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A Single D_H Gene Segment Creates Its Own Unique CDR-H3 Repertoire and Is Sufficient for B cell Development and Immune Function¹

Robert L. Schelonka^2,*, Ivaylo I. Ivanov^2,3,*, David H. Jung, Gregory C. Ippolito^*, Lars Nitschke, Yingxin Zhuang^*, G. Larry Gartland^*, Jukka Pelkonen, Frederick W. Alt, Klaus Rajewsky and Harry W. Schroeder, Jr^4,*,

^* Departments of Pediatrics, Microbiology, Medicine, and Genetics, University of Alabama, Birmingham, AL 35294; The Center for Blood Research, Harvard Medical School, Boston, MA 02115; Department of Genetics, Universität Erlangen-Nürnberg, Erlangen, Germany; and Department of Clinical Microbiology, University of Kuopio, Kuopio, Finland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
To test the contribution of individual D gene segments to B cell development and function, we used gene targeting to create mice that contain only DFL16.1 in the D_H locus. We term this D-limited IgH allele D-DFL. Although the absolute number of IgM⁺IgD^– B cells in the bone marrow was decreased, homozygous D-DFL BALB/c mice contained normal numbers of IgM⁺IgD⁺ B cells in bone marrow and spleen and normal numbers of B1a, B1b, and B2 cells in the peritoneal cavity. Bone marrow IgM⁺IgD⁺ B cells express a CDR-H3 repertoire similar in length and amino acid composition to the DFL16.1 subset of the wild-type BALB/c repertoire but divergent from sequences that do not contain DFL16.1. This similarity in content is the product of both germline bias and somatic selection, especially in the transition to the mature IgM⁺IgD⁺ stage of development. Serum Ig concentrations and the humoral immune response to a T-dependent Ag ([4-hydroxy-3-nitrophenyl]acetyl hapten) were nearly identical to wild-type littermate controls. A greater variance in the immune response to the T-independent Ag ((13)-dextran) was observed in D-DFL homozygotes, with half of the mice exhibiting levels below the range exhibited by controls. Although limited to a repertoire specific to DFL16.1, the presence of a single D_H gene segment of normal sequence was sufficient for development of normal numbers of mature B cells and for robust humoral immune function.

	Introduction

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A highly diversified Ig repertoire enables the adaptive immune system to recognize a wide array of ancient and novel pathogens and toxins (1, 2, 3, 4, 5). For the H chain, the focus for combinatorial diversification is CDR-3 (CDR-H3),⁵ which is created de novo by the rearrangement and juxtaposition of individual variable (V_H), diversity (D_H), and joining (J_H) gene segments. The location of CDR-H3 at the center of the Ag binding site emphasizes its often fundamental role in Ag recognition (6, 7, 8).

Mammalian IgH loci typically contain multiple D_H gene segments (6, 9). BALB/c mice, for example, contain 13 functional D_H gene segments per haploid genome. Twelve of these D_H are the progeny of an ancestral D_H that has undergone multiple cycles of duplication to create the DFL, DSP, and DST families (10, 11). Highly similar in sequence, these D_H primarily encode tyrosine and glycine in reading frame 1 (RF1), which is preferentially used in the expressed CDR-H3 repertoire (12, 13). DQ52, the 13th gene segment, encodes glycine in all three reading frames by deletion.

CDR-H3 is also the focus for somatic, nongermline junctional diversification of the repertoire. At the VDJ junctions, combinatorial variability is amplified by imprecision at the VD and DJ joining sites, which allows exonucleolytic loss as well as palindromic (P junction) gain of germline sequence. In addition, TdT-catalyzed N nucleotides provide an exponential opportunity to create random CDR-H3 sequence (1, 2), potentially freeing the repertoire from germline constraints.

