Predominance of VH-D-JH junctions occurring at sites of short sequence homology results in limited junctional diversity in neonatal antibodies.

Sequence diversity at the junctions of Ig genes differs between newborn and adult mice in two respects: 1) fetal/newborn Ig lack N regions; and 2) these N- junctional sequences very often contain 1 to 6 nucleotides that could have been encoded by either of the two joined gene segments. We address the hypothesis that such short homologies preferentially direct recombination to that site, and we analyze the effect of such homology-directed recombination upon the neonatal Ig repertoire. We examined 546 CDR3 sequences that were generated from polymerase chain reaction-amplified DNA from fetal and newborn liver using primers from three different VH families: S107, 7183, and J558. All junctional sequences using 14 frequently occurring IgH V-D and D-J gene combinations were analyzed. In 12 of the 14 combinations analyzed, there were 1 to 3 short sequence homologies, and the junctional sequences that would be created by those homologies were observed with high frequency. The D-J junctions often had two to three predominant junctional sequences, whereas the V-D junctions had one dominant junctional sequence. The only exceptions were the VHJ558-D junctions, where homology-directed recombination using the sequence homology between VHJ558 genes and most D genes would result in an out-of-frame join, and most of our sequences were productive. This latter result further suggests that homology-directed recombination may play a role in the nonrandom VH gene usage observed in fetal and newborn mice. Thus, most neonatal IgH junctions show limited diversity, not only due to the lack of N regions, but also because of nonrandom junctional sequences. Inasmuch as the few adult N- junctions also show a high frequency of homology-directed junctional sequences, V-D-J recombination throughout life may involve pairing via short homologies, with addition of N regions obscuring its role in the formation of adult IgH junctions.     

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 (D_H) 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 D_H 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 D_H 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 D_H sequence did not. Thus, germline bias against the use of arginine enriched D_H sequence helps to reduce the likelihood of producing self-reactive antibodies.     

The V<Subscript>H</Subscript> gene repertoire of splenic B cells and somatic hypermutation in systemic lupus erythematosus

In systemic lupus erythematosus (SLE) it has been hypothesized that self-reactive B cells arise from virgin B cells that express low-affinity, nonpathogenic germline V genes that are cross-reactive for self and microbial antigens, which convert to high-affinity autoantibodies via somatic hypermutation. The aim of the present study was to determine whether the V_H family repertoire and pattern of somatic hypermutation in germinal centre (GC) B cells deviates from normal in SLE. Rearranged immunoglobulin V_H genes were cloned and sequenced from GCs of a SLE patient's spleen. From these data the GC V gene repertoire and the pattern of somatic mutation during the proliferation of B-cell clones were determined. The results highlighted a bias in V_H5 gene family usage, previously unreported in SLE, and under-representation of the V_H1 family, which is expressed in 20–30% of IgM⁺ B cells of healthy adults and confirmed a defect in negative selection. This is the first study of the splenic GC response in human SLE.     

Antibody Expression from the Core Region of the Human IgH Locus Reconstructed in Transgenic Mice Using Bacteriophage P1 Clones

Mice carrying transgenic human immunoglobulin gene miniloci can be used for the production of human monoclonal antibodies. The human variable region (V) gene segments in these miniloci undergo productive rearrangement in mouse lymphoid tissue to yield a population of B lymphocytes expressing a repertoire of antibodies. Many of the miniloci studied to date have included only a small number of germline gene segments in an artificially compact configuration. Here we describe the use of the bacteriophage P1 cloning system to create mice carrying the core region of the human immunoglobulin heavy chain (IgH) locus. Three P1 clones carrying overlapping regions of the human IgH locus (spanning the five J_H-proximal V_Hsegments, the entire D_Hand J_Hclusters, and the Cμ and Cδ constant regions) were injected into mouse eggs and appear to have reconstituted the core region of the locus (>180 kb) following homologous recombination with each other. While this translocus yielded a titer of serum immunoglobulin similar to that obtained with a smaller plasmid-based minilocus, the P1-based locus gave rise to substantially greater diversification by somatic hypermutation. Such diversification is important for obtaining high-affinity antibodies. The results show the usefulness of the P1 system in facilitating the manipulation and recreation of large transgenes.     

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human V_H repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the V_H3 (41%), V_H4 (39%), and V_H1 (14%) subgroups used more frequently than the V_H5 (3.9%) or V_H7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human V_H genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.     

