Preferential Use of DH Reading Frame 2 Alters B Cell Development and Antigen-Specific Antibody Production

Robert L. Schelonka, Michael Zemlin, Ryoki Kobayashi, Gregory C. Ippolito, Yingxin Zhuang, G. Larry Gartland, Alex Szalai, Kohtaro Fujihashi, Klaus Rajewsky and Harry W. Schroeder Jr.
J Immunol December 15, 2008, 181 (12) 8409-8415; DOI: https://doi.org/10.4049/jimmunol.181.12.8409

Abstract

All jawed vertebrates limit use of DH reading frames (RFs) that are enriched for hydrophobic amino acids. In BALB/c mice, DFL16.1 RF2 encodes valine and isoleucine. To test whether increased use of RF2 affects B cell function, we examined B cell development and Ab production in mice with an IgH allele (ΔD-DμFS) limited to use of a single, frameshifted DFL61.1 gene segment. We compared the results of these studies to wild-type mice, as well as those previously obtained in mice limited to use of either a single normal DH or a single inverted DH that forces use of arginine in CDR-H3. All three of the mouse strains limited to a single DH produced fewer immature B cells than wild type. However, whereas mice limited to a single normal DH achieved normal B cell numbers in the periphery, mice forced to preferentially use RF2 had reduced numbers of mature B cells in the spleen and bone marrow, mirroring the pattern previously observed in mice enriched for charged CDR-H3s. There were two exceptions. B cells in the mice using RF2 normally populated the marginal zone and peritoneal cavity, whereas mice using inverted RF1 had increased numbers of marginal zone B cells and decreased numbers of B1a cells. When challenged with several T-dependent or T-independent Ags, Ag-specific Ab titers in the mice forced to use RF2 were altered. These findings indicate that B cell development and Ag-specific Ab production can be heavily influenced by the global amino acid content of the CDR-H3 repertoire.

The Ig H chain CDR 3 (CDR-H3)4 is created by the imprecise joining of individual V, D, and J gene segments and the variable inclusion of germline-encoded P (palindromic) and nongermline-encoded N nucleotides (1, 2, 3). Coupled with its location at the center of the Ag binding site, the diversity created by this process permits CDR-H3 to play an often decisive role in Ag recognition and binding (4, 5, 6).

One property unique to the DH gene segment is its ability to undergo rearrangement into any one of six distinct DH reading frames (RFs), each with its own peptide sequence. From shark to mouse to human, the pattern of amino acid usage within each of these RFs has been held remarkably constant. RF1 by deletion tends to encode tyrosine and glycine, RF1 by inversion tends to include positively charged amino acids, and RF2 and RF3 by deletion and inversion tend to encode hydrophobic amino acids. Paradoxically, DH RF choice in vivo has been shown to be tightly controlled by genetic means (7, 8, 9, 10), with RF1 used more frequently than all other RFs combined. This germline-encoded DH RF bias effectively limits diversity, which is the opposite of what might be expected for a gene segment named for its potential to increase diversity.

The genetically conserved bias for the use of only one of the six potential RFs raises the possibility that natural selection acts to limit diversity, using RF choice as a means to optimize immune function. This then raises the question of whether the use of RF1 enhances immune function or whether the use of the other RFs degrades it, a process termed D-disaster (11). To begin to distinguish between these possibilities, we previously created a strain of mice forced to use an inverted RF1 sequence that enriched CDR-H3 for positively charged amino acids. These mice exhibit altered patterns of B cell development and have diminished Ab production (9). However, inverted RFs are rarely used in vivo and the DH allele that we created to force this change did not easily permit access to a tyrosine and glycine-enriched RF1 sequence. Thus, it remains unclear whether the alterations in B cell numbers and Ab production that result from the use of the inverted DH sequence reflect the presence of charged CDR-H3s or the absence of tyrosine and glycine-enriched CDR-H3s.

Inverted DH RFs are very rarely used to encode CDR-H3 amino acids; however, amino acids derived from a RF2 sequence are a common component of the mature repertoire, albeit in the minority. Thus, the benefits, if any, of minimizing the use of DH in RF2 are much less clear (12). This issue has recently taken on added significance with the appreciation that the CDR-H3 regions of the most effective neutralizing Abs against HIV, which are difficult to elicit (13, 14), tend to contain hydrophobic amino acids such as those encoded by RF2 (15, 16, 17). This only emphasizes the question of why RF1 is so predominant when limiting the use RF2 could have a detrimental effect on host immunity by limiting the recognition of potentially critical epitopes.

