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The Journal of Immunology, 2005, 175: 7425-7436.
Copyright © 2005 by The American Association of Immunologists

Developmental Changes in the Human Heavy Chain CDR3

M. Margarida Souto-Carneiro*,{dagger}, Gary P. Sims*, Hermann Girschik{ddagger}, Jisoo Lee§ and Peter E. Lipsky1,*

* Repertoire Analysis Group, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; {dagger} Instituto Gulbenkian de Ciência, Oeiras, Portugal; {ddagger} Division of Rheumatology, Children’s Clinic, Faculty of Medicine, University of Würzburg, Würzburg, Germany; and § Division of Rheumatology, Department of Internal Medicine, Ewha Womans University College of Medicine, Seoul, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CDR3 of the Ig H chain (CDR3H) is significantly different in fetal and adult repertoires. To understand the mechanisms involved in the developmental changes in the CDR3H of Ig H chains, sets of nonproductive VHDJH rearrangements obtained from fetal, full-term neonates and adult single B cells were analyzed and compared with the corresponding productive repertoires. Analysis of the nonproductive repertoires was particularly informative in assessing developmental changes in the molecular mechanisms of VHDJH recombination because these rearrangements did not encode a protein and therefore their distribution was not affected by selection. Although a number of differences were noted, the major reasons that fetal B cells expressed Ig H chains with shorter CDR3H were both diminished TdT activity in the DJH junction and the preferential use of the short JH proximal D segment D7–27. The enhanced usage of D7–27 by fetal B cells appeared to relate to its position in the locus rather than its short length. The CDR3H progressively acquired a more adult phenotype during ontogeny. In fetal B cells, there was decreased recurrent DJH rearrangements before VH-DJH rearrangement and increased usage of junctional microhomologies both of which also converted to the adult pattern during ontogeny. Overall, these results indicate that the decreased length and complexity of the CDR3H of fetal B cells primarily reflect limited enzymatic modifications of the joins as well as a tendency to use proximal D and JH segments during DJH rearrangements.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Ab repertoire during fetal life and at the time of full-term birth is restricted and markedly distinct from that of the adult because there is no exposure to environmental Ags during the early stages of ontogeny (1, 2, 3, 4, 5, 6, 7, 8). Although it remains unresolved whether there is restricted usage of particular IgVH segments (1, 9, 10, 11, 12, 13, 14, 15, 16), it is generally accepted that the greatest differences between fetus, full-term neonate, and adult reside within the CDR3 of the Ig H chain (CDR3H).2 Particularly noteworthy is the increase in length of the CDR3H during ontogeny. (6, 11, 13, 15, 17, 18, 19, 20, 21). This increase in length seems to be related to several factors, such as the choice of IgDH and IgJH segments, the level of junctional TdT activity, the use of homology-directed recombination, and the level of excision in the coding ends (2, 6, 11, 17, 18, 19, 20, 21, 22). However, the use of different sampling and sequence analysis methods and the relatively small number of rearrangements analyzed in most studies have made it difficult to determine accurately the extent to which those factors influence the ontological changes in the CDR3H. Additionally, it is not clear whether these changes relate to alterations in molecular mechanisms of V(D)J recombination throughout ontogeny or to positive or negative selection by endogenous Ags because previous studies concentrated solely on the analysis of productive V(D)J rearrangements. We have found that the impact of molecular mechanisms and selection can be separately assessed by analyzing the nonproductive as well as the productive repertoire. The former comprises sequences that do not encode a protein and, therefore are not influenced by selection, whereas the latter is influenced by both the molecular mechanisms of V(D)J recombination and selection.

In an effort to clarify the role of the contributing factors to developmental changes in CDR3H, we analyzed several sets of both productive and nonproductive VHDJH rearrangements obtained from single B cells from fetal, full-term neonatal, and adult samples. By analyzing nonproductive rearrangements along with the productive repertoire, the present study sought to identify the developmentally related changes that result from the molecular events governing rearrangement separately from the influence of selection on CDR3H expression over the course of human development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Amplification of the IgV H chain by single cell PCR

