Wild-type female BALB/c (H-2^d) mice (Harlan-Winkelmann; Borchen, Germany) and BALB/c D-altered homozygous ΔD-DμFS (7) and ΔD-iD (6) mice were reared under clean conventional conditions the University of Kiel animal house. Immunizations and experimental procedures were approved by the “Ministry of Agriculture, Environment and Rural Areas” of the local government of Schleswig-Holstein, permission no. V 312-72241.121-3 (35-3/06).

BALB/c WT and D-altered ΔD-DμFS and ΔD-iD mice were immunized with the TD hapten–protein complex phOx-coupled chicken serum albumin (phOx-CSA) (molar ratio ~11) (9, 10). At 3–4 months of age, animals of all three strains received 80 μg of phOx-CSA adsorbed to Al(OH)₃ as a primary intraperitoneal immunization. Venous blood was serially obtained by tail vein phlebotomy and serum antibody concentrations were determined after primary or secondary immunization. PhOx-binding IgM and IgG titers were determined with ELISA using class-specific secondary antisera. Spleen cells of phOx-CSA-immunized WT or D-altered mice were fused with the non-secretor Ag8.653 myeloma cells by means of the conventional PEG-mediated hybridization technique. Fusions were performed 7 or 14 days after primary immunization and 3 days after each secondary (memory) immunization. Resulting mAb reactive with phOx-BSA but not with BSA alone were selected for further study.

The relative affinities of mAb antibodies to phOx were determined with a hapten-inhibition test as previously described (9). These affinities were assessed in comparison to that of two prototypic Ox1-idiotypic mAb: H11.5 (μ,κ) for IgM and NQ2/16.2 (γ,κ) for IgG Ab. Briefly, the binding of comparable amounts of anti-phOx mAb to surface-bound phOx-BSA was inhibited with graded concentrations of soluble phOx-caproic acid. Concentration values giving 50% inhibition were taken as relative affinity measures. An affinity factor was generated as the quotient of the relative affinity of H11.5 (μ,κ) for IgM and NQ2/16.2 (γ,κ) for IgG mAb divided by that of a particular mAb. MAb with higher affinity than the controls exhibited an affinity factor greater than 1, and those with a lower affinity exhibited an affinity factor of less than 1. Independent confirmation of these measures of affinity was obtained by fluorescence quenching (10).

Sequence analysis of the variable regions of mAb was performed as previously described (11) with some modifications. Briefly, total RNA of anti-phOx Ab secreting hybrid cell lines was isolated with TRIZOL^® (GIBCO-BRL, Eggenstein, Germany) and transcribed into cDNA with SuperScript^TM II RNAse H reverse transcriptase (GIBCO-BRL, Eggenstein, Germany) using pd(N)6 random and pd(T)_12–18 primers. The V_H and V_L mRNA sequences were first amplified by PCR using 10 primer sets for each of the V_H-regions, and 7 primer sets for each of the V_L-regions and two forward primers specific for the 3′-end for the first domain of the V_H and V_L constant regions, respectively. A second semi-nested amplification at 3′-end was then performed with relevant primers coupled to M13 oligonucleotides. This amplimer product was then used for sequencing (MWG Biotech; Ebersberg, Germany). V region sequences were analyzed with the integrative database VBASE2 (http://www.vbase2.org/).

One-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparison post test, using Graphpad Prism for Windows (version 4.02, GraphPad Software Inc., San Diego, USA) was used for statistical comparisons of individual IgM and IgG end-point titers.

GenBank accession numbers for all antibodies are indicated in the supplementary table legends of each group of antibodies.

The humoral Ab response to phOx in BALB/c WT mice was compared to that of homozygous D-altered ΔD-DμFS and ΔD-iD mutant mice. The D_H locus in these strains was first simplified by deleting 12 of the 13 D_H gene segments by means of cre/loxP gene-targeting (12); and then modifying the sequence of the remaining D_H. In the ΔD-DμFS strain, the WT sequence of the DFL16.1 D_H segment was modified by introducing two frame-shift mutations that flipped the normal preference for reading frame 1 (RF1), which encodes neutral tyrosine and glycine, to RF2, which encodes hydrophobic valine. In the ΔD-iD strain, the center of the DFL16.1 segment was replaced with the DSP2.2 D_H gene segment in inverted form. This leads to CDR-H3 enriched for charged arginine, asparagine, and histidine in place of tyrosine and glycine (6, 8).

In both mutant strains, primary immunization with the TD Ag phOx-CSA-induced significantly lower IgM and IgG anti-phOx Ab titers than WT (Figures 1A,B). Following secondary (memory) immunization, the IgG response remained suppressed while IgM production was meager and indistinguishable between all three strains.

Study of the molecular events during immune maturation of the TD anti-phOx response classically focused on analysis of mAb (9, 13, 14) secreted by hybridomas produced by fusion of permanently growing myeloma cells with immune splenocytes obtained after sequential primary and memory immunization (9, 13, 14). In order to directly compare the effect of altering D_H locus content in the same classic context, we generated anti-phOx mAb-secreting hybridomas from immune splenocytes harvested after primary, secondary, and tertiary immunizations of D-altered ΔD-DμFS and ΔD-iD mutant mice.