To address this issue, we have evaluated immune function in mice forced to use DH RF2 sequence in preference to RF1 (10). We find that even though one-fifth of the CDR-H3 repertoire in these mutant mice incorporates RF1-encoded tyrosine and glycine-enriched sequence, mice bearing an RF2-enriched repertoire have reduced numbers of mature B cells in the bone marrow and spleen, and Ag-specific Ab production is impaired. To avoid D-disaster, a proper mix of tyrosine and glycine in CDR-H3 may be required for optimal immune function.

Materials and Methods

Flow cytometric analysis and cell sorting

Flow cytometric analysis and cell sorting was performed as previously described on cells from bone marrow, spleen, and peritoneal cavity (9, 10, 18, 19). A MoFlo instrument (DakoCytomation) was used for cell sorting. Cells were independently sorted from the bone marrow, spleen, and peritoneal cavity of two homozygous ΔD-DμFS (depleted DH locus (ΔD) with a single frameshifted DFL16 gene segment (DμFS)) and two wild-type siblings (8-wk old). Developing B lineage cells in the bone marrow were identified on the basis of surface CD19, CD43, IgM, BP-1, and IgD (10, 20). In the spleen, transitional T1(A), T2(A), and T3 subsets were identified by the surface expression of CD19, AA4.1, IgM, and CD23 as described by Allman et al., which is what the “A” in parentheses stands for (21) (Fig. 1⇓A). Mature follicular and marginal zone (MZ) B cell populations were determined on the basis of surface expression of CD19, CD21, and CD23 (22) (Fig. 1⇓B). The transitional T1(L) subset was differentiated by the surface expression of CD19, IgM, and CD21 as described by Loder et al., which is what the “L” in parentheses stands for (23) (Fig. 1⇓B). In the peritoneal cavity; B1a, B1b, and B2 populations were identified on the basis of surface expression of CD19, CD5, and Mac-1 (20) (Fig. 1⇓C).

FIGURE 1.
FIGURE 1.

Representative flow cytometric analyses and gates. The average total numbers of cells in each of these subpopulations are given in Table I⇓. A, Transitional (Allman et al.; Ref. 21 ) B lineage subpopulations from the spleen of homozygous ΔD-DμFS and wild-type (WT) littermates. Cells within the lymphocyte gate in the spleen cells were first differentiated on the basis of CD19+ and AA4.1 expression. T1(A), T2(A), and T3(A) cell populations were then distinguished on the basis of CD23 and IgM expression (21 ). B, Transitional (Loder et al.; Ref. 23 ), MZ, and mature B cells from the spleen of homozygous ΔD-DμFS and wild-type (WT) littermates. Cells within the lymphocyte gate in the spleen cells were first differentiated on the basis of CD19+ expression. Transitional 1 (T1(L)) and 2 (T2(L)) subpopulations were differentiated by the surface expression of CD19, IgM, and CD21 (23 ). The marginal zone and mature B cell subpopulations were identified on the basis of CD23 and CD21 expression (22 ). C, Peritoneal cavity B lineage cells from homozygous ΔD-DμFS and (wild-type) WT littermates. Cells within the lymphocyte gate in the peritoneal cavity were first distinguished on the basis of CD19+ expression and then into B1a, B1b, and B2 subsets on the basis of surface CD5 and Mac-1 expression.

Immunizations

To survey the effect of altering the CDR-H3 repertoire on Ab production, we measured serum IgM, IgG subclass, and IgA Ig levels. We challenged the mice with α(1→3)-dextran (DEX), which in wild-type BALB/c mice elicits a T-independent response that is dominated by λ1 L chain-bearing Abs that express a diverse range of Ag binding sites with heterogeneous CDR-H3 sequences (24, 25). We measured the response to the 4-hydroxy-3-nitrophenyl)acetyl hapten (NP) of NP-chicken gamma globulin (NP19CGG), a T-dependent response that in BALB/c mice elicits a large fraction of IgG1λ anti-NP Abs (26). Finally, we evaluated the response to a fragment of tetanus toxoid (TT), which was expressed by a recombinant Salmonella typhimurium BRD 847 (aroA−, aroD−) (27, 28). ELISA determinations of basal levels of Ig isotypes in unimmunized 8-wk-old ΔD-DμFS and wild-type littermates using DEX, anti-NP, and anti-TT responses were performed as previously described (9, 19).

