Primary Antibody Repertoire Research Articles

Hindlimb unloading, a physiological model of microgravity, modifies the murine bone marrow IgM repertoire in a similar manner as aging but less strongly

BackgroundThe spaceflight environment is an extreme environment that affects the immune system of approximately 50% of astronauts. With planned long-duration missions, such as the deployment of the Lunar Gateway and possible interplanetary missions, it is mandatory to determine how all components of the immune system are affected, which will allow the establishment of countermeasures to preserve astronaut health. However, despite being an important component of the immune system, antibody-mediated humoral immunity has rarely been investigated in the context of the effects of the space environment. It has previously been demonstrated that 30 days aboard the BION-M1 satellite and 21 days of hindlimb unloading (HU), a model classically used to mimic the effects of microgravity, decrease murine B lymphopoiesis. Furthermore, modifications in B lymphopoiesis reported in young mice subjected to 21 days of HU were shown to be similar to those observed in aged mice (18–22 months). Since the primary antibody repertoire composed of IgM is created by V(D) J recombination during B lymphopoiesis, the objective of this study was to assess the degree of similarity between changes in the bone marrow IgM repertoire and in the V(D)J recombination process in 2.5-month-old mice subjected to 21 days of HU and aged (18 months) mice.ResultsWe found that in 21 days, HU induced changes in the IgM repertoire that were approximately 3-fold less than those in aged mice, which is a rapid effect. Bone remodeling and epigenetics likely mediate these changes. Indeed, we previously demonstrated a significant decrease in tibial morphometric parameters from day 6 of HU and a progressive reduction in these parameters until day 21 of HU, and it has been shown that age and microgravity induce epigenetic changes.ConclusionThese data reveal novel immune changes that are akin to advanced aging and underline the importance of studying the effects of spaceflight on antibody-mediated humoral immunity.

Enhancer-instructed epigenetic landscape and chromatin compartmentalization dictate a primary antibody repertoire protective against specific bacterial pathogens.

Antigen receptor loci are organized into variable (V), diversity (D) and joining (J) gene segments that rearrange to generate antigen receptor repertoires. Here, we identified an enhancer (E34) in the murine immunoglobulin kappa (Igk) locus that instructed rearrangement of Vκ genes located in a sub-topologically associating domain, including a Vκ gene encoding for antibodies targeting bacterial phosphorylcholine. We show that E34 instructs the nuclear repositioning of the E34 sub-topologically associating domain from a recombination-repressive compartment to a recombination-permissive compartment that is marked by equivalent activating histone modifications. Finally, we found that E34-instructed Vκ-Jκ rearrangement was essential to combat Streptococcus pneumoniae but not methicillin-resistant Staphylococcus aureus or influenza infections. We propose that the merging of Vκ genes with Jκ elements is instructed by one-dimensional epigenetic information imposed by enhancers across Vκ and Jκ genomic regions. The data also reveal how enhancers generate distinct antibody repertoires that provide protection against lethal bacterial infection.

IL-7R signaling activates widespread VH and DH gene usage to drive antibody diversity in bone marrow B cells.

SummaryGeneration of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. The role of interleukin-7 receptor (IL-7R) signaling in Igh recombination has been difficult to partition from its role in B cell survival and proliferation. With a detailed description of the Igh repertoire in murine IL-7Rα−/− bone marrow B cells, we demonstrate that IL-7R signaling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing toward 3′ VH genes during de novo VH-to-DJH recombination more severe than the fetal liver (FL) repertoire and uncover a role for IL-7R signaling in DH-to-JH recombination. Transcriptome and accessibility analyses suggest reduced expression of B lineage transcription factors (TFs) and targets and loss of DH and VH antisense transcription in IL-7Rα−/− B cells. Thus, in addition to its roles in survival and proliferation, IL-7R signaling shapes the Igh repertoire by activating underpinning mechanisms.

