J.. 1990. Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences. J. Exp. Med. 172: 1377
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Schroeder, H. W., Jr, F. Mortari, S. Shiokawa, P. M. Kirkham, R. A. Elgavish, F. E. Bertrand, III. 1995. Developmental regulation of the human antibody repertoire. Ann. NY Acad. Sci. 764: 242
    OpenUrlPubMed
  9. ↵
    Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, C. Foeller. 1991. Sequences of Proteins of Immunological Interest U.S. Department of Health and Human Services, Bethesda, MD.
  10. ↵
    Padlan, E. A.. 1994. Anatomy of the antibody molecule. Mol. Immunol. 31: 169
    OpenUrlCrossRefPubMed
  11. ↵
    Desiderio, S. V., G. D. Yancopoulos, M. Paskind, E. Thomas, M. A. Boss, N. R. Landau, F. W. Alt, D. Baltimore. 1984. Insertion of N regions into heavy-chain gene is correlated with expression of terminal deoxytransferase in B cells. Nature 311: 752
    OpenUrlCrossRefPubMed
  12. ↵
    Kirkham, P. M., H. W. Schroeder, Jr. 1994. Antibody structure and the evolution of immunoglobulin V gene segments. Semin. Immunol. 6: 347
    OpenUrlCrossRefPubMed
  13. ↵
    Kabat, E. A., T. T. Wu. 1991. Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. J. Immunol. 147: 1709
    OpenUrlAbstract
  14. ↵
    Lafaille, J. J., A. DeCloux, M. Bonneville, Y. Takagaki, S. Tonegawa. 1989. Junctional sequences of T cell receptor γδ genes: implications for γδ T cell lineages and for a novel intermediate of V-(D)-J joining. Cell 59: 859
    OpenUrlCrossRefPubMed
  15. ↵
    Schroeder, H. W., Jr, G. C. Ippolito, S. Shiokawa. 1998. Regulation of the antibody repertoire through control of HCDR3 diversity. Vaccine 16: 1363
    OpenUrlCrossRefPubMed
  16. ↵
    Sanz, I.. 1991. Multiple mechanisms participate in the generation of diversity of human H chain CDR3 regions. J. Immunol. 147: 1720
    OpenUrlAbstract
  17. ↵
    Yamada, M., R. Wasserman, B. A. Reichard, S. S. Shane, A. J. Caton, G. Rovera. 1991. Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes. J. Exp. Med. 173: 395
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Haire, R. N., Y. Ohta, S. J. Strong, R. T. Litman, Y. Liu, J. T. Prchal, M. D. Cooper, G. W. Litman. 1997. Unusual patterns of exon skipping in Bruton tyrosine kinase are associated with mutations involving the intron 17 3′ splice site. Am. J. Hum. Genet. 60: 798
    OpenUrlPubMed
  19. ↵
    Bertrand, F. E., III, L. G. Billips, P. D. Burrows, G. L. Gartland, H. Kubagawa, H. W. Schroeder, Jr. 1997. Immunoglobulin DH gene segment transcription and rearrangement prior to CD19 expression in normal human bone marrow. Blood 90: 736
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Chomczynski, P., N. Sacchi. 1987. Single step method of RNA isolation of acid guanidinium-thiocynate-phenol-chloroform extraction. Anal. Biochem. 162: 156
    OpenUrlCrossRefPubMed
  21. ↵
    Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  22. ↵
    Ravetch, J. V., U. Siebenlist, S. Korsmeyer, T. Waldmann, P. Leder. 1981. Structure of human immunoglobulin mu locus: characterization of embryonic and rearranged J and D genes. Cell 27: 583
    OpenUrlCrossRefPubMed
  23. ↵
    Schroeder, H. W., Jr, J.-Y. Wang. 1990. Preferential utilization of conserved immunoglobulin heavy chain variable gene segments during human fetal life. Proc. Natl. Acad. Sci. USA 87: 6146
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Ichihara, Y., H. Matsuoka, Y. Kurosawa. 1988. Organization of human immunoglobulin heavy chain diversity gene loci. EMBO J. 7: 4141
    OpenUrlPubMed
  25. ↵
    Corbett, S. J., I. M. Tomlinson, E. L. Sonnhammer, D. Buck, G. Winter. 1997. Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, “minor” D segments or D-D recombination. J. Mol. Biol. 270: 587
    OpenUrlCrossRefPubMed
  26. ↵
    Schroeder, H. W., Jr, J. L. Hillson, R. M. Perlmutter. 1987. Early restriction of the human antibody repertoire. Science 238: 791