Given the potential for diversity provided by N addition, the need for multiple highly similar D_H gene segments is unclear. Still, the sequences are not identical. DFL16.1, for example, encodes serine in RF1 (see Fig. 1a) but lacks aspartic acid and asparagine, two amino acids that are prominent in separate subsets of the DSP family. DFL16.1 is also longer than the other D_H by 6–12 nt, equivalent to two to four additional codons.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Generation of a single DH-containing IgH locus by use of the Cre-loxP system. a, A comparison of the RF sequence of the 13 DH gene segments illustrates two critical differences in sequence between DFL16.1 and its DH companions. First, DFL16.1 is two to four codons longer in sequence than the other DH. Second, DFL16.1 and DST4, which is rarely used, are the only D gene segments that encode serine RF1. Neither DFL16.1 nor DFL16.2 encodes aspartic acid or asparagine, which are common in the remaining eleven gene segments. b, Allele A illustrates the DH locus on one of the two IgH alleles before recombination (germline) and after the targeted introduction of a neo-LoxP cassette downstream of DFL16.1 (DFL neo-LoxP). Allele B illustrates the sister allele of the ESDQ52-KO cell line (17 ) that contains a loxP site in place of the DQ52 gene segment (DQ52). Cre-mediated recombination between the 5' loxP of the neo-loxP cassette and the loxP in the DQ52 allele will result in an 80-kb deletion that eliminates 11 of the 12 remaining DH gene segments and the neo gene with its 3' loxP. Shown at the bottom is the locus on the recombinant chromosome after Cre-mediated deletion has left a single DFL16.1 gene juxtaposed to the JH locus (D-DFL). The letters V and C denote the full set of germline variable gene segments and constant region exons, respectively. c, Two chimeric males (310 and 304) obtained from the injection of C57BL/6 blastocysts with the targeted C-33 ES clone were bred to BALB/cJ females to establish germline transmission. F1 albino pups were identified as the progeny of the targeted ES clone and genotyped by PCR. Chimeric male 310 yielded two progeny (310#0 and 310#1), which contained the DFL neo-loxP and germline DQ52 only. Chimeric male 304 yielded three mice, one of which contained the DFL neo-loxP and germline DQ52 (304#2) and one which contained only germline DFL16.1 and the DQ52 allele. These data show that the DFL16.1-neo-loxP transgene had recombined in trans relative to DQ52.

To test whether the presence of multiple D gene segments is necessary for robust B cell development and function and to determine whether the sequence of the D_H can influence the mature B cell CDR-H3 repertoire, we used gene-targeting to delete 12 of the 13 D_H gene segments from the D_H locus. We term this simplified IgH allele D-DFL for a depleted D_H locus limited to DFL16.1. In homozygous D-DFL BALB/c mice, the amino acid composition and average length of CDR-H3 repertoire in the mature IgM⁺IgD⁺ B cell population does not recapitulate the wild-type (WT) repertoire as a whole. Instead, it primarily matches that component of the WT repertoire, which contains DFL16.1. Despite lacking those components of the repertoire created by the other 12 D_H gene segments, the D-limited DFL16.1 repertoire is sufficiently plastic to allow development of normal numbers of mature B cells, to achieve normal serum Ig levels, and to mount robust humoral immune responses to T-dependent and T-independent Ags.

	Materials and Methods

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Generation of a mouse with a single germline DH gene segment (D-DFL)

The RI-2 charon phage containing the BALB/c DFL16.1 locus was a gift from Dr. Y. Kurosawa (Fujita Health University, Toyoake, Aichi, Japan) (10). An 800-bp BglII fragment containing DFL16.1 was modified by PCR-based, site-directed mutagenesis to contain NotI and KpnI cloning sites 50-bp downstream of the 3' recombination signal sequence. The integrity of the DFL16.1 gene segment and its flanking recombination signal sequences as well as the new cloning sites was confirmed by DNA sequencing. Additional germline sequence was added 5' and 3' of the modified BglII fragment, creating a targeting construct with a 4.2-kb 5' long arm and a 0.7-kb 3' short arm. A loxP-neo-loxP cassette was inserted into the NotI/KpnI cloning sites, and a HSV-thymidine kinase selection cassette was inserted into the tip of the long arm.

ESDQ52-knockout (KO), derived from wt/wt BALB/c-I ES cells of the IgH^a haplotype, had been targeted to insert a loxP site in lieu of a 240-bp XhoI-SacI fragment containing the DQ52 gene and a putative 5' cis-regulatory element (17). The allele containing the loxP replacement is termed DQ52 for deleted DQ52 (see Fig. 1a). PCR amplification of genomic DNA with primers flanking the DQ52 gene segment (5'KOcre, 5'-CAAGTGAATGACAGATGGACCTCC-3'; and DQ/R2, 5'-CAAGCCTCTCTACTTCCTCATA-3') yields a 307-bp amplicon (see Fig. 1b). A similar PCR amplification of the DQ52 allele yields a 200-bp amplicon (see Fig. 1b).