Assessment of autoreactive Abs

Anti-nuclear Abs (ANA) were measured by ELISA using a mouse ANA BioAssay ELISA kit from United States Biological. In this assay, IgG ANA level is quantitated based on the ability of mouse serum (diluted 100-fold) to react with nuclear Ag immobilized on microtiter wells. After a washing step, a goat anti-mouse IgG-HRP conjugate is added and ANA-bound conjugate is detected with the chromogenic substrate tetramethylbenzidine. The extent of the enzymatic reaction is measured as absorbance at 450 nm and is directly proportional to the amount of IgG ANA present in the sample. ANA negative and ANA positive mouse sera (United States Biological) were used as controls.

Statistical analysis

Statistical analysis was performed with JMP version 6.0 (SAS Institute) as previously described (9, 18, 19). Means are accompanied by the SEM.

Results

Enhanced use of RF2 alters B cell development

To test whether increased use of RF2-encoded amino acids in CDR-H3 influences B cell development, we compared the average absolute number of B lineage cells by developmental stage in a cohort of homozygous ΔD-DμFS female mice to a companion cohort of wild-type female littermate controls (Fig. 2⇓ and Table I⇓). Among developing ΔD-DμFS B cells, an initial increase in the number of pro-B (fraction B) cells was followed by normalization of the early pre-B (fraction C) population. The late pre-B (fraction D) and immature B (fraction E) compartments had a 40% decrease in numbers when compared with controls (p = 0.001 and p = 0.0002, respectively). This pattern of impairment matched that previously observed in ΔD-DFL (ΔD with a single DFL16.1 gene segment) and ΔD-iD mice (DD with single mutated DFL16.1 gene segment with inverted DSP 2.2 sequence) (9, 19).

FIGURE 2.
FIGURE 2.

Divergence in the absolute numbers of B lineage subpopulations from the bone marrow, spleen, and peritoneal cavity of homozygous ΔD-DFL, ΔD-DμFS, and ΔD-iD mice relative to their littermate controls. A, Percentage of loss or gain in homozygous ΔD-DFL, ΔD-DμFS, and ΔD-iD mice relative to wild-type littermate controls in the average absolute number of cells in the bone marrow fractions B (CD19+CD43+IgM−BP-1−), C (CD19+CD43+IgM−BP-1+), D (CD19+CD43−IgM−IgD−), and E (CD19+CD43−IgM+IgD−); transitional T1(A) (CD19+AA4.1+sIgMhighCD23−), T2(A) (CD19+AA4.1+sIgMhighCD23+), and mature follicular (CD19+CD21lowCD23high) B cell subsets (21 ,22 ,23 ); and in bone marrow mature recirculating fraction F (CD19+CD43−IgMlowIgDhigh) (Table I⇓). B, Percentage of loss or gain in homozygous ΔD-DFL, ΔD-DμFS, and ΔD-iD mice relative to wild-type littermate controls in the average absolute number of cells in splenic transitional T1(L) (CD19+IgMhighCD21low), T3 (CD19+ AA4.1+sIgMlowCD23+), MZ (CD19+CD21highCD23low), and in peritoneal cavity B1a (CD19+CD5+), B1b (CD19+CD5−Mac-1+), and B2 (CD19+CD5−Mac-1−) (Table I⇓). In both panels the SEM of each B lineage subpopulation for the littermate controls averaged ∼11% of the absolute number of cells in each subpopulation (gray area). For ΔD-DFL, ΔD-DμFS, and ΔD-iD the SEM is shown as an error bar. ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001.

View this table:
Table I.