Cord blood mononuclear cells from six full-term neonates were isolated using either a Ficoll gradient (Amersham) or using RosetteSep (StemCell Technologies) and stained with a PE-labeled CD19 mAb (BD Biosciences). Single-cell preparations were made from three fetal spleens and livers at 18 wk of gestation by mechanical disruption of tissue fragments, followed by filtration through nylon mesh. Fetal mononuclear cells were enriched by Ficoll density gradient centrifugation. The fetal cells were then stained with a PE-labeled anti-CD19 mAb (BD Biosciences) and an FITC-labeled anti-human IgM mAb (Caltag Laboratories). All tissue collections and processing were done in accordance with policies established by the Institutional Review Board for human subjects at the National Institutes of Health (Bethesda, MD). The CD19+/IgM+ or IgM– fetal B cells and the CD19+ full-term neonatal B cells were sorted using a FACSDiva (BD Biosciences) or a Dako Cytomation MoFlo (DAKO) into 5-ml tubes containing 500 µl of 1x PBS. The total sorted cells were diluted to a final concentration of 1–1.5 cells/5 µl of PBS, and then aliquoted into 96-well PCR plates containing 10 µl of lysis buffer (2x PCR buffer plus 0.4 mg/ml proteinase K; Sigma-Aldrich). Cells were lysed for 60 min at 56°C followed by a denaturation step of 95°C for 10 min to isolate genomic DNA. The total genomic DNA was amplified with an initial untemplated primer extension preamplification PCR (23). A total of 5 µl amplified genomic DNA were added to 75 µl of external PCR mixture (55 µl of double distilled H2O, 2.5 mM Mg2+, 260 µM dNTP, 1x PCR buffer, 4 U Taq Polymerase (Promega)) plus 100 µM external family specific IgVH forward primers including primer sequences: IgVH1 5'-RCC YAC KCC SAR RTB CAG CTG-3'; IgVH2 5'-ATG GAC ACA CTT TGC TMC ACR-3'; IgVH3 5'-CCA TGG AGT TTG GGC TGA G-3'; IgVH4 5'-GAA ACA CCT GTG GT CTT C-3'; IgVH5 5'-ATG GGG TCA ACC GCC ATC CT-3'; IgVH6 5'-CTC ATC TTC CTG CCC GTG CTG-3' plus a 200 µM external reverse IgJH primer 5'-ACC TGA GGA GAC GGT GAC-3'. An external touchdown PCR was conducted on a DNA Engine Tetrad peltier thermal cycler (MJ Research). A total of 5 µl of the external PCR product were used to set up the nested PCR (55 µl double distilled H2O, 2.5 mM Mg2+, 260 µM dNTP, 1x PCR buffer, 4 U Taq Polymerase (Promega)) plus 100 µM internal family specific IgVH forward primers including primer sequences: IgVH1 5'-CCT ACG TGA GGT YTC CTG CAA GGC-3'; IgVH2 5'-CAG RTC ACC TTG AAG GAG TCT-3'; IgVH3 5'-GTC CAG TGT SAG GTG CAG C-3'; IgVH4 5'-GGT GCA GCT GCA GGA GTC G-3'; IgVH5 5'-AAA AAG CCC GGG GAG TCT CTG ARG A-3'; IgVH6 5'-CAG GTA CAG CTG CAG CAG TCA-3') plus 100 µM internal reverse IgJH1, IgJH2, IgJH4, and IgJH5 primer 5'-GTG ACC RTK GTC CCT TGG CCC-3' plus the 100 µM internal reverse IgJH3 and IgJH6 primer 5'-TGA CCA GGG TKC CM GGC CC-3'. PCR products were purified using the Performa 96-Well Standard Plate kit (Edge BioSystems) and sequenced on a model 3100 capillary sequencer (Applied Biosystems) using the Big DyeTerminator v.1.1 Cycle Sequencing kit (Applied Biosystems) (GenBank accession numbers AY429730-430046 and AY607317-AY607564).

Compilation of the database

The following Ig H chain sequences were analyzed (Tables I and II). These sequences included: 1) 494 sequences from full-term neonatal B cells; 2) a control set of 651 sequences from genomic DNA (n = 400) and cDNA (n = 251) obtained from individual healthy human adult B cells (GenBank accession numbers Z80363-770, AY003749-869, and Z68345-487) (23, 24, 25); 3) a set of 138 sequences from cDNA obtained from healthy human preterm neonate cord blood B cells (GenBank accession number AF235505-642) (11); 4) a set of 46 sequences from cDNA from human fetal B cells (GenBank accession numbers X62954-72, X63080-1, M34022-32, and M18508-21 (9, 16, 26); along with 5) 75 sequences from genomic DNA of individual human fetal B cells (GenBank accession numbers AF297151-70, AY013306-9, and AY582384-435).