Using standard protocols (18), the ESDQ52-KO cell line was transfected with AscI-linearized DFL16.1 targeting vector (pHWS30). ES clone C-33 was identified by Southern blot analysis, PCR amplification, and DNA sequencing to contain a homologous DFL knock-in with a 3' loxP-neo-loxP cassette. Using a primer derived from within the neo-loxP cassette (BK23, 5'-TGTGGTTTCCAAATGTGTCAG-3') in conjunction with a reverse primer derived from genomic sequence 3' of the EcoRI site that defines the 3' boundary of the targeting construct (BK32, 5'-CATGCCCACAGTTACTATCCC-3'), PCR amplification of the allele containing the DFL knock-in yields a 994-bp amplicon (see Fig. 1). The C-33 clone was injected into C57BL/6J blastocysts. A DFL chimeric male was bred to yield BALB/cJ DFL mice.

In a parallel set of experiments, the C-33 ES clone was transfected with a Cre-expressing construct in vitro using standard techniques (18). Successful Cre-mediated recombination between the loxP immediately adjacent to DFL16.1 and the loxP site that has replaced DQ52 yields a novel D-DFL D_H locus (see Fig. 1a). PCR amplification with primers that flank the DFL16.1 (BK35, 5'-AGCAGACTCTTGAGCTCCAAA-3') and DQ52 (BK59, 5'-TCCTCGGCGCGCCTCCTTGGCAGGACATTGCA-3') loci yields a D-DFL amplicon 326 bp in length. By PCR analysis, three subclones were identified that contained the expected 80-kb deletion of the D locus by PCR (see Fig. 1). One of these subclones, C-33-11, was used to inject blastocysts with subsequent germline transmission. The integrity of the mutant D-DFL allele in albino F₁ pups was confirmed by Southern blot analysis, PCR amplification, and DNA sequencing.

The mice were maintained in a specific pathogen-free barrier facility. All experiments with live mice were approved by and performed in compliance with Institutional Animal Care and Use Committee regulations.

Flow cytometry, cell sorting, RNA preparation, RT-PCR, and sequencing

For FACS analysis of B cell development, single-cell suspensions of spleen, peritoneal cavity mononuclear cells, and the bone marrow from both femurs of individual mice were extracted and prepared as described previously (16, 19). From each source, 1 x 10⁶ cells were stained and analyzed on a FACSCalibur (BD Biosciences) using the following sets of mAbs: for spleen, anti-IgM (Cy5) (Southern Biotechnology Associates), anti-CD19 (SPRD), anti-CD21 PE, and anti-CD23 FITC (20); and for peritoneal cavity, anti-CD19 (streptavidin SpectraRed) (Southern Biotechnology Associates), anti-CD5 (PE) (BD Pharmingen), and anti-Mac-1 (FITC) (BD Pharmingen). For bone marrow, Hardy fractions B, C, D, E, and F (19) were analyzed as described previously (16).

For sequence analysis of Cµ cDNA transcripts from sorted cells, we obtained bone marrow B cell fractions from two D-DFL/D-DFL mice (8 wk of age) and two wt/wt littermates (8 wk of age) on two separate occasions. The eight mice analyzed were the progeny of heterozygous D-DFL/wt mating. Bone marrow cells were extracted from the two tibias and femurs of the individual mice (16, 19). From each bone marrow fraction, 2 x 10⁴ cells were sorted directly into RLT lysing buffer (Qiagen RNeasy minikit; Qiagen). Total RNA isolation, Cµ-primed cDNA generation, V_H7183VDJCµ PCR amplification, subcloning, and sequencing were performed as described previously (16).

Sequence analysis

CDR-H3 was identified as the region between (but not including) the 3' V_H encoded conserved cysteine (TGT) at Kabat position 92 (IMGT 104) and the 5' J_H-encoded conserved tryptophan (TGG) at Kabat position 103 (IMGT 118) (6, 9, 16). CDR-H3 was separated into two components: the base (Kabat 93 and 94, IMGT 105 and 106), typically alanine and arginine, and (Kabat 100–102, IMGT 115–117), typically phenylalanine, aspartic acid, and tyrosine; and the loop (the intervening amino acids). The average hydrophobicity of each CDR-H3 interval was calculated as described previously (13, 21).

We sequenced 264 D-DFL and 295 littermate V_H7183DJCµ cDNA transcripts (5) from sorted cells of Hardy fractions B through F of which 252 (95%) D-DFL and 277 (94%) WT cDNA transcripts were unique. Of these, 243 (96%) D-DFL and 255 (92%) WT clones contained open, in-frame rearrangements (see Table II). These unique sequences have been placed in the GenBank database (AY923258–AY923748).⁶ The frequency of D-DFL CDR-H3 sequences that contained four or less nucleotides of identity with DFL16.1 were 0, 7, 9, 7, and 2% in fractions B through F, respectively (p = 1.0, ²).