The characteristic parameters of these mAb, including isotype, relative affinity, usage of V_H/V_L genes and D_H and J_H gene segments, amino acid sequence, and reading frame usage in CDR-H3, and correspondence with the normally dominating Id_Ox1 are summarized Tables S1 and S2 in Supplementary Material. A comparison of these mAb with those of previously published mAb (10) from WT mice revealed crucial differences induced by the change in D_H sequence content. Unlike WT mice, where the majority of hybridomas ( >80%) isolated during late primary responses and on day 3 of secondary and tertiary responses produce IgG, and thus reflect T cell aided CSR (15); only half of the hybridomas obtained from the D-altered ΔD-DμFS and ΔD-iD mice produced IgG. This included both the hybridomas generated after the late primary response as well as hybridomas harvested from splenocytes 3 days after memory immunizations (Figures 1D,E). Thus, the normal pattern of favoring production of IgG secreting B cells failed to occur in mice lacking a WT D_H locus, and therefore, a WT CDR-H3 repertoire. A full depiction of these antibodies is provided in Tables S1 and S2 in Supplementary Material.

To assess the quality of these mAb, we compared their relative affinities to those of prototypic IgM and IgG Id_Ox1 mAb. Early primary IgM mAb of both mutant strains, as well as those from secondary and tertiary responses, were mostly of rather low affinity (Figures 1F,G). However, three IgM mAb from ΔD-DμFS mice exhibited higher affinities than the IgM Id_Ox1 mAb H11.5 (Figure 1F), even though none of them included the Id_Ox1 V gene combination of V_HOx1/V_κOx1, which is designated V_H171/V_κ072 in the integrative database VBASE2.

Early primary IgG mAb generally exhibited higher affinities than the IgM mAb, with some IgG mAb displaying even higher affinities than the IgG Id_Ox1 mAb NQ16.2. However, none of the mAb expressed the Id_Ox1 V_HOx1/V_κOx1 gene combination. IgG of higher affinity were found among secondary and tertiary response mAb. However, immune maturation did not progress in the steady way that characterizes the classic response of WT mice (see below).

In accordance with historical studies, in WT mice the anti-phOx response exhibited a drastic CSR-associated reduction in the variability of the V_H and V_L repertoires (10) (Figures 2A,D), especially in the class switched secondary response. No such reduction in V_H or V_L variability was observed in the D-altered ΔD-DμFS and ΔD-iD mice (Figures 2B,E and C,F). The classic secondary phOx response in WT mice is associated with a shift away from use of the V_H1 family (10). However, in the D-altered mice hybridomas secreting V_H1 family-encoded IgM and IgG mAb continued to be isolated at all phases of immune maturation (Tables S1 and S2 in Supplementary Material).

In WT mice, the TD response to phOx-CSA is dominated by V_H171/V_κ072-encoded Id_Ox1 Ab (4) whose idiotypic determinant is located in CDR-H3 (5). A compilation of previous data (9, 10) shows that mAb with the gene combination V_H171/V_κ072 (the “genetic” Id_Ox1) first dominate after CSR among IgG mAb (Figure 2G). This gene combination is counter-selected in secondary response mAb, but increases again in tertiary and quaternary responses. However, in both of the D-altered strains the Id_Ox1 gene combination V_H171/V_κ072 only represented a minority of clones. Among ΔD-DμFS mAb, the V_H171/V_κ072 gene combination was not identified among the IgM hybridomas. It was detected in IgG mAb drawn from the primary response, but at a low level. A plurality of the secondary IgG response used this combination, but this was again lost in the tertiary response (Figure 2H). In ΔD-iD mice, anti-phOx antibodies with the V_H171/V_κ072 gene combination were not observed in either the primary or secondary responses. It was found among tertiary IgM and IgG, but again at a low level (Figure 2I).

In WT mice, CSR and affinity maturation are strongly associated with a focusing of CDR-H3 content. For example, whereas the CDR-H3 of anti-phOx IgM hybridomas varied between 15 and 51 nucleotides, 70–100% of primary and memory IgG mAb exhibited a clearly dominating length of 21 nucleotides (10). This CSR-associated restriction of CDR-H3 lengths was not observed in D-altered ΔD-DμFS and ΔD-iD mice (Figure 3). CDR-H3 lengths with 21 and 36 nucleotides were slightly increased among ΔD-DμFS mAb (Figure 3A) and to an even lesser degree among ΔD-iD mAb (Figure 3B). Otherwise, IgM as well as IgG mAb from both strains of mice demonstrated a wide distribution of lengths.