DNA cleavage of a cryptic recombination signal sequence by RAG1 and RAG2. Implications for partial V(H) gene replacement

Antibody and T cell receptor genes are assembled from gene segments by V(D)J recombination to produce an almost infinitely diverse repertoire of antigen specificities. Recombination is initiated by cleavage of conserved recombination signal sequences (RSS) by RAG1 and RAG2 during lymphocyte development. Recent evidence demonstrates that recombination can occur at noncanonical RSS sites within Ig genes or at other loci, outside the context of normal lymphocyte receptor gene rearrangement. We have characterized the ability of the RAG proteins to bind and cleave a cryptic RSS (cRSS) located within an Ig V(H) gene segment. The RAG proteins bound with sequence specificity to either the consensus RSS or the cRSS. The RAG proteins nick the cRSS on both the top and bottom strands, thereby bypassing the formation of the DNA hairpin intermediate observed in RAG cleavage of canonical RSS substrates. We propose that the RAG proteins may utilize an alternative mechanism for double-stranded DNA cleavage, depending on the substrate sequence. These results have implications for further diversification of the antigen receptor repertoire as well as the role of the RAG proteins in genomic instability.     

TCR delta gene rearrangements revealed by fine structure of the recombination junction in mice

The standard products of V(D)J recombination of immunoglobulin and T cell receptor genes are two kinds of DNA junction, a coding joint and a signal joint. TCR delta V-D and D-D signal joints in adult mouse thymocytes were sequenced following PCR amplification. We observed differential nucleotide insertions at the V delta-D delta signal joints, depending on the V delta and D delta gene usage in the developmental stage. Nucleotide insertions at the V delta-D delta 1 signal joints were less frequent for the V delta 4, 5 genes preferentially utilized in adult thymocytes than for the V delta 3, 6 genes, infrequently rearranged to D delta 1. In addition to standard signal joints, unexpectedly, novel nonstandard products, "replacement joints" of D delta 1 substituted downstream by the recombination signal sequence of V delta were also found. However, no D delta 2-associated replacement joints other than V delta 5 were found. The other replacement joints of D delta 1-D delta 2 recombination were also observed. The mutation in TCR beta gene affected the frequency of nucleotide insertions at the V delta-D delta signal joints and inhibited the formation of replacement joint. Recombination mechanism generating the replacement joint and the possible role of TCR beta in up-regulation of TCR delta gene rearrangements are discussed.     

High RAD51 mRNA levels in young rabbit appendix. A role in B-cell gene conversion?

 The rabbit has a limited number of V _H genes that rearrange. As in the chicken, the 3′-most V _H 1 gene is rearranged in most B lymphocytes. This laboratory reported that by 6 weeks after birth, diversification of rearranged V _H genes occurs, at least in part, by gene conversion-like events in the appendix, suggesting that this organ is a homologue of the avian bursa of Fabricius. Rad51 contributes to the repair of double-strand breaks in DNA during somatic and meiotic recombination. The gene was first identified in lower eukaryotes, and later in vertebrates including chicken, as encoding an Escherichia coli RecA-like protein. We report the cloning and sequencing of RAD51 from the rabbit. Because the chicken bursa was shown to express high levels of RAD51 message, we investigated the expression of RAD51 in the rabbit appendix and other tissues. Using a quantitative polymerase chain reaction mimic assay and conventional northern analyses, we found high RAD51 expression in young rabbit appendix comparable to levels in testis where there is an abundance of meiotic recombination. RAD51 levels were three times higher in appendix B lymphocytes compared with T lymphocytes and were lower in adult appendix, as well as in spleen and Peyer's patches of young rabbits. We measured the levels of message in several appendix cell sub-populations obtained by fluorescence-activated cell sorting and found that sub-populations of B lymphocytes corresponding to different stages of B-cell development as well as B cells undergoing isotype switch did not have significantly different mRNA levels. Received: 18 October 1997 / Revised: 10 December 1997     

Human Immunoglobulin Repertoires against Tetanus Toxoid Contain a Large and Diverse Fraction of High-Affinity Promiscuous VH Genes

To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (V_H) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (V_L) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the V_H regions in this V_L-restricted panel with a previously published repertoire of anti-TT V_H regions with cognate V_H-V_L pairing showed a very similar distribution of V_H, D_H and J_H gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-V_L anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-V_L anti-TT V_H repertoire was combined with a collection of naïve V_L regions and again selected for binding to TT, many of the V_H genes were recovered in combination with a diversity of V_L regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT V_H combined with 15 diverse V_L chains were determined and found to be identical to each other and to the original isolate restricted to a single-V_L chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding V_H regions from an immunized repertoire have promiscuous features. These V_H regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, V_H region diversity and affinity of a natural repertoire.     

Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a γ2a variable region

To examine germ line genes of the heavy chain variable region (V_H) that might contribute to formation of antibodies of the NP^b family, we have derived cDNA clones from two hybridomas making NP^b antibodies. One, B1–8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG_2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the V_H region, had very different D segments and had identical J segments (J₂). A set of closely related germ line V_H genes was then cloned from a partial Eco RI library of C57BI/6 DNA. By comparing the germ line V_H regions to the cDNA V_H regions, we identified seven potential candidates for encoding the V_H regions of NP^b antibodies. The seven V_H regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1–8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1–8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other V_H genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their V_H regions from the same germ line gene. Certain of the sequenced V_H genes contain crippling mutations; the repertoire of germ line V_H genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hybridization.     

Regeneration of the immunoglobulin heavy-chain repertoire after transient B-cell depletion with an anti-CD20 antibody

B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis. Nevertheless, the role of B cells in the pathogenesis of the disease is not clear. In particular, it is not known how the regeneration of the B-cell repertoire takes place. Two patients with active rheumatoid arthritis were treated with rituximab, and the rearranged immunoglobulin heavy-chain genes (Ig-V_H) were analysed to follow the B-cell regeneration. Patient A was treated with two courses of rituximab, and B-cell regeneration was followed over 27 months by analysing more than 680 Ig-V_H sequences. Peripheral B-cell depletion lasted 7 months and 10 months, respectively, and each time was accompanied by a clinical improvement. Patient B received one treatment course. B-cell depletion lasted 5 months and was accompanied by a good clinical response. B cells regenerated well in both patients, and the repopulated B-cell repertoire was characterised by a polyclonal and diverse use of Ig-V_H genes, as expected in adult individuals. During the early phase of B-cell regeneration we observed the expansion and recirculation of a highly mutated B-cell population. These cells expressed very different Ig-V_H genes. They were class-switched and could be detected for a short period only. Patient A was followed long term, whereby some characteristic changes in the V_H2 family as well as in specific mini-genes like V_H3–23, V_H 4–34 or V_H 1–69 were observed. In addition, rituximab therapy resulted in the loss of clonal B cells for the whole period.     

Beyond the 12/23 rule of VDJ recombination independent of the Rag proteins

The combinatorial repertoire of AgRs is established through somatic recombination of V, D, and J gene segments during lymphocyte development. Incorporation of D segments into IgH, TCRbeta, and TCRdelta chains also contributes to junctional diversification by substantially extending the length of the third CDR. The V, D, and J gene segments are flanked by recombination signals (RS) of 12- or 23-mer spacer length that direct recombination according to the 12/23 rule. D genes in the TCRbeta and TCRdelta loci are flanked by a 12RS and 23RS, and their incorporation is controlled by mechanisms "beyond the 12/23 rule." In the TCRbeta locus, selective interactions between Rag proteins and the RS flanking the V-D and D-J genes, respectively, are sufficient to enforce D gene usage. In this article, we report that in the TCRdelta locus, the Rag proteins are not the major determinant of D gene incorporation. In developing mouse and human thymocytes, the two Ddelta genes rearrange predominantly to form D-D coding joints. In contrast, when tested in ex vivo transfection assays in a nonlymphoid cell line, the flanking RS mediate deletion, rather than incorporation, of the two D genes on both exogenous recombination substrates and the endogenous locus. These results suggest that selective Rag-RS interactions are not the sole regulators of D gene segment incorporation, and additional, perhaps lymphocyte-specific, mechanisms exist that allow proper shaping of the primary AgR repertoire.     

A single VH gene segment encodes the immune response to phosphorylcholine: Somatic mutation is correlated with the class of the antibody

The immune response in BALB/c mice to phosphorylcholine is highly restricted in its heterogeneity. Of the 19 immunoglobulins binding phosphorylcholine for which complete V_H-segment amino acid sequences have been determined, 10 employ a single sequence, denoted T15 after the prototype V_H sequence of this group of antibodies. The remaining 9 of these V_H segments are variants differing by 1 to 8 residues from the T15 sequence. Using a cloned V_H cDNA probe complementary to the T15 sequence, we isolated from a mouse sperm genomic library clones corresponding to four V_H gene segments that by DNA sequence analysis are >85% homologous to one another. These four V_H gene segments have been denoted the T15 V_H gene family. These V_H gene segments are most, if not all, of the germline V_H gene segments that could encode the V_H sequences of antibodies that bind phosphorylcholine. One of these four genes contains the T15-V_H-coding sequence. When the T15-family V_H gene segments were compared with the complete V_H protein sequences of 19 hybridoma and myeloma immunoglobulins that bind phosphorylcholine, several striking conclusions could be drawn. First, all of these V_H regions must have arisen from the germline T15 V_H gene segment. Thus virtually the entire immune response to phosphorylcholine is derived from a single V_H-coding sequence. Nine of the 19 V_H regions were variants differing from the T15-V_H-coding sequence and, accordingly, must have arisen by a mechanism of somatic diversification. Second, the variants appear to be generated by a somatic mutation mechanism. They cannot be explained by recombination or gene conversion among members of the T15 gene family. Third, somatic mutation is correlated with the class of the antibody. All of the somatic variation is found in the V_H regions derived from antibodies of the IgA and IgG classes. The IgM molecules express the germline T15 V_H gene segment exclusively.     