To determine basal levels of Ig isotypes in unimmunized 8 wk old D-DFL/D-DFL and wt/wt littermates, standard ELISA was performed with class-specific unlabeled and alkaline phosphatase-labeled Abs (Southern Biotechnology Associates). For the (13)-dextran (DEX) response, D-DFL/D-DFL and wt/wt littermates (8 wk) were immunized i.v. with 100 µg of Dextran B-1355S in saline (gift from J. F. Kearney, University of Alabama, Birmingham, AL) and bled 7 and 14 days later. For the [4-hydroxy-3-nitrophenyl]acetyl-chickengammaglobulin (NP₁₉-CGG) response (22), D-DFL/D-DFL and wt/wt littermates (8 wk) were immunized i.p. with 10 µg of NP₁₉-CGG (Biosearch Technologies) precipitated in potassium aluminum sulfate (alum) in saline. Mice were bled from the tail vein at weekly intervals. Quantitative ELISA were performed using DEX-BSA (gift of J.F. Kearney) and NP-BSA (Biosearch Technologies) as Ags coated at 10 µg/ml in PBS onto Costar EIA/RIA plates. Assays of IgM anti-DEX sera were performed as previously described using MOPC 104E as a standard (23). Assays of anti-NP sera were performed using mAb B1-8 as a standard. Following overnight 4°C incubation with serum samples and isotype-matched Ab standards, plates were blocked and then incubated with alkaline phosphatase-labeled goat anti-mouse IgM, IgG, or Ig as required (Southern Biotechnology Associates); plates were developed using p-nitrophenyl phosphate substrate (Sigma-Aldrich). For all ELISAs, 2-fold serial dilutions of individual serum samples were made in 1% BSA in PBS, and all ELISAs were read at 405 nm using a Bio-Rad Benchmark microplate reader and software (Bio-Rad).

Statistical analysis

Differences between populations were assessed where appropriate by the two-tailed Student t test, the two-tailed Fisher’s exact test, the ² test, Levene’s test for the homogeneity of variance, the Wilcoxon/Kruskal-Wallis (rank sums) test, or the nonparametric median test. Analysis was performed with JMP IN version 5.1 (SAS Institute). Means are accompanied by the SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Generation of the D-DFL mouse

A targeting construct containing a loxP-neo cassette downstream of the DFL16.1 gene segment (Fig. 1b) was created from BALB/c genomic sequence. The construct was targeted into the BALB/cJDQ52 ES cell line in which the most J_H-proximal D_H gene segment, DQ52, had been previously replaced by a separate loxP site (DQ52) (17). After targeting and selection, a single positive clone (C-33) containing the loxP-Neo cassette in the appropriate genomic location was identified. Because the original ES cell line was heterozygous for DQ52, the knocked-in DFL neo-loxP cassette could have replaced the germline DFL16.1 locus in an orientation either cis or trans to the DQ52 deletion. Analysis of the progeny of two chimeric males (Fig. 1c) revealed that the recombination had occurred in trans.

Subclones of C33 that had undergone in vitro Cre-mediated recombination were then evaluated by Southern hybridization (data not shown) and PCR (Fig. 1c) for evidence of recombination between the loxP immediately adjacent to DFL16.1 and the loxP site that had replaced DQ52 (DQ52). C3-11 was identified as a clone that likely contained the deletion. After blastocyst injection, germline transmission was identified in the F₁ progeny of one chimera. Sequence analysis confirmed the integrity of DFL16.1 and its flanking recombination signal sequences (data not shown). The D-DFL allele that resulted from this process was thus the product of Cre-mediated interchromosomal recombination.

Perturbations in the absolute number of B cell progenitors in D-DFL bone marrow are followed by normalization in the periphery

We used the scheme of Hardy (19) to evaluate developing B lineage cells in the bone marrow and the scheme of Loder et al. (20) to evaluate surface IgM⁺ B cell populations in the spleen (Table I). Compared with WT littermate controls, in the bone marrow we observed a 24% increase in the number of fraction B pro-B cells (p = 0.05), equivalence in the number of fraction C early pre-B cells, a 20% decrease in the numbers of fraction D late pre-B cells (p = 0.07), and a 22% decrease in the numbers of fraction E immature B cells (p = 0.01). Despite the decreased number of immature B cells, the number of mature IgM⁺IgD⁺ fraction F B cells proved identical to WT littermate controls. The absolute numbers of transitional (T1 and T2), marginal zone, and follicular cells in the spleen and the absolute numbers of B1a, B1b, and B2 cells in the peritoneal cavity were also equivalent to WT (Table I).