The distribution of charged, neutral, and hydrophobic amino acids dictates the average hydropathicity of the CDR-H3 loop. During immune maturation among WT mice, highly hydrophobic amino acids found among IgM anti-phOx CDR-H3 are gradually supplanted by arginine, aspartic acid, glycine, and tryptophan (Figures S3A–H in Supplementary Material). In contrast, from the primary to tertiary responses in both ΔD-DμFS mice (Figure 4) as well as ΔD-iD mice (data not shown), hydrophobic amino acids were found to a similar extent in the CDR-H3 of both IgM and IgG anti-phOx antibodies. The content of hydrophobic amino acids in the CDR-H3 loops was also reflected in the corresponding hydrophobicity values. In anti-phOx mAb from WT mice, about 25% of IgM mAb exhibited CDR-H3 with positive average hydropathicity values, whereas only 6% of IgG antibodies (with the exception of secondary IgG) belonged to this category (Figures S4A–H in Supplementary Material). In contrast, IgM as well as IgG mAb produced by ΔD-DμFS B cells expressed similar proportions of CDR-H3 loops with positive average hydropathicity values (Figure 5). [The numbers of mAbs generated from the ΔD-iD mice were insufficient to allow a firm conclusion on whether average hydrophobicity was maintained or altered in this strain (data not shown).]

Since idiotypic determinants defining Id_Ox1 antibodies are located in CDR-H3, we analyzed antibodies bearing this idiotype to assess, which D_H gene segments could be used to generate the typical Id_Ox1 CDR-H3 sequence C_ARDRGAY. In solely phOx-CSA-induced mAb from WT mice (10), this sequence could be generated from the five D_H gene segments DSP2.2, DSP2.9, DSP2.11, DST4, and DQ52 (Table 1). In contrast, after a primary immunization with the TI-2 Ag phOx-Ficoll and a subsequent TD immunization (primary to quaternary) with phOx-CSA, CDR-H3 sequences from anti-phOx mAb were drawn from a larger set of eight D_H segments (DSP2.2, DSP2.3, DSP2.5, DSP2.6, DSP2.9, DSP2.10) plus two sequences from inverted DFL16.1 and DFL 16.2, respectively (9). Moreover, in half of the mAb, the D_H segment could not be identified due to extensive nibbling (9). This raised the issue of whether ΔD-DμFS mutant mice bearing a frameshifted DFL16.1 D_H segment or ΔD-iD mice with an inverted DSP2.2 gene sequence would be able to generate a typical Id_Ox1 CDR-H3 sequence in association with the VH171 gene.

C_ARDRGAY-related sequences were identified (Table 2A). The majority of these CDR-H3s lacked identifiable D_H sequences and instead was largely the product of N and P nucleotide addition. Among the mAbs drawn from the ΔD-DμFS mice, two contained CDR-H3 amino acids coded by the frameshifted DFL16.1 gene segment. Five additional ΔD-DμFS mAb (FS1°14/05, FS3°/08, FS3°/09, FS3°/22, FS3°/18) also exhibited CDR-H3 sequences already found in Id_Ox1 antibodies from WT mice, but these mAbs used N and P nucleotides to recreate the sequence. Among the mAbs drawn from the ΔD-iD mice, one early primary mAb, iD1°7/06, exhibited the correct Id_Ox1 sequence in CDR-H3. These findings made it clear that both D-altered strains were able to generate Id_Ox1-typical or related CDR-H3 sequences in spite of the alteration in D_H sequence content. Thus, differences in the CDR-H3 repertoire in these D-altered strains cannot be simply explained by an inability to generate the classic Id_Ox1 sequence.

Short CDR-H3 with 6–8 amino acids (i.e., in the range of CDR-H3 of Id_Ox1 antibodies) were also found in association with V_H genes other than V_H171. These alternative V_H belonged to non-V_H2 families (Table 2B). Two of the V_H171-encoded antibodies exhibited longer CDR-H3. Thus, the great majority of anti-phOx mAbs produced in these D-altered mice used H chains that differed greatly from the characteristic Id_Ox1 sequence, even though classic Id_Ox1-like CDR-H3s could be generated by both D-altered mice.

It has been argued that diversity at CDR-H3 is sufficient for the creation of Ag-specificity and that a particular specificity can be correlated with identical or very similar amino acids sequences in CDR-H3 (16). Our anti-phOx mAb from the two D-limited mouse strains offered the opportunity to check this hypothesis in another experimental system. A survey of all anti-phOx antibodies from ΔD-DμFS and ΔD-iD mice is depicted in Tables S3A,B in Supplementary Material, respectively. From the 83 mAb of ΔD-DμFS mice, 58 (70%) made use of varying lengths of the genomic D_H sequence, 2 (2.4%) used inverted sequences, and in 23 (28%) the genomic D_H sequence was not detectable. From the 44 mAb of ΔD-iD mice, 23 (52%) used varying lengths of the D_H segment, 5 (11%) used variable lengths of the inverted sequence, and in 16 mAb (36%), no portion of the D_H segment could be identified. This large variability of CDR-H3-coding sequences led to an equal variability of 0–13 amino acid sequences in CDR-H3 loops. The average hydropathicity values of CDR-H3 loops varied from strongly hydrophobic to strongly hydrophilic (Table 3). Similar findings were obtained in WT mice (data not shown). Thus, since common sequences or sequence motifs in CDR-H3 could not be detected, we conclude that Ag-specificity in this model, immune response is not limited by either this parameter or by other, readily apparent physicochemical properties of the CDR-H3 interval.