The little brown bat, M. lucifugus, displays a highly diverse VH, DH and JH repertoire but little evidence of somatic hypermutation

Myotis lucifugus populations in Northeastern US are being decimated by a fungal disease. Since almost nothing is known about the immune system of bats, we are characterizing the immunoglobulin genes of bats. We show that M. lucifugus has a diverse V_H gene repertoire comprised of five of the seven human V_H gene families and an estimated 236 V_H3 genes. 95% of these germline VH3 genes differ in FR3. A comparison of 67 expressed V_H3 genes with 75 germline V_H3 genes revealed a mutation frequency similar to fetal piglets never exposed to environmental antigens. Analysis of CDR3 regions identified at least 13 putative J_H segments and a large D_H repertoire. The low mutation frequency, highly diverse V_H, D_H, and J_H germline repertoire suggests that this species may rely more on combinatorial and junctional diversity than on somatic hypermutation raising questions about the ability of M. lucifugus to respond rapidly to emerging pathogens.     

Pattern of usage and somatic hypermutation in the VH5 gene segments of a patient with asthma: Implications for IgE

The V_H5 family contains two functional genes, V_5–51 and V_H32, and appears to be over-represented in IgE antibodies from patients with allergic disease. Previous sequence analysis of V_H5 gene segments in IgE has revealed a substantial level of somatic hypermutation, with evidence for hotspots. To assess characteristics of V_H5 gene behavior, V_H5 gene segments in combination with Cμ, Cγ, Cα, and Cε have been amplified from blood B lymphocytes of a patient with atopic asthma. Sequence analysis revealed strong preferential usage of one of the two V_H5 gene segments (V_5–51) by IgM, IgG, and IgA. In contrast, IgE used both genes equally. Levels of somatic mutation were higher following all isotype switches, particularly to IgA. Mutational hotspots were identifiable in all isotypes, leading to several common replacement amino acids. The dominant mutational site in IgM was a common hotspot at Ser31. IgG, IgA, and IgE-derived sequences had mainly common hotspots, with few distinct sites. The results indicate that mutational hotspots are a feature of the V_H5 gene, are identifiable at an early stage of somatic hypermutation, and are not a unique feature of IgE. Generation of IgE antibodies appears to involve three processes: the preferential use of V_H5 genes, consistent with superantigen stimulation; the accumulation of somatic mutations in common hotspots, some of which are in complementarity-determining regions (CDR); and the acquisition of non-hotspot mutations in CDR, accounting for ～ 50% of replacement amino acids in these sites, and presumably contributing to affinity maturation.     

The VH repertoire and clonal diversification of B cells in inflammatory myopathies

The contribution of antigen‐driven B‐cell adaptive immune responses within the inflamed muscle of inflammatory myopathies (IMs) is largely unknown. In this study, we investigated the immunoglobulin V_H gene repertoire, somatic hypermutation, clonal diversification, and selection of infiltrating B cells in muscle biopsies from IM patients (dermatomyositis and polymyositis), to determine whether B cells and/or plasma cells contribute to the associated pathologies of these diseases. The data reveal that Ig V_H gene repertoires of muscle‐infiltrating B cells deviate from the normal V_H gene repertoire in individual patients, and differ between different types of IMs. Analysis of somatic mutations revealed clonal diversification of muscle‐infiltrating B cells and evidence for a chronic B‐cell response within the inflamed muscle. We conclude that muscle‐infiltrating B cells undergo selection, somatic hypermutation and clonal diversification in situ during antigen‐driven immune responses in patients with IMs, providing insight into the contribution of B cells to the pathological mechanisms of these disorders.     