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  		Table I. Rearrangements analyzed in this study

 

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  		Table II. Rearrangements analyzed (details)

 
Sequence analysis

All rearrangements were matched to their closest germline counterparts using the Web-based program JOINSOLVER (27). Rearrangements were considered productive if the VHDJH junction maintained the reading frame into the JH segment and contained no stop codons in the germline D segment or CDR3H junctions. Otherwise rearrangements were considered nonproductive. Junctional nucleotide additions between the VH and D or between the D and JH segments were scored as either P nucleotides, if they were inverted repeats of germline encoded ends, or N nucleotides, if they were nontemplated junctional additions. The junctions without N additions that contained base pairs that could have been encoded by either of the approximated coding ends, and therefore, could represent short regions of homology between the VH and D or the D and JH regions were considered to be microhomologies. D segment matches and D segment fusions were only accepted when they fulfilled the criteria established previously, based on a Monte Carlo analysis designed to distinguish actual matches from random chance (27). In cases in which the nucleotide sequence between the VH and JH coding ends had the same number of matches with a DIR family member or a D segment encoded on chromosome 15 and a conventional D segment, the latter was accepted as the D element used. Rearrangements using DD fusions, inverted D segments, or DIR segments were excluded from the D segment reading frame analysis.

Statistical analysis

To determine significant differences in distributions in productive and nonproductive rearrangements, the {chi}2 test was used. Values of p ≤ 0.05 were considered to be significant. The statistical significance between observed and expected frequencies in D genes and D gene reading frames was calculated using the {chi}2 goodnessoffit test. Student’s t test was used to analyze CDR3H length, VHJH distance, D segment match length, P and N nucleotides, and VH, D, and JH excision.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CDR3H length and D segment match increase with developmental stage

As shown in Tables III and IV (with details for each individual sample), mean CDR3H length of the nonproductive rearrangements increased significantly (p < 0.05) between the early and later stages of human development (fetal 42.1 ± 2.2 bp < full-term neonates 51.3 ± 1.4 bp ≤ adult 53.8 ± 1.9 bp). A similar trend was evident with the productive repertoire, where significant differences between fetal and full-term, and full-term and adult were present (fetal 38.6 ± 0.9 bp ≤ preterm neonates 39.4 ± 0.9 bp < full-term neonates 44.0 ± 0.6 bp < adults 46.7 ± 0.5 bp). Notably, at each post-fetal stage of human development, the CDR3H length in the productive repertoire was significantly (p < 0.05) shorter than in the nonproductive. Moreover, when calculating the mean match length of the assigned D segments (Table V), the fetal nonproductive repertoire had significantly (p < 0.05) shorter consecutive matches than the full-term and adult B cells (14.6 ± 1.3 bp vs 18.4 ± 0.8 bp vs 17.6 ± 0.7 bp). Finally, the nonproductive repertoire of full-term and adult B cells had significantly (p < 0.05) longer consecutive matches (18.4 ± 0.8 bp and 17.6 ± 0.7 bp, respectively) than the productive rearrangements (16.5 ± 0.3 bp and 14.6 ± 0.2 bp, respectively). Similar results were noted when the VH-JH distance was calculated, indicating that neither the VH or JH segment contributed to the differences in the CDR3H length.


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  		Table III. Mean CDR3H length and VH-JH distance of the productive and nonproductive rearrangements (± SE)

 

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  		Table IV. Mean CDR3H length of the productive and nonproductive rearrangements for each individual sample (±SD)

 

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  		Table V. Frequency of rearrangements with unmatched D segments and mean length of the D segment match (±SE)