Accumulation of Self-Reactive Na?ve and Memory B Cell Reveals Sequential Defects in B Cell Tolerance Checkpoints in Sj?gren’s Syndrome

Sjögren’s syndrome (SS) is an autoimmune disease characterised by breach of self-tolerance towards nuclear antigens resulting in high affinity circulating autoantibodies. Although peripheral B cell disturbances have been described in SS, with predominance of naïve and reduction of memory B cells, the stage at which errors in B cell tolerance checkpoints accumulate in SS is unknown. Here we determined the frequency of self- and poly-reactive B cells in the circulating naïve and memory compartment of SS patients. Single CD27−IgD+ naïve, CD27+IgD+ memory unswitched and CD27+IgD− memory switched B cells were sorted by FACS from the peripheral blood of 7 SS patients. To detect the frequency of polyreactive and autoreactive clones, paired Ig V_H and V_L genes were amplified, cloned and expressed as recombinant monoclonal antibodies (rmAbs) displaying identical specificity of the original B cells. IgV_H and V_L gene usage and immunoreactivity of SS rmAbs were compared with those obtained from healthy donors (HD). From a total of 353 V_H and 293 V_L individual sequences, we obtained 114 rmAbs from circulating naïve (n = 66) and memory (n = 48) B cells of SS patients. Analysis of the Ig V gene repertoire did not show significant differences in SS vs. HD B cells. In SS patients, circulating naïve B cells (with germline V_H and V_L genes) displayed a significant accumulation of clones autoreactive against Hep-2 cells compared to HD (43.1% vs. 25%). Moreover, we demonstrated a progressive increase in the frequency of circulating anti-nuclear naïve (9.3%), memory unswitched (22.2%) and memory switched (27.3%) B cells in SS patients. Overall, these data provide novel evidence supporting the existence of both early and late defects in B cell tolerance checkpoints in patients with SS resulting in the accumulation of autoreactive naïve and memory B cells.     

Deletion mapping of the mouse ornithine decarboxylase-related locus Odc-rs8 within Igh-V

The Odc-rs8 locus belongs to a family of mouse DNA sequences related to the gene encoding ornithine decarboxylase (ODC). Odc-rs8 was mapped by recombinant inbred (RI) strain analysis to the region of Chromosome (Chr) 12 occupied by the variable region genes of the immunoglobulin heavy chain (Igh) complex. In the present study, alleles at Odc-rs8 were shown to cosegregate with those for Igh variable region (Igh-V or V _H) genes among 37 inbred mouse strains that had been characterized previously for their haplotypes at Igh. For a more precise definition of the location of Odc-rs8 relative to Igh-V, DNAs from 17 Abelson murine leukemia virus (A-MuLV)-transformed pre-B cell lines cultured from mice heterozygous at Igh and Odc-rs8 were analyzed for the presence of DNA restriction fragments (RFs) derived from each parental Odc-rs8 allele. These cell lines, each of which has rearranged one or both Igh genes, previously were employed in mapping members of nine V _H gene families by deletion analysis (Brodeur et al. 1988). Comparing the deletion profiles of the cell lines for Odc-rs8 with those for the V _H gene families has located Odc-rs8 ^b within the V_HJ558/V_H3609 gene cluster and Odc-rs8 ^c either within or upstream of the 5-most 9% of V_HJ558, identifying Odc-rs8 as a potentially useful marker for the 5 end of the Igh complex.     

How DNA loop extrusion mediated by cohesin enables V(D)J recombination

‘Structural maintenance of chromosomes’ (SMC) complexes are required for the folding of genomic DNA into loops. Theoretical considerations and single-molecule experiments performed with the SMC complexes cohesin and condensin indicate that DNA folding occurs via loop extrusion. Recent work indicates that this process is essential for the assembly of antigen receptor genes by V(D)J recombination in developing B and T cells of the vertebrate immune system. Here, I review how recent studies of the mouse immunoglobulin heavy chain locus Igh have provided evidence for this hypothesis and how the formation of chromatin loops by cohesin and regulation of this process by CTCF and Wapl might ensure that all variable gene segments in this locus (V_H segments) participate in recombination with a re-arranged DJ_H segment, to ensure generation of a maximally diverse repertoire of B-cell receptors and antibodies.     

Antibody repertoires in humanized NOD-scid-IL2Rγ(null) mice and human B cells reveals human-like diversification and tolerance checkpoints in the mouse

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγ^null engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγ^null mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential D_H-J_H pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.