Rheumatoid factors in primary Sjögren's syndrome (pSS) use diverse VH region genes,the majority of which show no evidence of somatic hypermutation

Rheumatoid factor (RF) is the most common autoantibody found in patients with Sjögren's syndrome (SS). To study the genetic origin and the mechanisms acting behind its generation we have characterized and sequenced the immunoglobulin V_H genes used by 10 IgM RF MoAbs derived from peripheral blood of six female patients with pSS. We compared the structure of the RF immunoglobulin V_H genes with those obtained previously from rheumatoid arthritis (RA) patients and healthy immunized donors (HID). V_H1 and V_H4 were each used by four RF clones, one clone was encoded by V_H3 family gene and one by V_H2 family gene. This distribution frequency was different from that observed in RA, where V_H3 was the dominant family, followed by V_H1. Eight different germ-line (GL) genes encoded the clones and all of these genes were seen previously in RA and/or HID RF. Five clones rearranged to J_H6, four rearranged to J_H4 and one to J_H5, in contrast to RF from RA and HID, where J_H4 was most frequently used. D segment use and CDR3 structure were diverse. Interestingly, three out of four V_H4 clones used the GL gene DP-79 that was seen frequently in RA RF. The degree of somatic mutation in the pSS RF was very much lower than seen in RA and HID RF. All the pSS RF clones except three were in or very close to GL configuration. This indicates that there is little role for somatic hypermutation and a germinal centre reaction in the generation of RF from peripheral blood in pSS.     

Entry of B lymphocytes into the persistent cell pool in non-immunized mice is not accompanied by somatic mutation of VH genes

In this study we compare V_H-gene repertoires of short-lived and persistent B lymphocytes in normal nonimmunized mice. Enriched populations of persistent peripheral B cells were obtained in vivo either by (i) repeated injections with hydroxyurea or (ii) maintained ganciclovir administration to herpes simplex virus-1 thymidine kinase transgenic mice. Both approaches have previously been shown to deplete newly formed, short-lived B cells. V_H genes expressed by persistent or unselected B cell populations were amplified by polymerase chain reaction, cloned using the λ-ImmunoZAP system (Stratagene) and sequenced. The results presented here concern a total of 116 complete V_H sequences from two V_H gene families of established germ-line composition: V_H7183 and V_HX24. No differences were found between the two cell populations as to usage of D or J_H segments and to the presence of N sequence additions at D/J_H or V_H/DJ_H junctions and CDR3 length. Over 90% of the sequenced V_H genes were of germ-line arrangement with no evidence of somatic mutation. These results show that persistent B cells in normal mice are not of embryonic origin and that somatic hypermutation is not necessary for B cell survival. They also suggest that a significant fraction of persistent IgM⁺ B cells in normal mice are not generated by conventional antigenic stimulation and could represent a novel class of “memory” cells expressing germ-line repertoires.     

Somatic hypermutation of VHS107 genes is not associated with gene conversion among family members

Murine B cells proliferating in the germinal centers of peripherallymphoid tissue accumulate mutations in their rearranged variableregions, a diversification process which contributes to affinitymaturation of the antibody response. The highly targeted natureof the hypermutation process could be explained by a somaticgene conversion mechanism. Well characterized examples of suchan activity in B cells are seen during diversification of thechicken and rabbit Ig repertoires. The genomic organization,low complexity and high degree of homology exhibited by thefour members of the murine V_HS107 gene family suggested thatthese gene segments may be suitable candidates for the searchof gene conversion events derived from upstream V_HS107 counterparts.After an immune response to a complex T cell-dependent antigen(sheep red blood cells), rearranged V13, V11 and V1 genes wereisolated from splenic extrafollicular and germinal center Bcells. Extensive somatic mutation was evident in V11 and V1sequences. When these sequences were examined, as well as V1sequences isolated from phosphorylcholine-specific hybridomas,the observed nucieotide changes were not associated with anygene conversion between family members, suggesting instead thatthey arose by a mechanism which introduces point mutations.     

VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA)

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (V_H) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of V_H genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the V_H composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual V_H genes (eight V_H3 and three V_H4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these V_H genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of V_H3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged V_H4 genes was also observed in these patients. These data suggest that although usage of individual V_H genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.     