View this table: [in this window] [in a new window]   Table I. Cell numbers in bone marrow and spleen of D-DFL and WT littermate micea

We postulated that normalization of B cell numbers in the periphery might represent selection for randomized, predominantly N region-containing CDR-H3 intervals in the mature B cell population; but this did not occur. Of the 42 unique D-DFL CDR-H3 sequences cloned from fraction F cells, 95% contained five or more nucleotides of identity with DFL16.1 (Table II). Among WT sequences, 87% of fraction F sequences contained an identifiable D_H gene segment.

No significant differences were observed in the composition of the CDR-H3 repertoire between the sequences obtained from the WT littermate controls and the 619 IgM^a sequences that had been previously cloned from identically sorted WT BALB/c bone marrow (16). To maximize statistical power, we compared the composition of the D-DFL sequences to this combined database of 867 WT IgM^a sequences ("WT"; Table II). To control for potential D-specific patterns of repertoire development, we marked 184 CDR-H3 sequences that contained an identifiable DFL16.1 gene segment ("DFL16.1"; Table II) and 683 that did not ("Other"; Table II).

Diminished use of J_H4

Unlike WT mice where J_H4 is favored in fractions C through F, in D-DFL mice, J_H4 was least used (Fig. 2) (p < 0.001). In WT mice, J_H4 use increases between fraction B and fraction F (p < 0.05) (Fig. 2). There were insufficient numbers of fraction B sequences to assess the change from fraction B to F, but in D-DFL mice, no increase in J_H4 use was observed between fractions C and F. Instead, J_H1 use increased (p < 0.05). J_H4 use in WT fraction C and fraction F was similar among those sequences that contained DFL16.1 and those that did not (Fig. 2). Thus, the decreased use of J_H4 could not be attributed to a DFL16.1-specific bias.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Use of VH 7183 and JH gene segments as a function of B cell development. Shown on the left is the distribution of VH 7183 family gene segments with JH usage on the left. Both VH and JH segments are arranged in germline order. VH 7183 and JH use is reported as the percentage of the sequenced population of unique, in-frame, open transcripts for Hardy fractions B (top) through F (bottom). Use is reported for sequences from the D-DFL mice (D-DFL), for WT sequences that use DFL16.1 (DFL16.1; Table I), for WT sequences that do not use DFL16.1 (Other; Table I), and for all of the WT sequences (WT; Table II).

In mature B cells, D-DFL V_H7183 use proved equivalent to WT

The overall pattern of V_H7183 use in the D-DFL repertoire was statistically equivalent to WT in surface IgM⁺ bone marrow B cells (fractions E and F) but differed significantly from WT in the surface IgM^– populations (fractions B, C, and D) (p 0.05; ², 16 degrees of freedom) (Fig. 2). Variance from WT was primarily due to the increased use (p 0.05) in fractions B and D of V_H7183.10 (Fig. 2), the most commonly expressed V_H 7183 gene segment in immature and mature B cells (16), and the decreased use (p < 0.05) in fraction C of V_H7183.1 (V_H81X), the most frequently rearranged V_H gene segment in early B cell progenitors (16, 24, 25, 26, 27). Use of V_H7183.1 in fraction C was equivalent to that observed WT DFL16.1 sequences (Fig. 2). V_H 7183 use among D-DFL IgM⁺IgD⁺ bone marrow B cells was statistically indistinguishable from that observed in WT littermates.

Near identity of CDR-H3 amino acid use in D-DFL and WT DFL16.1

Throughout bone marrow development, D-DFL B lineage cells exhibited the same pattern of CDR-H3 tyrosine and glycine predominance with diminished use of hydrophobic or charged amino acids that had been previously observed in WT BALB/c mice (16) (Fig. 3 and data not shown). By fraction F, the pattern of use in D-DFL mice for 16 of the amino acids was equivalent to that observed in WT (Fig. 3). Serine, aspartic acid, asparagine, and alanine were the exception.