 
D segment usage

In the nonproductive repertoire, we could identify significantly (p < 0.05) fewer D segments in the fetal B cells than in the full-term or adult B cells (Tables V and VI). In contrast, in the productive repertoire we could identify significantly (p < 0.05) more D segments in the full-term B cells than in the fetal, preterm, and adult B cells. For the remaining rearrangements in the fetal, preterm, full-term, and adult B cells, no D segments could be unambiguously identified because the consecutive D match length was too short. As shown in Fig. 1, the use of D segments was not random. In general in the nonproductive repertoire, JH proximal D segments (with the exception of D6–6) were over-represented in the fetus (56.25% of the JH proximal half of the locus), whereas the distal half of the locus was more frequently used in the full-term neonates (33.33%) and the adult (43.86%). In the nonproductive repertoire, 4 of 25 genes were used significantly (p < 0.05) more than expected from random chance in the fetal (D6–6, D2–21, D1–26, D7–27) and in the adult (D2–2, D3–10, D2–21, D3–22) B cells, whereas 5 of 25 genes were used significantly (p < 0.05) more than expected from random chance in the full-term B cells (D2–2, D3–3, D6–13, D2–15, D3–22). Moreover, a number of D segments were not detected in the nonproductive repertoires. In the productive repertoire, 8 D segments were used more than expected from random chance in the preterm B cells (D2–2, D6–6, D2–8, D3–10, D6–13, D6–19, D1–26, D7–27), 7 D segments were used in the adult (D2–2, D3–3, D3–10, D6–13, D6–19, D3–22, D1–26), whereas fetal and full-term B cells used 5 D segments (D6–6, D1–7, D3–10, D6–13, D7–27) and 7 D segments (D3–3, D6–6 D3–10, D6–13, D6–19, D3–22, D1–26), respectively, more than expected from random chance. Notably, only 2 D segments (D1–14 and D6–25) were missing from the productive repertoire in the adult B cells, presumably because they cannot undergo recombination effectively. Those two segments were also absent from the fetal and preterm productive rearrangements, whereas D1–14 was found once in the full-term productive repertoire. When the distribution of D segments in the nonproductive and productive repertoires was compared, evidence of both positive and negative selection was found, but no clear developmental pattern could be observed. The fetal B cells showed significant (p < 0.05) negative selection of D6–6, D1–26, D2–21, and D6–19, whereas D3–3, D1–7, and D6–13 were positively selected. In the full-term B cells, D2–2, D6–13, and D7–27 were negatively selected, whereas D3–3, D6–6, and D6–19 were positively selected. The adult repertoire exhibited negative selection of D2–2, D2–8, and D2–21 and positive selection of D6–6 and D1–26.


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  		Table VI. Frequency of JH proximal 3' D (D1–26 and D7–27) segments for each individual sample

 


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  		FIGURE 1. Distribution of the IgDH segments in the fetal (a), preterm (b), full-term neonates (c), and adult (d) rearrangements. Frequency (%) of nonproductive ({blacksquare}) and productive ({square}) rearrangements is represented. D segments significantly over-represented ({blacktriangledown}) in the nonproductive repertoire are indicated. Significant (*, p < 0.05) difference between productive and nonproductive rearrangements is shown.

 
DIR family members could be identified in 1.6% of the adult nonproductive rearrangements and 2.3%, 1.1%, and 1.4% of the productive rearrangements from fetal, preterm, and adult B cells, respectively (Fig. 1), but were not found in nonproductive fetal or neonatal sequences. Inverted D segments could not be found in the nonproductive rearrangements of any developmental stage, but in the productive rearrangements they were found significantly (p < 0.05) more often in the preterm than in the fetal, full-term, and adult stages (7.5% vs 2, 1.6, and 3.8%, respectively). Inverted D segments were positively selected in the fetal and adult productive repertoires. Fetal B cells did not use multiple D segments. This was an infrequent occurrence in preterm, full-term, and adult B cells. All the preterm and full-term rearrangements with multiple D segments were organized as VHD5'D3'JH, which reflects the normal sequence of recombination events. The appearance of rearrangements with multiple D segment fusions was significantly more frequent in the full-term and adult nonproductive repertoires (1.3 and 3.9% of the total nonproductive rearrangements, respectively, p < 0.05), compared with that in productive repertoires (0.7 and 1% of total rearrangements, respectively), suggesting that these rearrangements were negatively selected.

JH segment usage

The six JH segment families were used differently in the rearrangements of each developmental stage (Fig. 2). In both the nonproductive and productive repertoires of all developmental stages, the JH1 family was the least common (p < 0.01) used. The JH3 family was used significantly (p < 0.01) more often in both nonproductive and productive repertoires of the early developmental stages than in the adult stage B cells. The JH2 family could be found significantly (p < 0.05) more often in the productive repertoire of early developmental stages than in the adult stage. The JH5 and JH6 families were used significantly (p < 0.01) less in the productive fetal repertoire than in any other developmental stage. No differences could be observed in the usage of the JH4 family. Because we obtained very similar frequencies for all JH families to previous studies analyzing cDNA with Cµ reverse primers (19), a potential bias in favor of any of the families, resulting from the use of separate JH primers, is unlikely.



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  		FIGURE 2. Distribution of the IgJH segments in the fetal (a), preterm (b), full-term neonates (c), and adult (d) rearrangements. Frequency (%) of nonproductive ({blacksquare}) and productive ({square}) rearrangements is represented. Significant (*, p < 0.05) difference between productive and nonproductive rearrangements is indicated. Significant (@, p < 0.05) difference is also determined when compared with adult rearrangements.