An in vivo method for diversifying the functions of therapeutic antibodies

V(D)J recombination generates mature B cells that express huge repertoires of primary antibodies as diverse immunoglobulin (Ig) heavy chain (IgH) and light chain (IgL) of their B cell antigen receptors (BCRs). Cognate antigen binding to BCR variable region domains activates B cells into the germinal center (GC) reaction in which somatic hypermutation (SHM) modifies primary variable region-encoding sequences, with subsequent selection for mutations that improve antigen-binding affinity, ultimately leading to antibody affinity maturation. Based on these principles, we developed a humanized mouse model approach to diversify an anti-PD1 therapeutic antibody and allow isolation of variants with novel properties. In this approach, component Ig gene segments of the anti-PD1 antibody underwent de novo V(D)J recombination to diversify the anti-PD1 antibody in the primary antibody repertoire in the mouse models. Immunization of these mouse models further modified the anti-PD1 antibodies through SHM. Known anti-PD1 antibodies block interaction of PD1 with its ligands to alleviate PD1-mediated T cell suppression, thereby boosting antitumor T cell responses. By diversifying one such anti-PD1 antibody, we derived many anti-PD1 antibodies, including anti-PD1 antibodies with the opposite activity of enhancing PD1/ligand interaction. Such antibodies theoretically might suppress deleterious T cell activities in autoimmune diseases. The approach we describe should be generally applicable for diversifying other therapeutic antibodies.

IL-7R Signalling Activates Widespread V <sub>H</sub> and D <sub>H</sub> Gene Usage to Drive Antibody Diversity in Bone Marrow B Cells

Generation of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. It has been proposed that interleukin-7 receptor (IL-7R) signalling is necessary for Igh recombination, but this has been challenging to partition from the receptor’s role in B cell survival and proliferation. By generating the first detailed description of the Igh repertoire of murine IL-7Rα-/- bone marrow B cells, we demonstrate that IL-7R signalling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing towards usage of 3’ VH genes during de novo VH-to-DJH recombination that is more severe than the fetal liver (FL) B cell repertoire, and we now show a role for IL-7R signalling in DH-to-JH recombination. Transcriptome and accessibility analyses suggests reduced expression of B lineage-specific transcription factors (TFs) and their targets, and loss of DH and VH antisense transcription in IL-7Rα-/- B cells. These results refute models suggesting that IL-7R signalling is only required for survival and proliferation, and demonstrate a pivotal role in shaping the Igh repertoire by activating underpinning epigenetic mechanisms.

Sequencing of VDJ genes in Lepus americanus confirms a correlation between VHn expression and the leporid species continent of origin

Leporid VH genes used in the generation of their primary antibody repertoire exhibit highly divergent lineages. For the European rabbit (Oryctolagus cuniculus) four VHa lineages have been described, the a1, a2, a3 and a4. Hares (Lepus spp.) and cottontail (Sylvilagus floridanus) express one VHa lineage each, the a2L and the a5, respectively, along with a more ancient lineage, the Lepus spp. sL and S. floridanus sS. Both the European rabbit and the Lepus europaeus use a third lineage, VHn, in a low proportion of their VDJ rearrangements. The VHn genes are a conserved ancestral polymorphism that is being maintained in the leporid genome.Their usage in a low proportion of VDJ rearrangements by both European rabbit and L. europaeus but not S. floridanus has been argued to be a remnant of an ancient European leporid immunologic response to pathogens. To address this hypothesis, in this study we sequenced VDJ rearranged genes for another North American leporid, L. americanus. Our results show that L. americanus expressed these genes less frequently and in a highly modified fashion compared to the European Lepus species. Our results suggest that the American leporid species use a different VH repertoire than the European species which may be related with an immune adaptation to different environmental conditions, such as different pathogenic agents.

Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination.

The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination.

Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells.

Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

Diversification of the Primary Antibody Repertoire by AID-Mediated Gene Conversion.

Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.