Molecular analysis of rheumatoid factor (RF)-negative B cell hybridomas from rheumatoid synovial tissue: evidence for an antigen-induced stimulation with selection of high mutated IgVH and low mutated IgVL/λ genes

The mutational pattern of IgV_H and IgV_L genes from synovial tissue B cell hybridomas (n = 8) of patients (n = 4) with rheumatoid arthritis (RA) was analysed, which had been produced by the electrofusion technique without prior in vitro stimulation. The molecular data were correlated with immunohistopathological data and parameters of local disease activity. The IgV_H genes of the B cell hybridomas belonged to the V_H3 family (DP42; DP47, n = 2; DP53), the V_H1 family (DP75), the V_H4 family (DP71) and the V_H5 family (DP73); 7/7 IgV_H genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 4/7 IgV_H genes and the mean R/S ratio of all IgV_H genes was 9.3 (CDR) and 1.0 (FR), suggesting an antigen-dependent selection. The IgV_L/λ genes belonged to the Vλ1 family (DPL2, DPL5, DPL8nf), the Vλ2 family (DPL11, n = 2) and to the Vλ6 family (IGLV6S1); 6/6 IgV_L genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 3/6 IgV_L genes and the mean R/S ratio of all IgV_L was 3.0 (CDR) and 2.3 (FR), suggesting an antigen-dependent selection. The synovial tissue exhibited germinal centres in the follicles (3/4), with the unique distribution of Ki-M4⁺ follicular dendritic cells and Ki-67⁺ proliferating cells and a dominance of IgA⁺ plasma cells (3/3). All patients were positive for RF in serum and exhibited severe local symptoms (swelling 4/4; warmth 4/4; effusion 2/4), whereas the hybridomas were negative for RF. Since B cell hybridomas showed hypermutation and affinity selection for IgV_H and IgV_L/λ genes and the patients exhibited severe local symptoms with germinal centres in synovial tissue, this study indicates that an antigen-driven process is behind the B cell expansion in the synovial tissue of clinically affected joints. These mutated B hybridomas were negative for RF, thus suggesting that antigens different from RF are also involved in the local B cell expansion and in the chronic synovitis of RA.     

Age‐associated B cells expanded in autoimmune mice are memory cells sharing H‐CDR3‐selected repertoires

Age‐associated B cells (ABCs) represent a distinct cell population expressing low levels of CD21 (CD21^?/low). The Ig repertoire expressed by ABCs in aged mice is diverse and exhibits signs of somatic hypermutation (SHM). A CD21^?/low B‐cell population is expanded in autoimmune diseases, e.g. systemic lupus erythematosus, as well as in lupus‐prone NZB/W mice and in mice lacking a pre‐B cell receptor (SLC^?/?). However, the nature of the CD21^?/low B cells (hereafter ABCs) in autoimmunity is not well understood. Here we show that in young SLC^?/? mice, the vast majority of the ABCs express memory B‐cell (MBC) markers in contrast to wild‐type controls. A similar population is present in lupus‐prone MRL mice before and at disease onset. In SLC^?/? mice, a majority of the ABCs are IgM⁺, their V_H genes have undergone SHM, show clonal diversification and clonal restriction at the H‐CDR3 level. ABC hybridomas, established from SLC^?/? mice, secrete typical lupus autoantibodies, e.g. anti‐Smith antigen, and some of those that bind to DNA comprise a H‐CDR3 that is identical to previously described IgM anti‐DNA antibodies from lupus‐prone mice. Together, these results reveal that ABCs in autoimmune mice are comprised of autoreactive MBCs expressing highly restricted H‐CDR3 repertoires.     

Molecular Ig gene analysis reveals that monocytoid B cell lymphoma is a malignancy of mature B cells carrying somatically mutated V region genes and suggests that rearrangement of the kappa-deleting element (resulting in deletion of the Ig kappa enhancers) abolishes somatic hypermutation in the human

Five cases of monocytoid B cell lymphoma (MBCL) were analyzed for somatic mutations in the rearranged V region genes. Somatic mutations were found in four of the five cases, whereas one unusual CD5⁺ lymphoma harbored unmutated V region genes. Since somatic mutations are introduced into V regiongenes of antigen-activated B cells in the course of T cell-dependent immune responses, these results suggest a derivation of the tumor B cells in MBCL from antigen-experienced mature B cells. An analysis of the ϰ-deleting element in two of the cases in which mutated V_H but unmutated and nonfunctional V_ϰ gene rearrangements were found suggests that somatic hypermutation does not take place in human rearranged V_ϰ region genes when the C_ϰ gene and the ϰ enhancers have been deleted in cis by rearrangement of the ϰ-deleting element.     

