HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence.

Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization.

Recirculating bone marrow B cells in C57BL/6 mice are more tolerant of highly hydrophobic and highly charged CDR-H3s than those in BALB/c mice.

To test whether mechanisms controlling the range of diversity of the developing antibody repertoire in C57BL/6 mice (IgH(b)) operate similarly to those identified in BALB/c mice (IgH(a)), we compared the sequences of VH 7183-containing H-chain transcripts from sorted adult bone marrow C57BL/6 B-cell subsets with those previously obtained from BALB/c mice. Patterns of VDJ gene segment utilization and CDR-H3 amino acid composition, charge, and average length in C57BL/6 pro-B cells were similar, although not identical, to BALB/c pro-B cells. However, C57BL/6 mature, recirculating B cells failed to demonstrate the reduction in the use of VH81X and the narrowing in the range of variance of CDR-H3 hydrophobicity that characterizes B-cell maturation in BALB/c mice. To further test the ability of the C57BL/6 strain to discard B cells expressing highly charged CDR-H3s, we introduced a mutant IgH(a) DH allele that forces use of arginine, asparagine, and histidine. Unlike BALB/c mice, C57BL/6 mice congenic for the charged DH maintained normal numbers of mature, recirculating B cells that were enriched for charged CDR-H3s. Together these findings indicate that the mature C57BL/6 B-cell pool permits expression of immunoglobulins with antigen-binding sites that are typically discarded during late-stage bone marrow B-cell development in BALB/c mice.

The peritoneal cavity B-2 antibody repertoire appears to reflect many of the same selective pressures that shape the B-1a and B-1b repertoires.

To assess the extent and nature of somatic categorical selection of CDR-3 of the Ig H chain (CDR-H3) content in peritoneal cavity (PerC) B cells, we analyzed the composition of V(H)7183DJCµ transcripts derived from sorted PerC B-1a, B-1b, and B-2 cells. We divided these sequences into those that contained N nucleotides (N(+)) and those that did not (N(-)) and then compared them with sequences cloned from sorted IgM(+)IgD(+) B cells from neonatal liver and both wild-type and TdT-deficient adult bone marrow. We found that the PerC B-1a N(-) repertoire is enriched for the signatures of CDR-H3 sequences present in neonatal liver and shares many features with the B-1b N(-) repertoire, whereas the PerC B-1a N(+), B-1b N(+), and B-2 N(+) repertoires are enriched for adult bone marrow sequence signatures. However, we also found several sequence signatures that were not shared with other mature perinatal or adult B cell subsets but were either unique or variably shared between the two or even among all three of the PerC subsets that we examined. These signatures included more sequences lacking N nucleotides in the B-2 population and an increased use of D(H) reading frame 2, which created CDR-H3s of greater average hydrophobicity. These findings provide support for both ontogenetic origin and shared Ag receptor-influenced selection as the mechanisms that shape the unique composition of the B-1a, B-1b, and B-2 repertoires. The PerC may thus serve as a general reservoir for B cells with Ag binding specificities that are uncommon in other mature compartments.

DH and JH usage in murine fetal liver mirrors that of human fetal liver.

In mouse and human, the regulated development of antibody repertoire diversity during ontogeny proceeds in parallel with the development of the ability to generate antibodies to an array of specific antigens. Compared to adult, the human fetal antibody repertoire limits N addition and uses specifically positioned VDJ gene segments more frequently, including V6-1 the most D(H)-proximal V(H,) DQ52, the most J(H)-proximal D(H), and J(H)2, which is D(H)-proximal. The murine fetal antibody repertoire also limits the incorporation of N nucleotides and uses its most D(H) proximal V(H), V(H)81X, more frequently. To test whether D(H) and J(H) also follow the pattern observed in human, we used the scheme of Hardy to sort B lineage cells from BALB/c fetal and neonatal liver, RT-PCR cloned and sequenced V(H)7183-containing VDJCµ transcripts, and then assessed V(H)7183-D(H)-J(H) and complementary determining region 3 of the immunoglobulin heavy chain (CDR-H3) content in comparison to the previously studied adult BALB/c mouse repertoire. Due to the deficiency in N nucleotide addition, perinatal CDR-H3s manifested a distinct pattern of amino acid usage and predicted loop structures. As in the case of adult bone marrow, we observed a focusing of CDR-H3 length and CDR-H3 loop hydrophobicity, especially in the transition from the early to late pre-B cell stage, a developmental checkpoint associated with expression of the pre-B cell receptor. However, fetal liver usage of J(H)-proximal D(H)Q52 and D(H)-proximal J(H)2 was markedly greater than that of adult bone marrow. Thus, the early pattern of D(H) and J(H) usage in mouse feta liver mirrors that of human.