View larger version (9K): [in this window] [in a new window]   FIGURE 3. Distribution of amino acids in the CDR-H3 loops of the VH7183DJCµ transcripts isolated from Hardy fraction F. The amino acids are arranged by relative hydrophobicity, as assessed by a normalized Kyte-Doolittle scale (40 41 ). Use is reported as the percentage of the sequenced population for sequences from the D-DFL mice (D-DFL), for WT sequences that use DFL16.1 (DFL16.1; Table I), for WT sequences that do not use DFL16.1 (Other; Table II), and for all of the WT sequences (WT; Table I). An asterisk marks a difference in distribution that achieves statistical significance at p 0.05.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Prevalence of serine, aspartic acid, asparagine, and alanine in the CDR-H3 loops of VH7183DJCµ transcripts as a function of B cell development. Use is reported as the percentage of the sequenced population for sequences from the D-DFL mice (D-DFL), for WT sequences that use DFL16.1 (DFL16.1; Table I), for WT sequences that use DSP gene segments, which include aspartic acid in RF1 (DSP-D; Table I), and WT sequences that use DSP gene segments which include asparagine in RF1 (DSP-N; Table II).

In mature IgM⁺IgD⁺ B cells, the average length and hydrophobicity of D-DFL CDR-H3 is nearly identical to that observed in WT DFL16.1 sequences

In the earliest stage of B cell development examined, fraction B, the average length of D-DFL CDR-H3 sequences, was similar to that observed in WT DFL16.1-containing sequences but diverged from both the total WT and the non-DFL16.1-containing CDR-H3 (p < 0.05). This difference in length from total WT and non-DFL16.1 sequences was also apparent in fraction C (p < 0.0001). However, among cells that had progressed to the late pre-B cell fraction D, the average length of the D-DFL CDR-H3 repertoire had converged to that observed in both total WT and non-DFL16.1 sequences, only to diverge away from those populations in fractions E (p < 0.05) and F (p < 0.01). In contrast, the average length of D-DFL fraction F CDR-H3 achieved near identity to that of fraction F WT DFL16.1 CDR-H3 (Fig. 5). The average hydrophobicity of D-DFL CDR-H3 loops was similar to total WT and to non-DFL16.1-containing CDR-H3 in fractions B, achieved near identity in fraction C, diverged in fraction D (p 0.01), converged toward the WT DFL16.1 average in fraction E, and then achieved equivalence to WT DFL16.1 in fraction F.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Change in average length and average CDR-H3 hydrophobicity in VH7183DJCµ transcripts isolated from Hardy fractions B through F. a, Change in the average length of CDR-H3 intervals from homozygous D-DFL mice (D-DFL), from CDR-H3 intervals from WT sequences that use DFL16.1 (DFL16.1; Table I), from WT sequences that do not use DFL16.1 (Other; Table II), and from all of the WT sequences (WT; Table I). b, Change in the average hydrophobicity index value of the CDR-H3 loops in the same populations reported in a.

Humoral immune responses in BALB/c D-DFL mice are similar, but not identical, to WT BALB/c littermates

Among 8-wk-old littermates, the average concentrations of IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were indistinguishable from WT (Table III). After primary and secondary i.p. challenge with NP₁₉-CGG, a T-dependent Ag (28), the anti-NP IgG response in the D-DFL mice was nearly identical to that observed in littermate controls (Fig. 6). In contrast, 7 days after challenge with DEX, a T-independent Ag (29, 30, 31), the average IgM and Ig anti-DEX serum levels in homozygous D-DFL mice (Fig. 7) were approximately half that observed in WT littermates (152 ± 58 vs 255 ± 40 µg/ml, p = 0.07; and 80 ± 37 vs 170 ± 42 µg/ml, p = 0.05).

View this table: [in this window] [in a new window]   Table III. Serum Ig levels in D-DFL mice and their WT littermates

View larger version (12K): [in this window] [in a new window]   FIGURE 6. The primary and secondary immune response to the T-dependent Ag NP19-CGG in D-iD/D-iD mice is indistinguishable from that observed in WT littermates. Shown are the anti-NP IgG Ab titers in preimmune sera (0 d) and in immune sera obtained 7, 14, and 21 days after primary i.p. immunization, and 7 and 14 days after secondary i.p. immunization.

View larger version (14K): [in this window] [in a new window]   FIGURE 7. The primary immune response to the T-independent bacterial polysaccharide DEX in half of the homozygous D-DFL mice is similar to the response observed in WT littermates but is diminished in the rest. Shown are anti-DEX IgM and Ig Ab titers in immune sera 7 days postimmunization. (Titers in preimmune sera were <1 µg/ml; data not shown.)

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In these D-limited mice, normal numbers of mature B cells were generated despite the deletion of 86 kb of DNA in the D_H locus and 12 of the 13 functional D_H. Effective VDJ recombination occurred in the absence of a cis-regulatory promoter and enhancer sequence upstream of DQ52 that is active early in B cell development (32), indicating that a single normal D_H with a non-DQ52 promoter and recombination signal sequences is sufficient for the initiation of DJ rearrangement and completion of VDJ recombination.