VH CDR3-dependent positive selection of murine VH12-expressing B cells in the neonate

Five to fifteen percent of peritoneal B1 (CD5⁺) cells from unmanipulated mice produce antibodies that bind bromelain-treated mouse red blood cells and the hapten phosphatidylcholine (PtC). The majority of these B cells express either of two V_H/V? gene combinations, V_H12/_V?4 or V_HH/V?9. Both the V_H11 and V_H12 genes are rearranged to J_H1 and encode third complementarity determining regions (CDR3) of restricted length and sequence. These and other observations argue strongly that PtC-specific Bl cells are antigen selected. To determine when selection of PtC-specific Bl cells begins in mice we have used the polymerase chain reaction to amplify V_H12-D-J_h1 rearrangements from livers of fetal and neonatal mice, and determined the CDR3 encoding sequences of individual clones. We find an unusually low ratio of productive (P) to non-productive (NP) rearrangements (0.4–1.0) at both developmental stages. P rearrangements in day 1 neonates are biased in D gene use and in the sequence and length of their deduced V_H CDR3. These biases are similar to those of PtC-specific B1 cells in the adult peritoneum. D gene use and CDR3 length and sequence are significantly less biased among V_H12 P rearrangements 2 to 3 days earlier in the day 18 fetal liver. We suggest that this rapid change in repertoire is due to positive ligand selection that is dependent on the sequence of V_H CDR3. We suggest further that the majority of V_H12-expressing cells are not ligand selected and consequently undergo programmed cell death. The evidence of restriction in day 1 neonatal livers and the low P/NP ratio in the fetus suggests that selection of V_H12-expressing cells begins before birth.     

Developing neonatal rabbit appendix, a primary lymphoid organ, is seeded by immature blood-borne B cells: evidence for roles for CD62L/PNAd, CCR7/CCL21, alpha4beta1 and LFA-1

Young rabbit appendix is a homologue of chicken bursa of Fabricius; both are crucial sites for preimmune B-cell repertoire development. We describe here some of the molecules involved in the multi-step recruitment of blood-borne B cells into neonatal rabbit developing appendix. Sialyl-Lewis-x, CD62L and integrins such as LFA-1 and alpha4beta1 were detected on B cells in peripheral blood. Peripheral lymph node addressin (PNAd), a CD62L counter-receptor was observed on appendix HEV. We also detected chemokine receptor CCR7 on peripheral blood B cells and one of the CCR7 ligands, CCL21, on appendix HEV but not in appendix follicles. Higher levels of CXCR5 expression compared to CCR7 on appendix B cells suggest that CXCR5 may be involved in recruitment of B cells into follicles. The proportions of appendix B cells expressing CD62L, sialyl-Lewis-x and alpha4beta1 declined between day 3 and 4 weeks after birth while percentages of Lewis-x+ appendix B cells increased. These changes correlate with the stage of repertoire diversification by gene conversion in both rabbits and chickens. The cross-reactivity of antibodies to mouse or human adhesion molecules described in this study indicates that some of the structures of these important molecules are conserved across species.     

Polymorphism of the functional immunoglobulin variable region genes in the chicken by exchange of sequence with donor pseudogenes

We have isolated a number of new allelic variants of the unique functional genes encoding chicken immunoglobulin heavy and light chain variable regions (V_H1 and V_L1, respectively). The distribution and nature of nucleotide variation among these and previously identified V_H1 and V_L1 alleles demonstrates that random point mutations are likely not the predominant cause of allelic variation at these loci. Comparison of the variant nucleotides with sequences from the pseudo-V_H and pseudo-V_L gene families, which lie 5′ to V_H1 and V_L1, respectively, suggests that the great majority of allelic variants can be accounted for by segmental transfer of sequence from donor pseudogenes into the germ-line VH₁ and V_L1 genes. These results demonstrate that the chicken V_H1 and V_L1 genes are susceptible to sequence replacement at the germ-line level as well as somatically during antibody diversification. The limited repertoire of Bcell specificities produced by gene rearrangement in the chicken has led to speculation that these specificities may play a critical role in the progression of chicken B cell development. The results presented here do not support this hypothesis since many of the allelic variant nucleotides described here encode non-conservative amino acid substitutions within the antigen-binding sites of the Ig molecule.     