Preferential use of DH reading frame 2 alters B cell development and antigen-specific antibody production.

All jawed vertebrates limit use of D(H) 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 (DeltaD-DmicroFS) 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 D(H) or a single inverted D(H) that forces use of arginine in CDR-H3. All three of the mouse strains limited to a single D(H) produced fewer immature B cells than wild type. However, whereas mice limited to a single normal D(H) 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.

Regulation of repertoire development through genetic control of DH reading frame preference.

In jawed vertebrates most expressed Ig H chains use only one of six possible D(H) reading frames. Reading frame (RF)1, the preferred reading frame, tends to encode tyrosine and glycine, whereas the other five RFs tend to be enriched for either hydrophobic or charged amino acids. Mechanisms proposed to favor use of RF1 include a preference for deletion over inversion that discourages use of inverted RF1, RF2, and RF3; sequence homology between the 5' terminus of the J(H) and the 3' terminus of the D(H) that promotes rearrangement into RF1; an ATG start site upstream of RF2 that permits production of a truncated Dmicro protein; stop codons in RF3; and, following surface expression of IgM, somatic, presumably Ag receptor-based selection favoring B cells expressing Igs with tyrosine- and glycine-enriched CDR-H3s. By creating an IgH allele limited to the use of a single, frameshifted DFL16.1 D(H) gene segment, we tested the relative contribution of these mechanisms in determining reading frame preference. Dmicro-mediated suppression via an allelic exclusion-like mechanism dominated over somatic selection in determining the composition of the CDR-H3 repertoire. Evidence of somatic selection for RF1-encoded tyrosine in CDR-H3 was observed, but only among the minority of recirculating, mature B cells that use D(H) in RF1. These observations underscore the extent to which the sequence of the D(H) acts to delimit the diversity of the Ab repertoire.

Analysis of TACI mutations in CVID & RESPI patients who have inherited HLA B*44 or HLA*B8.

BACKGROUND: Recent reports have suggested that Common Variable Immunodeficieny (CVID) can present as an autosomal dominant trait dependent on the inheritance of a set of uncommon mutations/alleles of TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) involving exons 3 or 4. Penetrance, however, appears to be incomplete. Among our clinic population, the greatest genetic linkage for CVID is to the major histocompatibility complex (MHC) on chromosome 6. The majority of our patients have inherited HLA *DQ2, *DR7, *DR3(17), *B8, and/or *B44. Of these, HLA*B44 was present in almost half of the patients and was thus the most common susceptibility allele. HLA *B44 was also found to be over-represented among patients who presented to our clinic with adult-onset recurrent sinopulmonary infections (RESPI) and normal serum immunoglobulin levels, a cohort that included first and second degree relatives of patients with CVID. One of the two original reports of the association between TACI and CVID also reported Human Leukocyte Antigen (HLA) haplotypes. Of 13 affected subjects, nine had inherited HLA *B8 and six had inherited HLA B44. This raised the possibility that TACI mutations might synergize with MHC class I alleles to enhance susceptibility to humoral immune deficiency. METHODS: We identified 63 CVID patients irrespective of HLA status and 13 RESPI patients who had inherited HLA*B44. To evaluate for mutations in the gene for TACI, we PCR amplified and sequenced TACI exons 3 and 4 from these patients. RESULTS: Of the 76 patients, eleven proved heterozygous for a previously reported, silent T->G polymorphism [rs35062843] at proline 97 in exon 3. However, none of the 13 RESPI patients and only one of the 63 CVID patients inherited a TACI allele previously associated with CVID. This patient was heterozygous for the TACI A181E allele (exon 4). She did not carry *DQ2, *DR7, *DR3(17), *B8, or *B44. CONCLUSION: These findings suggest that TACI mutations are unlikely to play a critical role in creating susceptibility to CVID among patients with previously recognized MHC class I and class II susceptibility alleles. Supported by NIH/USIDNET N01-AI30070, NIH R21 AI079741 and NIH M01-RR00032.