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Ponte, P., S. Y. Ng, J. Engel, P. Gunning, L. Kedes. 1984. Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human β-actin cDNA. Nucleic Acids Res. 12: 1687
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Sanger, F., S. Nicklen, A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Suh, S. W., T. N. Bhat, M. A. Navia, G. H. Cohen, D. N. Rao, S. Rudikoff, D. R. Davies. 1986. The galactan-binding immunoglobulin Fab J539: an X-ray diffraction study at 2.6 A resolution. Proteins 1: 74
    OpenUrlCrossRefPubMed
  30. ↵
    Abola, E. E., F. C. Bernstein, S. H. Bryant, T. F. Koetzle, and J. Weng. 1987. Crystallographic Databases: Information Content, Software Systems, Scientific Applications. F. H. Allen, G. Bergerhoff and R. Sievers, eds. Data Commission of the International Union of Crystallography, Bonn/Cambridge/Chester, p. 107.
  31. ↵
    Bernstein, F. C., T. F. Koetzle, G. J. B. Williams, E. F. Meyer, Jr, M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimaouchi, M. Tasumi. 1977. The Protein Data Bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112: 535
    OpenUrlCrossRefPubMed
  32. ↵
    Gathings, W. E., A. R. Lawton, III, M. D. Cooper. 1977. Immunofluorescent studies of the development of pre-B cells, B lymphocytes and immunoglobulin isotype diversity in humans. Eur. J. Immunol. 7: 804
    OpenUrlPubMed
  33. ↵
    Gu, H., D. Kitamura, K. Rajewsky. 1991. B cell development regulated by gene rearrangement: arrest of maturation by membrane-bound Dmu protein and selection of DH element reading frames. Cell 65: 47
    OpenUrlCrossRefPubMed
  34. ↵
    Feeney, A. J.. 1992. Predominance of VH-D-JH junctions occurring at sites of short sequence homology results in limited junctional diversity in neonatal antibodies. J. Immunol. 149: 222
    OpenUrlAbstract
  35. ↵
    Vetrie, D., I. Vorechovsky, P. Sideras, J. Holland, A. Davies, F. Flinter, L. Hammarstrom, C. Kinnon, R. Levinsky, M. Bobrow. 1993. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature 361: 226
    OpenUrlCrossRefPubMed
  36. ↵
    Tsukada, S., D. C. Saffran, D. J. Rawlings, O. Parolini, R. C. Allen, I. Klisak, R. S. Sparkes, H. Kubagawa, T. Mohandas, S. Quan, et al 1993. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72: 279
    OpenUrlCrossRefPubMed
  37. ↵
    Campana, D., J. Farrant, N. Inamdar, A. D. Webster, G. Janossy. 1990. Phenotypic features and proliferative activity of B cell progenitors in X-linked agammaglobulinemia. J. Immunol. 145: 1675
    OpenUrlAbstract
  38. ↵
    Cooper, M. D., N. Nishimoto, K. Lassoued, C. Nunez, T. Nakamura, H. Kubagawa, J. E. Volanakis. 1993. Antibody deficiencies reflect abnormal B cell differentiation. J. Gergely, III, and M. Benezur, III, and A. Erdei, III, and A. Falus, III, and G. Fust, III, and G. Medgyezi, III, and G. Petranyi, III, and E. Rajnavolegyi, III, eds. Progress in Immunology 8th ed.535 Springer-Verlag Publishing Co., New York.
  39. ↵
    Raaphorst, F. M., C. S. Raman, J. Tami, M. Fischbach, I. Sanz. 1997. Human Ig heavy chain CDR3 regions in adult bone marrow pre-B cells display an adult phenotype of diversity: evidence for structural selection of DH amino acid sequences. Int. Immunol. 9: 1503
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Dorner, T., H. P. Brezinschek, R. I. Brezinschek, S. J. Foster, R. Domiati-Saad, P. E. Lipsky. 1997. Analysis of the frequency and pattern of somatic mutations within nonproductively rearranged human variable heavy chain genes. J. Immunol. 158: 2779
    OpenUrlAbstract
  41. ↵
    Taylor, L. D., C. E. Carmack, S. R. Schramm, R. Mashayekh, K. M. Higgins, C.-C. Kuo, C. Woodhouse, R. M. Kay, N. Lonberg. 1992. A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins. Nucleic Acids Res. 6287: 6295
    OpenUrl
  42. ↵
    Nadel, B., A. J. Feeney. 1997. Nucleotide deletion and P addition in V(D)J recombination: a determinant role of the coding-end sequence. Mol. Cell. Biol. 17: 3768
