Serum IgM repertoire of antigen specificities have been screened using a semi-quantitative immunoblot assay that enables accurate en bloc analysis of the self-reactivity of IgM present in the serum (actual repertoire) of unmanipulated mice (26–28). We used the same assay to analyze the serum IgM reactivities of D_H-altered mice with self-antigens from brain tissue. Shown in Figure 1A is a comparison of the pattern of self-reactivity expressed by the WT BALB/c, ΔD-DFL, and ΔD-iD strains. To test whether the reactivity levels correlate with immunoglobulin specificity or with the relative abundance of the proteins, we compared protein staining with immunoreactivity densities in the same membrane. The comparison shown in Figure 1B reveals highly abundant proteins that are not recognized by natural serum IgM, as well as highly IgM-recognized proteins that are not abundant in the tissue extract. With a few notable exceptions, the mean sera IgM reactivity profiles were remarkably conserved among mouse lineages that generate very different primary CDR-H3 repertoires (22, 23) (Figure 1C).

It is noteworthy that these data revealing restriction of self-antigen recognition comply with the idea of the immunological homunculus, as proposed by Cohen (29). Furthermore, these data demonstrate that the reactivities found in the immunoblot assay cannot be attributed to simple non-specific interactions.

The results shown above suggest that strong mechanisms of antigen-driven somatic selection may operate at the cellular level to recreate a normal WT NAb repertoire of specificities in genetically modified mice. In mice, the peritoneal cavity (PerC) is the major site for both B-1 and B-2 cells. We, thus, sought to investigate whether these somatic selection pressures would generate similar repertoires among the PerC B-1 and B-2 B cells (available repertoire) from the different mouse strains. The reactivity profiles of these B cell populations provided a privileged opportunity for comparison between different B cell populations that share the same anatomical niche. After sorting each PerC B cell population (B-1a, B-1b, and B-2), we cultured the cells under LPS stimulation to induce polyclonal IgM secretion and tested the supernatants using the immunoblot against brain extract assay.

All three subsets from D_H-altered PerC B cells demonstrated frequencies of response to LPS that were similar to their corresponding WT B cell subsets (Figure 2A). Production of IgM in culture was also similar (Figure 2B). Thus, D_H alteration and restriction of the CDR-H3 repertoire did not affect the B cell response to polyclonal stimulus from LPS.

We then performed the same analysis of self-reactivities shown for serum IgM with the supernatants from the B-1 and B-2 cultures (Figure 3). As there is a difference in the frequency of response to LPS among B cell subsets, we diluted each supernatant to normalize all to the same IgM concentration per growing clone (30). We performed an immunoblot analysis of the reactivities against syngeneic brain extract from seven separate culture supernatants from the three strains of mice, for a total of 21 supernatants for each B cell subset.

The mean intensity of reactivities present in B-1a cells culture supernatants remained almost completely invariant despite the genetic alteration of the D_H gene sequences (Figure 3A, right). The mean reactivity profiles for B-1b cells supernatants (Figure 3B, right) also demonstrated similarities in self-antigen reactivities irrespective of genotype, although the replicates in the membrane suggested a greater degree of variation around the mean values (Figure 3B, left). Conversely, the mean reactivity profiles of the B-2 cells were divergent both within and across the different mouse strains. This corresponded well with the different sets of reactivities of the B-2 cells supernatants (Figure 3C, right). The individual variability of immunoreactivities of the 21 B-2 cell culture supernatants were also highly increased when compared to the B-1 reactivity profiles (Figure 3C, left). These findings suggested that differences in the variance of the reactivities were intrinsic to the B cell subset, and not dependent on the germline sequence of the immunoglobulin repertoire.

To address the issue of variance in depth, we divided the immunoreactivity profiles into 36 sections, which corresponded to the major bands of reactivity. We plotted the magnitudes of reactivity against these sections for each supernatant from the B-1a, B-1b, and B-2 subsets (Figures 4A–C, respectively). The solid lines indicate the mean profile of all supernatants for each B cell subset. In this format, the variance of the distribution around the mean profile can be properly visualized. Despite few exceptions, we observed that the variance around the mean was much lower for the B-1a supernatants when compared to B-1b and B-2 (Figure 4).

To further confirm that each B cell subset displays a unique repertoire, we analyzed the reactivity profiles within each mouse strain. Irrespective of D_H content, the B-1a individual culture supernatants profiles proved very homogeneous, with little variance around their mean (Figure 5A, top; and Figure S1 in Supplementary Material). The reactivity profiles from B-1b subset were observed to differ from the B-1a for all mouse strains (Figure 5A, middle). The B-2 reactivity profiles displayed increased variance of the distribution in WT mice and especially for the ΔD-iD strain (Figure S1 in Supplementary Material), whereas the single normal D_H control (ΔD-DFL) displayed less variability and intensity of reactivity (Figure 5A, bottom).

To substantiate these observations, we performed principal component analysis (PCA) for each mouse strain. This multivariate approach takes in consideration all the reactivities together, providing an unbiased evaluation of the data (26, 31). All PerC B cell subset repertoires were discriminated within each mouse strain, suggesting distinct repertoire signatures for each population.