The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions

Targeted diversification of immunoglobulin variable regions is induced by activation-induced deaminase and may occur by either somatic hypermutation or gene conversion. MRE11-RAD50-NBS1 (MRN) is a ubiquitous and conserved nuclease complex critical for DNA break repair and is essential in class-switch recombination. Here we show that ectopic expression of NBS1, the regulatory subunit of MRN, accelerated hypermutation in the human B cell line Ramos and accelerated gene conversion in the chicken B cell line DT40. In both cases, accelerated diversification depended on MRN complex formation. These data suggest that MRN promotes DNA cleavage and/or mutagenic repair of lesions initiated by activation-induced deaminase, acting in the shared pathway of immunoglobulin gene diversification.     

Discriminating intrinsic and actigen-selected mutational hotspots in immunoglobulin V genes

Studies of the antibody hypermutation mechanism have revealed that it is not a random process but exhibits characteristic nucleotide substitution preferences. Here, Alexander Betz and colleagues show that these innate nucleotide substitution preferences can be used to examine databases of antigen-selected V gene sequences and thereby distinguish intrinsic from antigen-selected hotspots. This analysis reveals intrinsic mutational hotspots in both V_H and V_I genes, reflecting innate features of the hypermutation machinery which may give clues to the enzymatic mechanism.     

Analysis of rearranged immunoglobulin heavy chain variable region genes obtained from a bone marrow transplant (BMT) recipient

Haematopoietic stem cell transplantation has been used for the treatment of many different malignant and non-malignant diseases. The immune system of transplant recipients must be regenerated from the transplant inoculum, and it is not surprising that many transplant recipients are deficient in generating specific antibody responses to exogenous stimuli. This B cell immunodeficiency in these patients is associated with clinically significant infections, although the underlying mechanism remains unknown. We have previously shown that the pattern of usage of V_H genes was similar between healthy subjects and BMT recipients, indicating that the immunodeficiency was not due to a dramatic imbalance in V_H utilization. However, motif-specific hybridization analysis indicated that the accumulation of somatic mutations was much greater among rearrangements in controls than in BMT recipients. The failure of BMT recipients to accumulate somatic mutations in rearranged V_H genes correlates with an absence of IgD⁻ B cells, and is consistent with a defect in antigen-driven B cell responses. In the current study, which extends those findings, we have determined the nucleotide sequences of 68 heavy chain rearrangements from one patient as well as 39 rearrangements from a healthy control. Analysis of these sequences made possible a more precise definition of variable region configuration and of the status of somatic mutation in this BMT recipient. The results validate the hybridization data and support the conclusion that, although somatic hypermutation and, by inference, antigen-driven responses are detected in BMT recipients, they are deficient compared with healthy subjects as late as 1 year after transplant.     

Human IgA- and IgM-secreting intestinal plasma cells carry heavily mutated VH region genes

Single IgA- or IgM-secreting plasma cells were isolated from histological sections of human jejunum and terminal ileum, and Ig heavy chain variable (V_H ) region genes were amplified and sequenced. Taken together, 62 of 63 cells analyzed harbored somatically mutated V_H region genes, indicating that the vast majority of both IgA- and IgM-secreting intestinal plasma cells derive from germinal center B cells. On average, rearranged V_H genes of IgA- and IgM-secreting plasma cells showed a mutation frequency of 9.0 % and 8.5 %, respectively, which exceeds the level of somatic mutation of V region genes carried by human memory B cells. Moreover, we detected deletions or insertions in the complementarity-determining regions of 5 of the 58 functional V_H region genes analyzed, suggesting that these alterations may contribute to the diversification of the human antibody repertoire in the course of an immune reaction.