Mohamed Khass

VpreB serves as an invariant surrogate antigen for selecting immunoglobulin antigen-binding sites

The VpreB component of the surrogate light chain serves as an invariant antigen that may affect the repertoire of antibody responses. Surrogate selection A broad yet specific antibody response is critical to fight off pathogens; however, it remains unclear why certain antibodies may be more easily induced than others. Now, Khass et al. report that a component of the surrogate light chain—VpreB—may also act as an invariant antigen that contributes to shaping the antibody repertoire. They demonstrated that progressively reducing the tyrosine content of the heavy chain antigen recognition site impaired the ability of B cells expressing the pre-BCR to pass the first checkpoint. VpreB selected for particular amino acids in the complementarity-determining region of the heavy chain, shaping the subsequent antibody response. Developmental checkpoints eliminate B cells that synthesize defective immunoglobulin (Ig) heavy (HC) and light (LC) chains. The first checkpoint tests μHCs paired with VpreB/λ5 in a pre-B cell receptor (pre-BCR) to determine whether the μHC will be able to bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with the μHC antigen-binding region complementarity-determining region (CDR)–H3; however, whether this site contributes to Ig repertoire selection and function is unknown. We analyzed the amino acid content of CDR-H3s from HCs cloned from living and apoptotic pre-B cells and from IgG-antigen structures. Using a panel of DH gene–targeted mice, we showed that progressively reducing CDR-H3 tyrosine content increasingly impaired pre-BCR checkpoint passage. Counting from cysteine at framework 3 position 96, we found that VpreB particularly selected for tyrosine at CDR-H3 position 101 and that Y101 also bound antigen in IgG-antigen structures. Therefore, in addition to its stabilization role in the pre-BCR, VpreB also acts as an early invariant antigen by selecting for particular CDR-H3 amino acids. These interactions shape the specificity of the IgG humoral response and may thus impose limitations on development of certain neutralizing antibodies.Developmental checkpoints eliminate B cells synthesizing defective immunoglobulin heavy (HC) and light (LC) chains. The first checkpoint tests for formation of a VpreB/λ5/µHC-containing preB-cell receptor (preBCR) and predicts whether µHCs will bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with µHC antigen-binding region CDR-H3, but whether it plays a role in immunoglobulin repertoire selection and function is unknown. On a position-by-position basis, we analyzed the amino acid content of CDR-H3s from H chains cloned from living and apoptotic preB cells and from IgG:Antigen structures. Using a panel of DH gene-targeted mice, we show that progressively reducing CDR-H3 tyrosine content increasingly impairs preBCR checkpoint passage. Counting from cysteine at Framework 3 position 96, we found that VpreB particularly selects for tyrosine at CDR-H3 position 101, and that Y101 also binds antigen in IgG:Antigen structures. VpreB thus acts as an early invariant antigen. It selects for particular CDR-H3 amino acids and shapes the specificity of the IgG humoral response. This helps explain why some neutralizing antibodies against pathogens are readily produced while others are rare.

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 (IgHb) operate similarly to those identified in BALB/c mice (IgHa), we compared the sequences of VH7183‐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 IgHa 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.

Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

In the Absence of Central pre-B Cell Receptor Selection, Peripheral Selection Attempts to Optimize the Antibody Repertoire by Enriching for CDR-H3 Y101

Sequential developmental checkpoints are used to “optimize” the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires μHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies.

Susceptibility Loci in C57BL/6 sle1, sle2 and sle3 Contain Genes that Alter Peripheral Selection of the CDR-H3 Sequences Enriched for Arginine