 
Preferential rearrangement of 5'D segments with 3'JH segments

We analyzed the combinatorial preferences of D and JH segments in the nonproductive rearrangements to determine whether there was a bias for particular DJH rearrangements. A significant bias (p < 0.01, {chi}2 test) could be observed in the pairing of D and JH segments, with 3'D segments (JH proximal) coupled preferentially to 5'JH segments (Fig. 3, a–c) throughout development. This bias was not found in any of the productive rearrangements (data not shown). However, there was a significant and progressive increase (p < 0.05) of 5'D segments (JH distal) pairing with 3'JH segments in the full-term neonates and adult rearrangements compared to the fetus. Additionally, in the fetal and full-term stages, there was an evident bias in the pairing of the two most 3'D segments (D1–26 and D7–27) with 5'JH segments, particularly JH2 and JH3 when compared with the adult (Fig. 3d). In the adult, D1–26 and D7–27 were exclusively paired with JH4.



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  		FIGURE 3. Pairing of IgDH and IgJH segments in the nonproductive repertoire of the fetal (a), full-term neonates (b), and adult (c) rearrangements. D proximal ({square}) JH genes (JH1, JH2, JH3, and JH4) and D distal ({blacksquare}) JH genes (JH5 and JH6) are indicated. The D genes are divided into five groups according to their position in the locus (D1–1 to D5–5 are the most JH distal and D1–26 and D7–27 are the most JH proximal). d, Pairing of the two most JH proximal D segments (D1–26 and D7–27) with the different JH segments in the nonproductive fetal ({square}), full-term neonates ({cjs2108}), and adult ({blacksquare}) rearrangements. Significant (*, p < 0.05) bias in the pairing of the D and JH segments. Significant (@, p < 0.05) difference also shown when compared with adult. Only the rearrangements for which a D segment could be assigned were analyzed.

 
TdT activity

In the fetal, preterm, and full-term nonproductive rearrangements, there were significantly (p < 0.05) more N nucleotides inserted between the VH and the D coding ends than inserted between the D and JH (Table VII). In contrast, the mean number of VHD and DJH N nucleotide additions was comparable in the adult nonproductive rearrangements. As a result, N nucleotide insertions in the DJH junction from both fetal and full-term nonproductive rearrangements were significantly (p < 0.05) shorter than the adult ones. This finding was particularly notable in the fetal DJH joins (4.6 ± 1.1 N nucleotide additions) vs adult DJH joins (9.9 ± 0.9 N nucleotide additions). Additionally, in the productive repertoire, the number of N nucleotides inserted in the VHD and DJH junctions of fetal, preterm, and full-term rearrangements was significantly (p < 0.05) less than the number for the adult rearrangements. When comparing the number of N nucleotides inserted in both junctions between the productive and nonproductive rearrangements, clear evidence of positive selection for shorter junctional N nucleotide additions was observed at all developmental stages.


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  		Table VII. TdT activity in the VHD or DJH junctions of productive and nonproductive rearrangements

 
The presence of N nucleotides was the most common situation in the adult rearrangements, although there were a few sequences that lacked N nucleotide additions at either the VHD (nonproductive 3.6%, productive 3.0%) or DJH (nonproductive 7.2%, productive 7.3%) junction. In the nonproductive repertoire, the VHD junction of fetal rearrangements lacked N additions significantly more often than the full-term and adult ones (6.3 vs 1.5% and 3.6%, respectively, p < 0.05). Moreover, a significantly (p < 0.05) larger number of rearrangements from the early developmental stages lacked N additions in the DJH junction when compared with the adult rearrangements, as found in both the nonproductive (fetus 43.8%, full-term 21.2%) and in the productive (fetus 59.1%, preterm 58.7%, and full-term 30.7%) repertoires.

In the adult productive repertoire, the DJH N nucleotide additions in rearrangements using the D1 through D6 families, were significantly (p < 0.05) greater than the additions using the D7 family (D1: 2.7, D2: 2.9, D3: 3.9, D4: 2.2, D5: 4.7, D6: 2.6 bp vs D7: 0.8 bp). However, this difference could be a result of the small number of adult rearrangements using the D7 family. Moreover, for the other developmental stages no significant differences in the number of N additions in rearrangements using different D families were noted, possibly because of the reduced number of sequences for each family.