As in the case of the single V_H mouse, although immune responses were similar to WT, they were not completely identical. For example, the frequency of J_H4 use was consistently depressed. Diminished use of J_H4 could not be attributed to an idiosyncratic effect of the individual DFL16.1 gene segment. A similar decrease in the rearrangement frequency of J_H4 was previously observed in mice that lacked a portion of cis-regulatory sequence upstream of DQ52 (32). Although our results, which focus on the expressed repertoire, cannot be directly compared with studies of VDJ rearrangements, deletion of the remaining portion of region upstream of DQ52 and of the promoter regions upstream of 11 of the 12 remaining D_H did not appear to result in any changes in J_H usage over and above those previously observed in mice that lacked DQ52 and flanking sequence.

Perturbations from WT patterns were also noted in the absolute numbers of developing B cells in the bone marrow. These perturbations began with an accumulation of fraction B IgM^–CD19⁺CD43⁺ pro-B cells. To minimize the risk that gene targeting might have introduced genetic alterations elsewhere in the genome that could have affected the numbers of developing B cell progenitors, we consistently compared the homozygous D-DFL mutant offspring of heterozygous D-DFL/WT mice to their homozygous IgH WT littermates. This strategy also controlled for age and environment. The increase in the numbers of pro-B cells in the D-DFL mutants when compared with these littermates can thus be most likely ascribed to the D_H locus deletion. An accumulation of IgM^–B220⁺CD43⁺ pro-B cells has also been observed in the context of a deletion of DFL16.1 through J_H1 in C57BL/6 mice (33). Preliminary analysis of hybridomas obtained from heterozygous D-DFL BALB/c mice has shown that the D-DFL allele, but not the WT allele, is frequently found in a germline unrearranged state (unpublished data). The increase in the number of fraction B cells from the D-DFL allele may thus reflect inefficiency in the initiation of DJ rearrangement due to the loss of cis-regulatory sequence.

Although an accumulation of pro-B cells was observed in the D-DFL mice, the absolute numbers of early pre-B cells were equivalent to WT. Fraction C is associated with VDJ rearrangement (34, 35, 36). A preliminary analysis of hybridomas obtained from heterozygous and homozygous D-DFL mice indicates that VDJ rearrangement on the D-DFL allele occurs in an unimpeded manner (unpublished data). This is to be expected because the entire D_H locus is typically deleted in cells undergoing V to DFL16.1-J rearrangement, which normally appears to be a highly favored rearrangement (10).

In support of the view that VDJ rearrangement is proceeding normally, V_H7183 use in D-DFL fraction C VDJCµ transcripts proved similar to WT. The single prominent exception, V_H81X, exhibited a similar reduced prevalence among WT DFL16.1-containing fraction C transcripts. Thus, diminished use of V_H81X may be a consequence of retaining the DFL16.1 gene segment rather than deleting the other 12 D_H. Because our analysis focused on evaluation of V_H7183-D-J-Cµ cDNA sequences with open reading frames, we were unable to determine whether the decreased prevalence of V_H81X reflects ineffective rearrangement to DFL16.1 or selection against cells that express V_H81X-DFL16.1-J-Cµ proteins.

Efficient transition from fraction C to fraction D requires successful assembly of a pre-BCR (34, 35, 36). Subsequent passage to fraction E then requires both in-frame L chain rearrangement and successful association of the rearranged L chain with its H chain partner (19). Fraction E cells may lose surface IgM expression during receptor editing or may be released from the bone marrow to undergo maturation in the periphery, which is associated with surface coexpression of IgD. Potential mechanisms for the decrease in the absolute numbers of cells in D-DFL fraction E include failure to undergo proper L chain rearrangement, leading to a block in the progression from D to E; a more rapid progression from E to the transitional cell population in the periphery due to enhanced success of H-L partnering; or, conversely, enhanced receptor editing (37) due to functional failure of H-L partnering, causing inflation in the numbers of cells identified as fraction D. This latter scenario seems less likely because fraction D numbers were also depressed, although it remains possible that a precipitous drop in fraction D numbers has been obscured by an accumulation of fraction E cells undergoing receptor editing. However, the numbers of cells in the transitional population in the periphery are similar to WT, which would support efficient transition from fraction E to the periphery. Formal testing of these hypotheses will require kinetic evaluation (38).