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Gauss, G. H., M. R. Lieber. 1996. Mechanistic constraints on diversity in human V(D)J recombination. Mol. Cell. Biol. 16: 258
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Ramsden, D. A., K. Baetz, G. E. Wu. 1994. Conservation of sequence in recombination signal sequence spacers. Nucleic Acids Res. 22: 1785
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Wasserman, R., Y. Ito, B. Galili, M. Yamada, B. A. Reichard, S. S. Shane, B. Lange, G. Rovera. 1992. The pattern of joining gene usage in the human IgH chain is established predominantly at the B precursor cell stage. J. Immunol. 149: 511
    OpenUrlAbstract/FREE Full Text
  46. ↵
    George, J. F., Jr, H. W. Schroeder, Jr. 1992. Developmental regulation of Dβ reading frame and junctional diversity in TCRβ transcripts from human thymus. J. Immunol. 148: 1230
    OpenUrlAbstract
  47. ↵
    Shimizu, T., H. Yamagishi. 1992. Biased reading frames of pre-existing DH-JH coding joints and preferential nucleotide insertions at VH-DJH signal joints of excision products of immunoglobulin heavy chain gene rearrangements. EMBO J. 11: 4869
    OpenUrlPubMed
  48. ↵
    Wasserman, R., N. Galili, Y. Ito, B. A. Reichard, S. S. Shane, G. Rovera. 1992. Predominance of fetal type DJH joining in young children with B precursor lymphoblastic leukemia as evidence for an in utero transforming event. J. Exp. Med. 176: 1577
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Steenbergen, E. J., O. J. Verhagen, L. E. van, H. Behrendt, P. A. Merle, M. R. Wester, A. E. von dem Borne, C. E. van der Schoot. 1994. B precursor acute lymphoblastic leukemia third complementarity-determining regions predominantly represent an unbiased recombination repertoire: leukemic transformation frequently occurs in fetal life. Eur. J. Immunol. 24: 900
    OpenUrlPubMed
  50. ↵
    Lassoued, K., C. A. Nunez, L. Billips, H. Kubagawa, R. C. Monteiro, T. W. LeBien, M. D. Cooper. 1993. Expression of surrogate light chain receptors is restricted to a late stage in pre-B cell differentiation. Cell 73: 73
    OpenUrlCrossRefPubMed
  51. ↵
    Wang, Y.-H., J. Nomura, O. M. Faye-Petersen, M. D. Cooper. 1998. Surrogate light chain production during B cell differentiation: Differential intracellular versus cell surface expression. J. Immunol. 161: 1132
    OpenUrlAbstract/FREE Full Text
  52. ↵
    ten Boekel, E., F. Melchers, A. G. Rolink. 1997. Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7: 357
    OpenUrlCrossRefPubMed
  53. ↵
    Wasserman, R., Y. S. Li, S. A. Shinton, C. E. Carmack, T. Manser, D. L. Wiest, K. Hayakawa, R. R. Hardy. 1998. A novel mechanism for B cell repertoire maturation based on response by B cell precursors to pre-B receptor assembly. J. Exp. Med. 187: 259
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Cramer, D. V., H. W. Kunz, T. J. Gill. 1974. Immunologic sensitization prior to birth. Am. J. Obstet. Gynecol. 120: 431
    OpenUrlPubMed
  55. ↵
    Billips, L. G., C. A. Nunez, F. E. Bertrand, III, A. K. Stankovic, G. L. Gartland, P. D. Burrows, M. D. Cooper. 1995. Immunoglobulin recombinase gene activity is modulated reciprocally by interleukin 7 and CD19 in B cell progenitors. J. Exp. Med. 182: 973
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Kabat, E. A.. 1988. Antibody complementarity and antibody structure. J. Immunol. 141: S25
    OpenUrlPubMed
  57. ↵
    Wu, T. T., G. Johnson, E. A. Kabat. 1993. Length distribution of CDRH3 in antibodies. Proteins Struct. Funct. Genet. 16: 1
    OpenUrlCrossRefPubMed
  58. ↵
    Schettino, E. W., S. K. Chai, M. T. Kasaian, H. W. Schroeder, Jr, P. Casali. 1997. VHDJH gene sequences and antigen reactivity of monoclonal antibodies produced by human B-1 cells: evidence for somatic selection. J. Immunol. 158: 2477
    OpenUrlAbstract
  59. Dersimonian, H., K. P. W. J. McAdam, C. G. Mackworth-Young, B. D. Stollar. 1989. The recurrent expression of variable region segments in human IgM anti-DNA autoantibodies. J. Immunol. 142: 4027
    OpenUrlAbstract
PreviousNext
Back to top