Characterization of rearrangements with DJH junction microhomologies

As shown in Tables VIII and IX, the percentage of rearrangements with microhomologies decreased significantly (p < 0.05) with increasing age in both nonproductive and productive repertoires. Moreover, the DJH junction had a significantly (p < 0.05) higher percentage of sequences with microhomology than the VHD junction in the fetal and neonatal repertoires. In addition, rearrangements with microhomologies appeared to be positively selected in the fetal and neonatal repertoires. Because rearrangements with DJH junction microhomologies were so common in the early developmental stages we characterized them in greater detail. There was no preferential DJH pairing in rearrangements with DJH junction microhomologies. Furthermore, the distribution of the different D and JH segments was similar to the pattern of usage at each developmental stage. Additionally, with the exception of the significantly (p < 0.05) elevated usage of reading frame 2 in the full-term rearrangements, no other significant reading frame differences were observed in the fetal and preterm rearrangements with DJH junction microhomologies (data not shown). Because the frequency of microhomology can be influenced by the use of TdT (28, 29), we assessed whether the frequency of microhomologies differed with developmental stage only in those sequences that exhibited no TdT activity (Tables VIII and IX). It was apparent that the frequency of microhomologies in the sequences that did not have evidence of TdT activity was significantly less (p < 0.05) in the adult than in any of the other stages of development.


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  		Table VIII. Frequency of microhomologies in the VHD or DJH junctions of productive and nonproductive rearrangements

 

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  		Table IX. Frequency of microhomologies in rearrangements without evidence of TdT activity

 
Exonuclease activity

The VH coding end had significantly (p < 0.05) less exonucleolytic excision when compared with the D and JH coding ends, both in the nonproductive and productive repertoires of B cells from all developmental stages (Table X). In all stages, D segment and JH coding end excision was similar in the nonproductive or productive repertoires. However, the D5' coding end excision was comparable between fetal, full-term, and adult rearrangements in both the productive and nonproductive repertoires, whereas the D3' and JH coding ends from fetal and full-term nonproductive rearrangements were excised significantly (p < 0.05) less than the adult counterparts. This was not corrected by selection because the same trend could be observed in the productive repertoire.


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  		Table X. Exonuclease activity in the VHD or DJH junctions (±SE)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study used the single cell PCR technique to obtain productive and nonproductive H chain rearrangements from human fetal, full-term cord blood and adult B cells, allowing a more comprehensive analysis of the development of the CDR3H characteristics throughout human ontogeny. By including the nonproductive rearrangements in our analysis, we could clearly separate the roles of selection from the molecular mechanisms of VHDJH rearrangements in determining the characteristics of the CDR3H.

As it has been previously reported in mice (1, 15, 17, 21), pigs (30), macaques (3), and humans (1, 2, 11, 13, 18, 19) the length of the CDR3H increased with developmental stage. In the current study, this finding was noted in the nonproductive repertoires indicating that the developmental changes in CDR3H length are largely dependent on the molecular mechanisms of VHDJH recombination. It is notable that selection altered the CDR3H length of term neonate and adult sequences, but not that of the fetus, resulting in a shorter mean CDR3H. The fetal CDR3H remained short because of the overuse of the shortest D segment, D7–27, and the reduced TdT activity, particularly in the DJH junction, even after selection. The abundant use of D7–27 was sufficient to cause the entire fetal repertoire to have a shorter mean CDR3H length because the mean length of the retained fetal D segments other than D7–27 was similar to that of the adult repertoire.

In our analysis, and contrary to previous reports (13, 17, 18, 31), we found similar low frequencies of DIR, inverted D segment, and multiple D segment fusions at all stages, suggesting that the molecular constraints imposed on their use during recombination are already present in early ontogeny.

The developmental changes in the length of the CDR3H appeared to have a direct influence on the capacity to identify D segments. Because of its shorter length, the fetal stage had the lowest frequency of matched D segments. Using an approach that permits identification of D segments by a statistical algorithm (27), we were able for the first time to assess the differences in D segment usage throughout ontogeny, and define whether they are the result of molecular or selection changes. The most notable difference in the fetal nonproductive rearrangements was the increased usage of the two most JH proximal segments D1–26 and D7–27, and decreased usage of the VH proximal segments such as D2–2 or D3–3. However, by the time the in utero gestational period was over, the D segment use shifted to the 5'VH proximal end of the locus, particularly increasing the frequencies of D2–2, D3–3, D3–10, and D6–13 in full-term neonates and adults. Moreover, the finding that in the fetus the two most JH proximal D segments, D1–26 and D7–27, preferentially rearranged with JH2 and JH3 further supports the hypothesis that proximity of the D segment and JH pairing is favored in the VHDJH rearrangement process in B lineage cells of the fetus (32, 33). Even though reports suggested that the reason for fetal D7–27 pairing with 5'JH segments was based on the D segment size (12, 19), we consider this unlikely because other small D segments, such as the ones in the D4 family, were not frequently used, whereas the relatively long D1–26, which is the second most JH proximal D segment, also paired preferentially with 5'JH segments. Therefore, it appears more likely that it is the locus position of the D segment and not the size that directs the DJH pairing in the fetus.