Differences in the composition of the human antibody repertoire by B cell subsets in the blood.

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V(D)J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N-region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V(D)J gene usage, hydrophobicity, length, DH reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD(+), memory IgD(-), and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse, the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.

Categorical selection of the antibody repertoire in splenic B cells.

In the bone marrow, the passage of developing B cells through critical checkpoints of differentiation is associated with a reduction of specific categories of CDR3 of the Ig heavy chain (CDR-H3), particularly those with excessive hydrophobic or charged amino acids and those with a length of eight or fewer residues. To gain insight into the role of CDR-H3 content in the development of B cells in the spleen, we compared the sequences of V(H)7183DJCmu transcripts from sorted transitional T1, marginal zone, and follicular B cell subsets to those expressed by immature IgM(+)IgD(-) and mature IgM(lo)IgD(hi) B cells in the bone marrow. Although differences in V(H) utilization were noted, the T1 CDR-H3 repertoire showed extensive similarity to that of immature bone marrow B cells, and the follicular CDR-H3 repertoire most resembled that of mature bone marrow B cells. Unlike the splenic follicular and bone marrow mature B cell CDR-H3 repertoires, the marginal zone B cell CDR-H3 repertoire retained both short and highly charged amino acid motifs, including those with two arginines. Our findings suggest that antigen binding sites containing specific categories of CDR-H3 sequence content may inhibit, permit, or even facilitate passage of the host B cell through critical checkpoints in peripheral as well as central development.

Development of the expressed Ig CDR-H3 repertoire is marked by focusing of constraints in length, amino acid use, and charge that are first established in early B cell progenitors.

To gain insight into the mechanisms that regulate the development of the H chain CDR3 (CDR-H3), we used the scheme of Hardy to sort mouse bone marrow B lineage cells into progenitor, immature, and mature B cell fractions, and then performed sequence analysis on V(H)7183-containing Cmu transcripts. The essential architecture of the CDR-H3 repertoire observed in the mature B cell fraction F was already established in the early pre-B cell fraction C. These architectural features include V(H) gene segment use preference, D(H) family usage, J(H) rank order, predicted structures of the CDR-H3 base and loop, and the amino acid composition and average hydrophobicity of the CDR-H3 loop. With development, the repertoire was focused by eliminating outliers to what appears to be a preferred repertoire in terms of length, amino acid composition, and average hydrophobicity. Unlike humans, the average length of CDR-H3 increased during development. The majority of this increase came from enhanced preservation of J(H) sequence. This was associated with an increase in the prevalence of tyrosine. With an accompanying increase in glycine, a shift in hydrophobicity was observed in the CDR-H3 loop from near neutral in fraction C (-0.08 +/- 0.03) to mild hydrophilic in fraction F (-0.17 +/- 0.02). Fundamental constraints on the sequence and structure of CDR-H3 are thus established before surface IgM expression.

A single DH gene segment creates its own unique CDR-H3 repertoire and is sufficient for B cell development and immune function.

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 DH locus. We term this D-limited IgH allele DeltaD-DFL. Although the absolute number of IgM+IgD- B cells in the bone marrow was decreased, homozygous DeltaD-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 (alpha(1-->3)-dextran) was observed in DeltaD-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 DH gene segment of normal sequence was sufficient for development of normal numbers of mature B cells and for robust humoral immune function.