Although the mean reactivity profiles were generally conserved among the three mouse strains (Figure 3A), individual differences between the repertoires were also apparent (Figures 2 and 3). We, thus, performed a PCA of the data presented in Figure 4, which are grouped by B cell subset. This multivariate analysis revealed the impact of the genetic alteration of D_H sequences on the B-1a available repertoire of specificities (Figure 6A, left), demonstrating distinct differences between the strains. To better evaluate the contribution of each factor to the resulting B cell subset repertoires, we collected all the data from the reactivity profiles (B-1a, B-1b, and B-2 subsets from the three mouse strains) on a single heat map (Figure 6B). This large data matrix was similarly subjected to PCA.

Both the immunoblot assay (Figures 2 and 3) and the heat map of the reactivities (Figure 6B) disclosed a set of major bands that were unique to each strain and were shared among individual mice from each strain. These shared similarities and differences were much less apparent in the B-1b and B-2 subsets (Figure 6A). When analyzed by PCA together, the characteristics common to the subsets appeared to exert a greater effect than the characteristics provided by the differences in D_H content (Figure 6C).

The panel of D_H-altered BALB/c mouse strains was bred in our mouse colony at the University of Alabama at Birmingham (UAB). Mice bearing ΔD-DFL (22) or ΔD-iD (23) D_H alleles were created by cre–loxP targeting of a BALB/c ES cell line. For each allele, 12 of the 13 BALB/c D_H gene segments were deleted and then the single, remaining DFL16.1 gene segment was either retained or altered. All WT and D_H-altered mice were studied at from 8 to 10 weeks of age. The mice were maintained in a SPF barrier facility and in a climate-controlled environment with a 12 h light/12 h dark cycle, with diet and water supplied ad libitum. Animal care was conducted in accordance with established guidelines and protocols approved by the UAB Animal Care and Use Committee.

Flow cytometric analysis and cell sorting were performed as previously described (36). Briefly, peritoneal washout cells were obtained from five different mice of each D_H-altered strain. The following mAbs were used to isolate PerC B cells into the B-1a, B-1b, and B-2 subpopulations: anti-B220 (RA 3.6B2) (BD Pharmingen) (Southern Biotechnology, Birmingham, AL, USA), anti-Mac-1 (BD Pharmingen, San Diego, CA, USA) and anti-CD5 (BD Pharmingen, San Diego, CA, USA). PerC B-1a cells were sorted as B220^loCD5⁺, B-1b as B220^loCD5⁻Mac-1^lo/+, and PerC B-2 cells were sorted as B220^hiCD5⁻Mac-1⁻. A MoFlo instrument (Dako) was used for cell sorting.

The preparation of brain extract, as well as determination of protein concentration, electrophoretic separation, blotting onto nitrocellulose membranes, and the test of immunoreactivities in the Mini-Cassette System, was performed as previously described (27) with secondary anti-IgM antibody coupled to alkaline phosphatase from Southern Biotechnology (Birmingham, AL, USA). The four gels used for limiting dilution assay (LDA) of reactivities were polymerized and run in parallel, such that profiles of total proteins blotted (revealed with colloidal gold) and the immunoreactivity profiles of lanes containing the standard pooled supernatant matched accurately between the gels, both qualitatively and quantitatively.

The densitometric profiles of immunoreactivities were analyzed as previously described (30). Briefly, profiles were acquired first by scanning (Silverscanner II). This was followed by colloidal gold staining in order to reveal the migration position of the proteins. A comparison between any two immunoreactivity profiles could subsequently be performed by referring to the corresponding protein profiles. Data analysis was performed using the software IGOR Pro (Wavemetrics, Lake Oswego, OR, USA). Special software packages were written by the authors for the analysis and statistical treatment of the data and can be obtained from the authors.

B cell cultures were performed as described previously (36). Sorted B-1a, B-1b, and B-2 cells were cultured in 250 μl of complete RPMI medium in 96 well flat-bottom plates in the presence of 30 μg/ml of LPS (Salmonella typhimurium, Sigma-Aldrich). All cultures contained 5 × 10³ S17 feeder cells/well for growth support as described by Ref. (37) with some modifications. Briefly, 1 day before the start of the LDA cultures, 5 × 10³ S17 cells were added per well and incubated overnight at 37°C with 5% CO₂. The next day, the S17 culture plates were irradiated with 3000 rad and various numbers of the sorted B cells were added to the cultures. Culture supernatants were used to determine the frequency of IgM secreting clones by ELISA according to the Poisson distribution (38, 39) and contained sorted B cells with 22 replicates for each cell number (18, 6, 2, and 0.66 B cells per well). Another culture set was performed to analyze reactivity profiles by immunoblot, with 22 replicates of 10,000; 3333; 1111; and 370 B cells added per well. Culture supernatants were typically harvested on the fifth day of culture, unless indicated otherwise in the text.

To determine IgM concentration in the supernatants, ELISA was performed as previously described (36), using anti mouse IgM-specific reagents (Southern Biotechnology). Standard curves, obtained by using polyclonal, serially threefold diluted, mouse IgM (Southern Biotechnology), were used to quantify IgM.

Differences between populations were assessed by Student’s t-test, two tailed; Fisher’s exact test, two tailed; χ2-test; or Levene’s tests for the homogeneity of variance, as appropriate. Analysis was performed with JMP version 7.0 (SAS Institute, Inc., Cary, NC, USA). Means are reported with the SE of the mean.