Previously, we have observed that in adults a significant bias was noted in the tendency for 5'D segments to rearrange with 3'JH segments without relation to the position of the VH gene (27). This tendency was consistent with the conclusion that during B cell development, multiple rounds of DJH rearrangement occurred before final VH to DJH recombination. The net result was the bias for 5'D segments to be rearranged to 3'JH genes with random VH usage. By analyzing the fetal and full-term nonproductive repertoires for VH, D, and JH segments, we were able to detect a strong bias away from the use of 5'D segments in the fetus (with the exception of D6–6), whereas in the full-term neonates a tendency toward the pairing of 5'D segments with 3'JH segments could be observed. This observation suggests that multiple DJH rearrangements in the fetus are less frequent than in full-term neonates and adults, and that there is progressive usage of recurrent DJH rearrangements with developmental age.

Although in the adult it is commonly accepted that the mean degree of TdT activity, which can be inferred from the number of N nucleotide additions, on the VHD and DJH junctions is similar in both productive and nonproductive repertoires (18, 27, 34, 35), there is still no absolute consensus on junctional TdT activity in early developmental stages. Some reports do not separate the TdT activity for each junction (17, 18, 20). Others indicate less N nucleotide additions in the VHD junction compared with the DJH junction (31, 36), contradicting other reports of more N nucleotide additions in the VHD than in the DJH junctions of mouse B cells precursors (21, 33, 37) and human preterm and full-term neonate mature B cells (11, 14). In our study after separating the number of N nucleotide additions for each junction, we confirmed that in the early developmental stages the VHD junction manifests significantly more TdT activity than the DJH junction in the nonproductive and productive repertoires. Moreover, whereas the TdT activity in the VHD junction is similar throughout human ontogeny, in the DJH junction, the TdT activity increased during in utero gestation (6), but in full-term neonatal sequences we observed that N nucleotide additions were still half that observed in adults. Additionally, in the present study we did not observe an increase in N nucleotide addition in the DJH junction when particular D segments were used, as reported previously for DH3 and DH7 (19).

Although in adult rearrangements only a small percentage of DJH junctions have no N additions, the frequency is much higher than 20% at all gestational ages. Because the DJH junction is the first to be formed and is already present in the common lymphoid progenitor cells before there is a lineage commitment, it is likely that the high frequency of DJH junctions lacking or with very little TdT activity in the early stages of ontogeny reflects the low levels, or even absence, of TdT activity present in human common lymphoid progenitor cells before there is B or T lineage commitment. For instance in cell lines from 13- to 15-day-old fetal mice, the DJH joins did not contain any N nucleotide additions, which was related to a complete absence of TdT expression, whereas fetal cell lines from later developmental stages contained both N additions and TdT expression and activity (22). A similar process may occur during human B cell development, with increased N nucleotide addition resulting from increased expression of TdT after lineage commitment. Thus, the increased number of N nucleotides in the VHD junction, which is formed after the DJH junction in rearranging B cells, could be related to a higher level of TdT activity after lineage commitment. However, an increase in TdT activity on the DJH junction seems to be the result of a higher TdT activity in later developmental stages. The increase may also result from a substitution of the first (early fetal) B cell repertoire with DJH joins lacking N additions with a more mature one in which the corresponding pre- and pro-B cells are formed at a developmental stage in which TdT expression and activity has increased considerably.

The reduced TdT activity in the DJH junction during the gestational period until full-term birth was closely related to the frequency of use of homology-targeted recombination. In neonate and adult mice, PMS2-deficient mice and in neonatal muscovy ducks (38, 39, 40), the use of homology is a common process to resolve coding end recombination. Homology-targeted recombination is also present in the majority of junctions in TdT–/– mice (41, 42). Notably in mice, the 3' end of D segments always terminates with an AC motif, whereas the JH segment has a TAC motif at the 5' end. The presence of these motifs causes DJH junction microhomologies to be very common events, ensuring that the D segment is set in reading frame 1 (21, 43). Even though we could observe microhomologies at the DJH junction during all gestational phases and at full-term birth, they neither favored a specific D segment reading frame nor a particular DJH pairing. Importantly, in humans the existence of D3' end and JH5' end motifs favoring homologous recombination is not very clear because of the 24 functional D segments, only 18 have an AC motif close to the 3' end, and the JH3 segment has no 5' TAC motif. Therefore, the formation of microhomologies may be less favored in human VHDJH rearrangements. However, only the rare participation of D7–27 in DJH microhomologies (2.3% of all microhomologies) can be explained by the absence of a D3' end AC motif. It is also notable that microhomology containing rearrangements appear to be positively selected.