Cell numbers in the bone marrow, spleen, and peritoneal cavity of normal and mutant micea

In the spleen and bone marrow, the pattern of conventional B cell production in ΔD-DμFS mice mirrored the reductions observed in ΔD-iD mice rather than in ΔD-DFL, which develop normal numbers of cells (Fig. 2⇑ and Table I⇑). The sizes of the transitional T1(A) (CD19+AA4.1+IgMhighCD23−) and T2(A) subsets (CD19+AA4.1+IgMhighCD23+) (21) were statistically indistinguishable from those of wild-type littermate controls (Fig. 2⇑ and Table I⇑). However, a decrease in the transitional splenic T1(L) (CD19+ IgMhigh CD21low) population of Loder et al. (23) was apparent, and the splenic follicular (CD19+CD23highCD21low) and bone marrow mature recirculating fraction F (CD19+CD43−IgM+IgD+) B cell compartments were almost halved (p = 0.001 and p = 0.0001, respectively) (Fig. 2⇑ and Table I⇑). MZ (CD19+CD23lowCD21high) B cell numbers and the numbers of peritoneal B-1a (CD19+CD5+), B-1b (CD19+CD5−Mac-1+), and B-2 (CD19+CD5−Mac-1−) cells were normal in ΔD-DμFS mice (Fig. 2⇑ and Table I⇑). Thus, the reduction in B cell numbers was primarily focused in the conventional mature B cell populations of the splenic follicles and bone marrow.

Ab production in BALB/c ΔD-DμFS mice is variably impaired

Although serum Ig levels in the ΔD-DμFS mice were similar to those in wild-type controls (Fig. 3⇓), the pattern of immune responses to specific Ags proved extremely variable. In wild-type BALB/c mice, i.v. challenge with DEX elicits a T-independent response that is dominated by λ1 L chain-bearing Abs that express a diverse range of Ag binding sites with heterogeneous CDR-H3 sequences (24, 25). Seven days after challenge with DEX, the geometric mean of IgM anti-DEX serum levels in homozygous ΔD-DμFS mice was approximately one-third that observed in wild-type littermates (74 ± 29 μg/ml vs 234 ± 35 μg/ml, p = 0.005) (Fig. 4⇓A). By comparison, the reduction in mean titer was greater than that observed in ΔD-DFL, but less than that observed in ΔD-iD (9, 19) (Fig. 4⇓A). This suggests that anti-DEX responsiveness is a function of the composition and complexity of the wild-type repertoire. The results suggest a direct correlation between the extent of divergence from the normal repertoire and the divergence of the host response to this T-independent Ag.

FIGURE 3.
FIGURE 3.

Serum Ig levels in homozygous ΔD-DμFS mice. Spontaneously produced IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA levels in ΔD-DμFS mice are indistinguishable from wild-type (WT) littermate controls under specific pathogen-free conditions.

FIGURE 4.
FIGURE 4.

Ab responses in homozygous ΔD-DμFS mice. A, Seven days after challenge with DEX, T-independent anti-DEX IgM serum levels were reduced in ΔD-DFL (n = 8) (19 ), ΔD-DμFS (n = 10) (this work), and ΔD-iD (n = 5) (9 ) relative to normalized wild-type (WT) littermate controls (n = 8, n = 9, and n = 4, respectively). In each experiment, the geometric mean titer was lower and the variance was greater in D-limited mice. Within experiments, the relative decrease in titer was positively correlated with the degree of divergence of the D-limited CDR-H3 repertoire from that expressed by wild-type mice, with mean titers decreasing by one-half in ΔD-DFL, one-third in ΔD-DμFS, and one-eighth in ΔD-iD. B, After primary and secondary challenge with NP19-CGG, the T-dependent production of IgG anti-NP was indistinguishable between ΔD-DμFS (n = 9) and wild-type (WT) (n = 10) mice.

In BALB/c, the primary response to the NP hapten of NP19-CGG requires T cell help and contains a large fraction of IgG1λ anti-NP Abs (26). Among those sequences that have been cloned from this population, many incorporate DFL16.1 in RF1. After primary and secondary i.p. challenge with NP19-CGG, the anti-NP IgG response in the ΔD-DμFS mice proved indistinguishable from the littermate controls (Fig. 4⇑B). This level of response was identical with that which we previously observed in ΔD-DFL mice, which exclusively express DFL16.1-RF1 encoded Igs (19). In comparison, we have previously shown that the anti-NP response in ΔD-iD, which requires somatic mechanisms to generate DFL16.1-like sequences, was 3-fold diminished when compared with wild type (9).