In this issue

The Journal of Immunology: 162 (10)
The Journal of Immunology
Vol. 162, Issue 10
15 May 1999
  • Table of Contents
Print
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for your interest in spreading the word about The Journal of Immunology.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
IgM Heavy Chain Complementarity-Determining Region 3 Diversity Is Constrained by Genetic and Somatic Mechanisms Until Two Months After Birth
(Your Name) has forwarded a page to you from The Journal of Immunology
(Your Name) thought you would like to see this page from the The Journal of Immunology web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
IgM Heavy Chain Complementarity-Determining Region 3 Diversity Is Constrained by Genetic and Somatic Mechanisms Until Two Months After Birth
Satoshi Shiokawa, Frank Mortari, Jose O. Lima, César Nuñez, Fred E. Bertrand, Perry M. Kirkham, Shigui Zhu, Ananda P. Dasanayake, Harry W. Schroeder
The Journal of Immunology May 15, 1999, 162 (10) 6060-6070;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
IgM Heavy Chain Complementarity-Determining Region 3 Diversity Is Constrained by Genetic and Somatic Mechanisms Until Two Months After Birth
Satoshi Shiokawa, Frank Mortari, Jose O. Lima, César Nuñez, Fred E. Bertrand, Perry M. Kirkham, Shigui Zhu, Ananda P. Dasanayake, Harry W. Schroeder
The Journal of Immunology May 15, 1999, 162 (10) 6060-6070;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Structure and Functional Characterization of a Humanized Anti-CCL20 Antibody following Exposure to Serum Reveals the Formation of Immune Complex That Leads to Toxicity
  • Loss of the Transfer RNA Wobble Uridine–Modifying Enzyme Elp3 Delays T Cell Cycle Entry and Impairs T Follicular Helper Cell Responses through Deregulation of Atf4
  • The Role of the HLA Class I α2 Helix in Determining Ligand Hierarchy for the Killer Cell Ig-like Receptor 3DL1
Show more MOLECULAR AND STRUCTURAL IMMUNOLOGY

Similar Articles

Navigate

  • Home
  • Current Issue
  • Next in The JI
  • Archive
  • Brief Reviews
  • Pillars of Immunology
  • Translating Immunology

For Authors

  • Submit a Manuscript
  • Instructions for Authors
  • About the Journal
  • Journal Policies
  • Editors

General Information

  • Advertisers
  • Subscribers
  • Rights and Permissions
  • Accessibility Statement
  • Public Access
  • Privacy Policy
  • Disclaimer

Journal Services

  • Email Alerts
  • RSS Feeds
  • ImmunoCasts
  • Twitter

Copyright © 2021 by The American Association of Immunologists, Inc.

Print ISSN 0022-1767        Online ISSN 1550-6606