Human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein induces DNA damages through Activation-Induced cytidine Deaminase (AID)

How T cells are transformed by HTLV-1 is still unclear, but it is well accepted that the viral oncoprotein Tax is associated with genomic instability of infected cells. Tax has recently been shown to directly induce, in T cells, the expression of AID (Ishikawa C et al., Carcinogenesis, 2011), a cytidine deaminase whose physiologic expression is usually restricted to B cells, in which it initiates class-switch recombination and somatic hypermutations to reshape the primary antibody repertoire after antigen encounter. It is also well established that AID-mediated mutations outside of immunoglobulin gene locus are involved in the oncogenic transformation of B lymphocytes...

Sequencing of modern Lepus VDJ genes shows that the usage of VHn genes has been retained in both Oryctolagus and Lepus that diverged 12 million years ago

Among mammals, the European rabbit (Oryctolagus cuniculus) has a unique mechanism of generating the primary antibody repertoire. Despite having over 200 VH genes, the VH1 gene, the most D-proximal VH gene, is used in 80-90 % of VDJ rearrangements, while the remaining 10-20 % is encoded by the VHn genes that map at least 100 Kb upstream of VH1. The maintenance of the VHn genes usage in low frequency in VDJ rearrangements has been suggested to represent a relic of an ancestral immunologic response to pathogens. To address this question, we sequenced VDJ genes for another leporid, genus Lepus, which separated from European rabbit 12 million years ago. Approximately 25 VDJ gene sequences were obtained for each one of three Lepus europaeus individuals. We found that Lepus also uses the VHn genes in 5-10 % of its VDJ rearrangements. Our results show that the VHn genes are a conserved ancestral polymorphism that has been maintained in the leporids genome and is being used for the generation of VDJ rearrangements by both modern Lepus and Oryctolagus.

Identifying the cellular interactions required for diversification of the primary antibody repertoire in rabbit GALT (P6101)

Abstract Rabbits generate an initial antibody repertoire of limited diversity due to preferential use of the 3’-most VH gene segment during V-D-J gene rearrangement. Around one week of age, rabbit B cells begin diversifying this antibody repertoire in gut-associated lymphoid tissue (GALT), by mutating their V-D-J genes in response to antigen-independent signals acquired from intestinal commensals. To identify the cell-cell and cell-microbe interactions required for V-D-J gene diversification, we examined B cell expression levels of four chemokine receptors that mediate homing to important sites within GALT. We stained the chemokine receptors CXCR4, CCR6, CXCR5 and CCR7 on appendix B cells from two- to eight-day-old rabbits with fluorescence-labeled antibodies or chemokine-Ig fusion proteins and analyzed them by flow cytometry. The changes in chemokine receptor expression we observed during the first week of life suggest that, after entering appendix follicles, B cells first home to the follicle-associated epithelium, where they likely encounter commensal bacteria sampled from the intestinal microbiota. B cells then home sequentially to the follicular dendritic cell network, the T cell area and finally to the basal region of the follicle, where they proliferate and diversify their V-D-J genes to generate the primary antibody repertoire.

Primary repertoire expansion by junctional diversity in Bos taurus (P1445)

Abstract B cell development varies significantly between species. In many domestic species fetal and neonatal gut-associated lymphoid tissue is essential for the generation of the primary antibody repertoire due to limited range of Ig genes. In fetal ruminants, the repertoire is expanded by somatic hypermutation (SHM) driven by activation-induced cytidine deaminase in the absence of external antigens. In addition to SHM also other mechanisms are likely to contribute to the repertoire expansion. Junctional diversity (JD) created during the recombination process might play a substantial role in bovine fetal IgH. To further characterize the significance of JD, we studied the tissue-specific expression of terminal deoxynucleotidyl transferase (TdT) using RT-qPCR and immunostaining. We also constructed and sequenced several IgH cDNA libraries of different tissues and analysed the junction formation between V(D)J sequences. Our work demonstrates that in fetal bovine B lymphocytes TdT is primarily expressed in the bone marrow. B lymphocytes in other lymphoid tissues did not show elevated TdT expression levels. V(D)J junctions were heterogeneous in sequence, suggesting the addition of N-nucleotides by TdT. In addition, spectratyping revealed significant differences in the distribution of CDR3 lengths between tissues. Our results suggest that along with SHM, TdT-mediated JD contributes significantly to the bovine primary antibody repertoire in fetal life.

Activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer's patch and spleen and is associated with expansion of the primary antibody repertoire in the absence of exogenous antigens.

Due to a limited range of immunoglobulin (Ig) genes, cattle and several other domestic animals rely on postrecombinatorial amplification of the primary repertoire. We report that activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer's patch and spleen but not in fetal bone marrow. The numbers of IGHV (immunoglobulin heavy chain variable) mutations correlate with AID expression. The mutational profile in the fetuses is similar to postnatal and immunized calves, with targeting of complementarity-determining region (CDR) over framework region (FR), preference of replacement over silent mutations in CDRs but not in FRs, and targeting of the AID hotspot motif RGYW/WRCY. Statistical analysis indicates negative selection on FRs and positive selection on CDRs. Our results suggest that AID-mediated somatic hypermutation and selection take place in bovine fetuses, implying a role for AID in the diversification of the primary antibody repertoire in the absence of exogenous antigens.

Diversification of the primary antibody repertoire begins during early follicle development in the rabbit appendix

Rabbits generate a diversified primary antibody repertoire by somatically mutating, in gut-associated lymphoid tissue (GALT), an initial repertoire that is limited by preferential rearrangement of the 3′-most IGVH gene segment. To determine when repertoire diversification begins in GALT, we performed in situ hybridization on neonatal rabbit appendix sections with an activation-induced cytidine deaminase (AID) riboprobe, because AID is required for the mutational processes that diversify the primary antibody repertoire. We first detected AID mRNA expression around 1 week of age, in the basal region of developing follicles. By PCR-amplifying V–D–J genes from AID mRNA+ B cells isolated by laser capture microdissection, we found evidence of somatic hypermutation, and one likely instance of somatic gene conversion. Our results suggest that V–(D)–J gene diversification begins during early postnatal appendix development, in B cells stimulated to enter a proliferative program by signals derived from select intestinal commensals.

Chemokine-mediated B cell trafficking in rabbit GALT during diversification of the primary antibody repertoire (160.15)

Abstract Rabbits preferentially rearrange the 3’-most VH gene segment during VDJ gene rearrangement. Early in life, rabbit B cells generate a diversified primary antibody repertoire by mutating their VDJ genes in gut-associated lymphoid tissue (GALT) in response to signals acquired from intestinal commensals. To gain insight into the cell-cell and cell-microbe interactions required for VDJ gene diversification, we examined B cell surface expression levels of four chemokine receptors that mediate trafficking to important sites within GALT during repertoire diversification. We stained the chemokine receptors CXCR4, CCR6, CXCR5 and CCR7 on appendix B cells from two- through seven-day-old rabbits with fluorescence-labeled antibodies or Ig-fusion proteins of their ligands and analyzed them by flow cytometry. The changes in relative chemokine receptor expression levels we observed during the first week of life suggest that B cells, upon entering appendix follicles, first traffic to the follicle-associated epithelium, where they likely encounter commensal bacteria sampled from the intestinal microbiota. B cells subsequently migrate to, and likely receive additional signals from, the follicular dendritic cell network. Upon becoming activated, B cells then traffic to the basal region of the follicle, where they proliferate and diversify their VDJ genes to generate the primary antibody repertoire.

Chemokine-directed B cell trafficking in rabbit GALT during diversification of the primary antibody repertoire (149.4)