In BALB/c mice, immunization with purified TT elicits a T-dependent response that is dominated by κ L chain-bearing Abs (29). Limitation to DFL16.1 and enrichment for use of DFL16.1 RF2 had a variable effect on this T-dependent response. After oral immunization with a recombinant strain of Salmonella that expresses the Tox C fragment of tetanus toxin (27), the IgM anti-TT response doubled in ΔD-DFL mice (p = 0.04). It underwent a 4-fold increase in the ΔD-DμFS mice (p = 0.008) (Fig. 5⇓). The total IgG anti-TT response in ΔD-DFL mice was slightly increased, although this did not achieve statistical significance. IgG1, IgG2a, and IgG2b levels were statistically indistinguishable from those of wild type. However, the IgG3 anti-TT response in ΔD-DFL mice was 16-fold increased (p < 0.0001). This pattern was reversed in ΔD-DμFS, where the IgG anti-TT titer was 16-fold reduced (p = 0.004) with a concomitant 6-fold reduction in IgG1 (p = 0.04) and IgG2a (p = 0.03), and a 34-fold decrease in IgG2b. This contrasted sharply with an IgG3 titer that was equivalent to wild type. The IgA anti-TT response in ΔD-DμFS mice was reduced relative to controls, but the extent of reduction did not achieve statistical significance.

FIGURE 5.
FIGURE 5.

Response to oral immunization with recombinant salmonella that expresses the Tox C fragment of tetanus toxin. A, The IgM anti-TT response was increased in ΔD-DFL and ΔD-DμFS, whereas the IgG anti-TT response was reduced in ΔD-DμFS mice. B, In ΔD-DFL mice the IgG1, IgG2a, and IgG2b anti-TT response was similar to that of wild type (WT), but IgG3 was 16-fold increased. In contrast, ΔD-DμFS mice produced lower levels of anti-TT IgG1, IgG2a, and IgG2b than wild-type mice, but similar IgG3 levels as wild-type mice. Thus, the production of Ig isotypes and subclasses is differentially regulated according to the preferred CDR-H3 composition. ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001.

To test whether altering DH RF bias might lead to the early appearance of autoreactive Igs, we examined sera from 15-member cohorts of 8-wk-old female ΔD-DμFS, ΔD-DFL, ΔD-iD, and wild-type mice for evidence of ANA. ANA reactivity was observed only in mice carrying the ΔD-iD allele, with elevations over background in the sera of two of the 15 cohort members.

Discussion

Although DH gene segments were named for their role in increasing the potential for diversity at the time of Ig rearrangement, molecular properties that promote a preference for specific RFs while preserving a particular pattern of amino acid usage by each RF are conserved (8, 30). In the present work, by taking a genetic approach we created a mouse model that allowed us to test in vivo the effects on immune function of overriding the evolutionarily conserved biologic prohibition against too frequent use of RF2. This permitted us to directly test the D-disaster hypothesis (11). Indeed, when viewed in conjunction with our previous studies of ΔD-DFL and ΔD-iD mice (9, 10, 18, 31), the addition of these new ΔD-DμFS mice created a stable of D-limited mutants with which we could perform a full spectrum examination of the effects of DH RF amino acid content on B cell development and Ab production.

All three D-limited strains exhibited a similar reduction of immature B cell numbers. This would suggest that global sequence-based selection in the bone marrow against progenitor B cells expressing Ig μ-chains that have incorporated CDR-H3s of disfavored hydrophobicity plays a relatively limited role in B cell production when compared with the effects of the loss of efficiency in DJ rearrangement forced by the deletion of the other 12 DH gene segments (19).

Previous studies by other investigators have shown that mice with a wild-type DH locus can achieve normal numbers of peripheral B cells in the face of a 70% reduction in immature B cell numbers (32). Among the three strains of D-limited mice, all of which exhibited an ∼40% reduction of immature B cell numbers, only ΔD-DFL mice with their tyrosine- and glycine-enriched CDR-H3s were able to achieve normal homeostasis by filling the splenic follicles, the MZ, the peritoneal cavity, and the mature, recirculating pool in the bone marrow with normal numbers of cells.