Divergence in the prevalence of serine, aspartic acid, and asparagine by D_H gene segment in WT mice suggests that each D gene segment generates its own unique subrepertoire. The near identity of the D-DFL CDR-H3 repertoire to that component of the WT repertoire, which contains DFL16.1, provides strong support for this hypothesis. This near identity is even more striking given the alteration in J_H1 and J_H4 use. The effect of decreased J_H4 use on CDR-H3 loop amino acid content appears limited to a decrease in the prevalence of alanine. This amino acid is primarily found adjacent to the J_H component of the base of the CDR-3 loop in a location less likely to allow direct contact with Ag. J_H1 shares a longer length and two terminal tyrosines with J_H4. Selection during development for a preferred DFL16.1 distribution of longer lengths and for the presence of tyrosine in the CDR-H3 loop may thus be contributing to the enhanced use of J_H1 in the D-DFL IgM⁺IgD⁺ B cell population in compensation for the loss of rearrangements to J_H4.

When examined on the basis of length, amino acid composition, and average hydrophobicity, the similarity between D-DFL and WT DFL16.1-containing sequences is perturbed in the late pre-B and immature B cell populations, both of which exhibited a decrease in the absolute numbers of cells in the D-DFL mice when compared with WT. The sequence characteristics of the CDR-H3 population in IgM⁺IgD⁺ D-DFL B cells are nearly identical to that observed in WT, as are the numbers of IgM⁺IgD⁺ B cells in the bone marrow and spleen, and the B1a, B1b, and B2 B cell subsets in the peritoneal cavity. These data suggest that while early B cell development may be perturbed and that a significant component of the repertoire that is normally generated in WT mice by the deleted D_H gene segments has not been recreated D-DFL B cells are responding normally to DFL16.1 sequence-specific selective pressures in the periphery. This response appears to be sufficient to allow all the peripheral B cell compartments that we examined to achieve the same level of population observed in WT littermate controls.

Although some of the D-DFL mice achieved serum anti-DEX levels of the same magnitude as those observed in littermate controls, the response in others was significantly depressed. The majority of previously published anti-DEX H chain sequences contain very short CDR-H3 whose progenitor D_H gene segments cannot be assigned (29, 30, 31). Although matched for long length with the WT DFL16.1 repertoire, the D-DFL IgM⁺IgD⁺ B cell repertoire is depleted of short CDR-H3 sequence. In WT mice, short CDR-H3 may preferentially derive from one or more of the deleted D_H gene segments.

In contrast to DEX, the primary and secondary responses to NP₁₉-CGG, a classic T-dependent Ag that typically elicits a H-chain repertoire enriched for DFL16.1 (28, 39), were indistinguishable from WT. If each D_H gene segment creates its own subrepertoire, then each D_H may make its own key contribution to the immunocompetence of the host. Proof of this hypothesis may require analysis of mice that bear altered D_H sequence.

	Acknowledgments

 
We thank P. Burrows, T. Carvalho, M. Cooper, and J. Kearney for their invaluable advice and support and Sarah Robinson for excellent technical assistance.

	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 This work was supported by National Institutes of Health Grants AI07051 (to G.C.I.), AI42732 (to H.W.S.), AI48115 (to H.W.S.), HD043327 (to R.L.S.), and TW02130 (to H.W.S.).

² R.L.S. and I.I.I. contributed equally to this article.

³ Present address: Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016.

⁴ Address correspondence and reprint requests to Dr. Harry W. Schroeder, Jr., Division of Developmental and Clinical Immunology, WTI 378, 1530 3rd Avenue South, University of Alabama, Birmingham, AL 35294-3300. E-mail address: harry.schroeder{at}ccc.uab.edu

⁵ Abbreviations used in this paper: CDR-H3, CDR-3 of the Ig H chain; RF1, D_H reading frame 1; WT, wild type; D-DFL, depleted DH locus with single DFL16.1 gene segment; DQ52, allele containing a loxP site in place of the DQ52 gene segment; KO, knockout; DEX, (13)-dextran; NP₁₉-CGG, [4-hydroxy-3-nitrophenyl]acetyl-chickengammaglobulin; DSP-D, DSP gene segments containing aspartic acid; DSP-N, DSP gene segments containing asparagine.

⁶ The online version of this article contains as supplemental material a Microsoft Excel worksheet with all of the unique CDR-H3 sequences reported in this article, as well as a set of 619 previously reported IgM^a unique sequences from wild-type BALB/c mice (16 ).

Received for publication May 18, 2005. Accepted for publication August 12, 2005.

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