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by deposition of ds- DNA binding autoantibodies in various body organs. These antibodies result from failure to control the composition of the B cell repertoire. Development of optimum B cell repertoire depends on the amino acid composition and the physicochemical characteristics at the center of the antigen binding site, the third complementarity determining region heavy chain (CDR-H3). Repertoire control involves positive selection for hydrophilic amino acids such as tyrosine and negative selection of hydrophobic and charged amino acids, specifically those containing arginine within the CDR-H3. Anti-dsDNA antibodies present in SLE patients exist in healthy individuals but at low levels, since dsDNA-specific B cells are deleted from the repertoire, but amplified in SLE patients. These antibodies contain arginine residues in CDR-H3, especially at positions 99-102, where they are positioned to bind negatively charged phosphate groups on the DNA backbone. Three genomic intervals, namely sle1 on chromosome 1, sle2 on chromosome 4, and sle3 on chromosome 7, were found to be associated with SLE susceptibility. We hypothesized that development of ds-DNA binding antibodies in SLE might result from failure to control CDR-H3 amino acid composition. We proposed that the SLE congenic loci might have unique effects in allowing survival/expansion of B cells expressing these auto-reactive antibodies. Our strategy was to change the composition of CDR-H3 by altering the germline composition of the DH gene segments. We created a ΔD-iD altered allele enriched for arginine while depleted of tyrosine at positions 99-102. We then monitored the influence of different SLE loci on the development and maintenance of B cells bearing CDR-H3 arginine. These findings support our hypothesis that peripheral B cell selection is altered by the presence of sle congenic alleles, allowing passage of B cells able to produce autoreactive antibodies binding ds-DNA. These findings may help in developing therapeutics to suppress autoimmunity in SLE.

The sequences encoded by immunoglobulin diversity (DH) gene segments play key roles in controlling B-cell development, antigen-binding site diversity, and antibody production

Although at first glance the diversity of the immunoglobulin repertoire appears random, there are a number of mechanisms that act to constrain diversity. For example, key mechanisms controlling the diversity of the third complementarity determining region of the immunoglobulin heavy chain (CDR‐H3) include natural selection of germline diversity (DH) gene segment sequence and somatic selection upon passage through successive B‐cell developmental checkpoints. To test the role of DH gene segment sequence, we generated a panel of mice limited to the use of a single germline or frameshifted DH gene segment. Specific individual amino acids within core DH gene segment sequence heavily influenced the absolute numbers of developing and mature B‐cell subsets, antibody production, epitope recognition, protection against pathogen challenge, and susceptibility to the production of autoreactive antibodies. At the tip of the antigen‐binding loop (PDB position 101) in CDR‐H3, both natural (germline) and somatic selection favored tyrosine while disfavoring the presence of hydrophobic amino acids. Enrichment for arginine in CDR‐H3 appeared to broaden recognition of epitopes of varying hydrophobicity, but led to diminished binding intensity and an increased likelihood of generating potentially pathogenic dsDNA‐binding autoreactive antibodies. The phenotype of altering the sequence of the DH was recessive for T‐independent antibody production, but dominant for T‐cell‐dependent responses. Our work suggests that the antibody repertoire is structured, with the sequence of individual DH selected by evolution to preferentially generate an apparently preferred category of antigen‐binding sites. The result of this structured approach appears to be a repertoire that has been adapted, or optimized, to produce protective antibodies for a wide range of pathogen epitopes while reducing the likelihood of generating autoreactive specificities.

Alterations in B cell development, CDR-H3 repertoire and dsDNA-binding antibody production among C57BL/6 ΔD−iD mice congenic for the lupus susceptibility loci sle1, sle2 or sle3

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease that reflects a failure to block the production of self-reactive antibodies, especially those that bind double-stranded DNA (dsDNA). Backcrossing the lupus-prone NZM2410 genome onto C57BL/6 led to the identification of three genomic intervals, termed sle1, sle2 and sle3, which are associated with lupus susceptibility. We previously generated a C57BL/6 strain congenic for an immunoglobulin DH locus (ΔD–iD) that enriches for arginine at dsDNA-binding positions. We individually introduced the ΔD–iD allele into the three sle strains to test whether one or more of these susceptibility loci could affect the developmental fate of B cells bearing arginine-enriched CDR-H3s, the CDR-H3 repertoire created by the DH and the prevalence of dsDNA-binding antibodies. We found that the combination of the ΔD–iD allele and the sle1 locus led to a decrease in mature, recirculating B cell numbers and an increase in marginal zone cell numbers while maintaining a highly charged CDR-H3 repertoire. ΔD–iD and sle2 had no effect on peripheral B cell numbers, but the CDR-H3 repertoire was partially normalized. ΔD–iD and sle3 led to an increase in marginal zone B cell numbers, with some normalization of hydrophobicity. Mice with ΔD–iD combined with either sle1 or sle3 had increased production of dsDNA-binding IgM and IgG by 12 months of age. These findings indicate that the peripheral CDR-H3 repertoire can be categorically manipulated by the effects of nonimmunoglobulin genes.