In previous studies with wild-type mice, we found that the splenic T1(L) transitional compartment housed a population of B cells that bore charged or hydrophobic CDR-H3s at a frequency similar to that observed in the immature B cell compartment from bone marrow (33). However, charged or hydrophobic CDR-H3s were rare among the mature IgM+IgD+ B cells that populated the splenic follicles or the recirculating bone marrow pool. Conversely, although hydrophobic CDR-H3s were rare among marginal zone B cells, nearly one in 10 of the VDJ sequences we obtained from the marginal zone incorporated charged CDR-H3s, suggesting that B cells might be distributed to the various compartments based, in part, on the composition of CDR-H3.

Combining the results we previously obtained in wild-type (18, 33), ΔD-DFL (19), and ΔD-iD (9) mice with these new results from ΔD-DμFS, we found that the differences in B cell numbers between the ΔD-iD and ΔD-DμFS mice and those observed in ΔD-DFL followed the same pattern of inclusion or exclusion of specific categories of CDR-H3 hydrophobicity that we had previously observed in splenic MZ and follicular B cells and in the mature, recirculating B cells of the bone marrow. For example, we observed a 30% increase in MZ cell numbers in the ΔD-iD mice with their charged CDR-H3s, but not in ΔD-DμFS mice with their hydrophobic Ag binding sites.

Previous studies from a number of laboratories (reviewed in Ref. 34) have shown that recruitment into the follicles, the MZ, and the B-1 compartment can be heavily influenced by the Ag specificity of the Ab. The findings from our three D-limited mice now show that the amino acid content of the CDR-H3 region at the center of the Ag binding site can also influence recruitment into these same compartments and that the achievement of wild-type numbers may require a repertoire enriched for a specific category of tyrosine and glycine-enriched CDR-H3s. The exact relationship between Ag specificity and CDR-H3 amino acid content remains to be determined. However, further study of the nature of this relationship may lead to a better understanding of the forces and Ags that determine the developmental fate of B cells in the periphery.

Without kinetic studies, which are currently in progress in our laboratories, we cannot yet say whether the decrease in B cell numbers reflects diminished longevity, anergy, or diminished cell division. However, the average number of cells in the splenic CD19+AA4.1+IgMlowCD23+ compartment, termed T3 by Allman et al. (21) and T3(A) by this work, was decreased by more than a third (p = 0.01) in both the ΔD-iD and ΔD-DμFS mice. This population has been associated with anergy (35); thus, its decline would tend to suggest that the decrease in B cell numbers most likely reflects one or both of the other two mechanisms.

To assess the effect of altering the CDR-H3 repertoire on Ab production, we measured total serum Ig levels. Among 8-wk-old ΔD-DμFS littermates, the average concentrations of IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were indistinguishable from those of wild type. This differed from ΔD-iD mice, where total IgG and IgG subclass levels were significantly depressed, but was similar to what was observed in ΔD-DFL mice, which achieved normal levels (9, 19).

However, although total serum Ig levels in the ΔD-DμFS mice were equivalent to controls, Ab production in response to specific Ags varied. IgM Abs in the anti-DEX response, which is T-independent, typically use CDR-H3s whose short length precludes identification of the parental DH (24, 25). When compared with controls, anti-DEX titers in the ΔD-DμFS mice were intermediate between ΔD-DFL and ΔD-iD, mirroring the extent of divergence in complexity and amino acid content from wild type.

IgG anti-NP Abs preferentially use DFL16.1 in RF1. Anti-NP titers were normal in both ΔD-DFL, which preferentially uses DFL16.1 in RF1, and in ΔD-DμFS, where RF1 sequences are present but in the minority. Anti-NP titers were significantly lower in ΔD-iD, which cannot incorporate a DFL16.1-like sequence into their Ig without the aid of a nongermline nucleotide (N) addition (9, 19). Unlike the hapten NP, anti-tetanus Tox C immunoglobulins have not been linked to use of a specific set of DH sequences. IgG anti-TT titers were differentially influenced by the global changes in the complexity and amino acid content of the CDR-H3 repertoire represented by ΔD-DFL, ΔD-DμFS, and ΔD-iD, with ΔD-DμFS again representing the intermediate state.