These findings suggest that in these early stages of human development, the presence of homology targeted recombination, and the associated constraints to nucleolytic processing (44, 45), is a consequence of the lack of TdT activity. It might also be a way to repair the double strand breaks in the absence of TdT, ensuring the formation of functional BCRs that allow B cell survival (46), while restricting diversity and perhaps preventing autoimmunity (47, 48). If the prevention of autoimmunity were an outcome of the use of microhomology for recombination, it would be expected that an alteration of amino acid sequence might be apparent (1, 49, 50). However, comparison of the amino acid composition of CDR3H from fetal, preterm, and full-term rearrangements with and without DJH microhomologies yielded no significant differences (data not shown). Interestingly, the frequency of arginine residues was similar to the one expected from random chance in both fetal and full-term rearrangements with DJH microhomology, and not reduced as reported for TdT-deficient MRL-Faslpr mice lacking junctional N nucleotide additions (47, 48). Therefore, we have no evidence to suggest that the use of microhomology for recombination alters either the amino acid composition or the number of arginine residues in the human CDR3H.

Previous works (1, 19) reported higher levels of exonucleolytic excision of the D3 family members than of D7–27 in fetal rearrangements. Even though we noticed family related differences in exonucleolytic excision at all developmental stages, it is important to note that the D3 family members are roughly three times longer than the 11 bp long D7–27 (34). Thus, whereas the D3 family members can undergo extensive excision and still be identified (27), a similar level of excision in D7–27 no longer permits a correct identification of the segment, making it difficult to compare excision levels between those two families. Furthermore, we could not confirm a significantly higher excision of the JH4 segment when paired with D7–27 (6.5 ± 2.5 bp excised) than when paired with D3 family members (4.6 ± 0.9 bp excised) as reported (1, 19). We therefore conclude that exonucleolitic activity is not D segment-dependent at any developmental stage

In all developmental stages the exonucleolytic activity was greater on the DJH junction than on the VHD join. However, in the fetal, preterm, and full-term rearrangements the excision of the D3' and JH coding ends was significantly lower than in the adults, contrasting to the similar levels of VH and D5' coding end excision throughout ontogeny. Thus, the low exonucleolytic activity on the fetal D3' and JH coding ends seems to be related to the formation of the DJH join during the common lymphoid progenitor stage, when exonuclease and TdT activity appear to be reduced. However, when the VH segment is joined in the fetal rearrangements, the exonucleolytic activity has already increased to an adult-like level, and therefore both the VH and the D5' coding ends present similar excision as the adults.

In this study we could confirm previous reports from cultured mouse B cell precursors (37), mouse B cells (51), and human cord blood pre-B cells (36) that the ratio between productive and nonproductive rearrangements increases significantly throughout development. Moreover, if both alleles are rearranged, the ratio between productive and nonproductive rearrangements should be 2.5:1, which in our study is closest to that noted with the fetal repertoire. This finding implies that the frequency with which both chromosomes are rearranged is greater in the fetal stage than in the adult. There is no mechanism to explain this difference although the possibility that B cell development is slower in the fetus or that RAG enzymes are less tightly regulated could contribute to the greater tendency to attempt VHDJH rearrangements on both loci.

In summary, using nonproductive VHDJH rearrangements, the present study demonstrates clearly, that differences in CDR3H length throughout human development are the direct results of ontological variation in the molecular events of the VHDJH recombination. Additionally we could document that the significant differences in CDR3H size between fetus and adult is solely the result of an over-representation of the shortest D segment, D7–27, and decreased TdT activity in the fetal VHDJH rearrangements.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Peter E. Lipsky, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 6D47C, Bethesda, MD 20892-1820. E-mail address: LipskyP{at}mail.nih.gov Back

2 Abbreviation used in this paper: CDR3H, CDR3 of the Ig H chain. Back

Received for publication June 28, 2005. Accepted for publication September 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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