Together, these findings imply that in addition to patterns of B cell development, the relative representation of favored vs nonfavored CDR-H3 repertoires can also have significant effects on Ag-specific Ab production. Regulation of the global composition of the primary Ab repertoire thus represents a mechanism whereby specific immune responses can be controlled.

Potential examples of the use of this mechanism to limit immune responses exist in human. In the first trimester of life, one-half of the expressed Ab repertoire uses the D7–27 (DQ52) gene segment (reviewed in Ref. 36). In both mouse and human, DQ52 differs from the other D gene segments by the absence of tyrosine (37, 38), making its CDR-H3 repertoire distinct from the rest. It remains to be determined whether the ontogenetic changes in immune responses observed between the human fetus and adult can be directly attributed to this change in DH preference or whether other human conditions, such as the relative immune deficiency associated with aging, will also prove to be associated with alterations in the regulation of CDR-H3 content.

Unlike T cell receptors, which bind to processed epitopes and thus have general access to linear peptides on the interior as well as the surface of Ags, Igs tend to bind to epitopes expressed by intact molecules in aqueous solution whose surfaces tend to be enriched for neutral or polar epitopes. The apparent bias against creating binding sites that bind nonpolar surfaces creates a potential “hole” in the immune response. The consequences of this “blind spot” may well be illustrated by the nature of the binding of 2F5 and 4E10 to the HIV gp41 membrane-proximal external region (MPER). 2F5 and 4E10 are among the most potent and broadly neutralizing anti-HIV Abs identified to date (39). Both anti-gp41 MPER Abs contain long CDR-H3 regions with a hydrophobic tip, which enables them to bind simultaneously to the water-soluble surface of the gp41 MPER and to the adjacent viral lipid membrane (39). It is possible that the difficulty that has been experienced in raising Abs like 2F5 and 4E10 reflects the relative paucity of Ig with hydrophobic CDR-H3s.

Our results document the existence of categories of Ig Ag binding sites whose sequence properties can influence B cell maturation in the bone marrow, regulate the population of peripheral B cell subsets, and even influence the efficiency of producing specific IgG subclass responses after antigenic challenge. It is possible that some infectious agents may evade proper immune surveillance by either avoiding the presentation of epitopes on their surface that can be recognized by the preferred spectrum of repertoires (39) or by suppressing immune function by enriching for the use of disfavored CDR-H3s (40). Given that constraints on CDR-H3 amino acid content and hydrophobicity appear to be present in all jawed vertebrates, the expression of genetically favored categories of Abs that influence B cell maturation and immune function may represent a reminiscence of the first Ig-like Ag receptor produced by a cartilaginous fish more than 500 million years ago that likely incorporated a CDR-H3 region enriched for tyrosine and glycine encoded by that portion of the molecule now contributed by the DH.

Acknowledgments

We thank P. Burrows, M. Cooper, and J. Kearney for invaluable advice and support.

Disclosures

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 in part by National Institutes of Health Grants AI07051, AI42732, AI48115, HD043327, and TW02130; by Deutsche Forschungsgemeinschaft Grant SFB/TR22-TPA17; and by Alexander von Humboldt-Stiftung FLF1071857.

  • ↵2 R.L.S. and M.Z. contributed equally to this work

  • ↵3 Address correspondence and reprint requests to Dr. Harry W. Schroeder, Jr., University of Alabama at Birmingham, Shelby Building 176, Third Avenue South, Birmingham, AL 35294. E-mail address: hwsj{at}uab.edu

  • ↵4 Abbreviations used in this paper: CDR-H3, Ig H chain CDR 3; ANA, antinuclear Ab; ΔD, depleted DH locus; DEX, α(1→3)-dextran; DFL, single DFL16.1 gene segment; DμFS, single frameshifted DFL15.1 gene segment; iD, mutated DFL16.1 gene segment with inverted DSP 2.2 sequence; MZ, marginal zone; MPER, membrane-proximal external region; NP, (4-hydroxy-3-nitrophenyl)acetyl hapten; NP19CGG, NP-chicken gamma globulin; RF, reading frame; TT, tetanus toxoid.

  • Received June 23, 2008.
  • Accepted October 8, 2008.

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