Abstract Rabbits preferentially rearrange the 3’-most VH gene segment during VDJ gene rearrangement. Early in life, rabbit B cells diversify these VDJ genes in gut-associated lymphoid tissue (GALT) in response to signals acquired from intestinal commensal bacteria, and thus generate a diversified primary antibody repertoire. To gain insight into the cell-cell and cell-microbe interactions required for VDJ gene diversification, we examined B cell surface expression levels of four chemokine receptors that mediate trafficking to important sites within GALT during repertoire diversification. We stained B cells from two- through seven-day-old appendix with fluorescence-labeled Ig-fusion proteins of the chemokines CXCL12, CCL20, CXCL13 and CCL21 and analyzed them by flow cytometry. The changes we observed in relative chemokine receptor expression levels during the first week of life suggest a model in which B cells, upon entering appendix follicles, first traffic to the follicle-associated epithelium, where they likely encounter commensal bacteria sampled from the intestinal microbiota. B cells subsequently migrate to, and likely receive additional signals from, the follicular dendritic cell network. Upon becoming activated, B cells then traffic to the basal region of the follicle, where they proliferate and diversify their VDJ genes, thereby generating the primary antibody repertoire.

B-cell development in bovine fetuses proceeds via a pre-B like cell in bone marrow and lymph nodes

The production of B cells and the primary antibody repertoire in mammalian species other than rodents or man appears to depend on gut-associated lymphoid tissue. Bovine B cells are generated in ileal Peyer's patch from late gestational to juvenile age. However, little is known about where and when the bona fide B lymphopoiesis takes place. We analyzed bovine fetuses for signs of ongoing B lymphopoiesis using a combination of immunohistochemistry, flow cytometry, real-time quantitative PCR and RNA in situ hybridization. In fetal bone marrow and lymph node, we could demonstrate pre-B like cells positive for intracellular Ig μ but negative for membrane IgM. Strong expression of immunoglobulin lambda-like polypeptide 1 and recombination activating genes was also detected in the same tissues. Similar analyses did not reveal pre-B like cells in the corresponding adult tissues. These results suggest that bovine fetal bone marrow and lymph node support B lymphopoiesis via a pre-B cell like stage before and in parallel to the development of the ileal Peyer's patch.

Role of CXCL12 and its receptor, CXCR4, in diversification of the rabbit primary antibody repertoire (133.3)

Abstract The antibody repertoire of early postnatal rabbits is of limited diversity due to preferential V gene usage during VDJ gene rearrangement. Early in life, rabbit B cells expand this repertoire by diversifying their VDJ genes in gut-associated lymphoid tissue. VDJ gene diversification begins around 1 week of age, in B cells located in the basolateral region of nascent follicles. Because these B cells share similarities with B cells in germinal center dark zones, we hypothesized that activated B cells localize to the basolateral region in response to the chemokine CXCL12. To test this hypothesis, we examined B cell expression of CXCR4, the CXCL12 receptor, by in situ hybridization. We found that B cells in the basolateral region, but not those located elsewhere in the follicle, expressed CXCR4. Because antibody repertoire diversification requires signals derived from intestinal commensals, we further examined CXCR4 expression in appendices that were kept sterile by surgical ligation at birth. We found that B cells in these appendices did not express CXCR4 and did not diversify their VDJ genes. By contrast, we detected CXCL12 expression in both conventional and sterile appendices, demonstrating that it is not dependent on commensal-derived signals. Our results suggest that B cells activated by microbial-derived signals upregulate CXCR4 and migrate to the basolateral region of the follicle, likely in response to CXCL12, where they proliferate and diversify their VDJ genes.

Onset of Antibody Repertoire Diversification in the Rabbit Appendix (81.17)

Abstract Unlike mice and humans, rabbits generate their primary antibody repertoire by somatically diversifying V(D)J genes in gut-associated lymphoid tissues (GALT). Rabbit V(D)J genes are diversified by somatic hyper-point mutation and somatic gene conversion, both of which require activation-induced cytidine deaminase (AID). To determine when repertoire diversification begins, we examined AID expression in neonatal rabbit appendix by in situ hybridization. We first detected AID mRNA expression at one week of age, in B cells in the basolateral region of appendix follicles. We asked whether AID+ B cells were undergoing V(D)J gene diversification by using laser capture microdissection (LCM) to isolate AID+ and AID- B cells from 1-week-old appendix follicles. We RT-PCR-amplified and cloned VDJ genes from the two B cell populations, and searched for evidence of somatic diversification by comparing their nucleotide sequences to those of the appropriate germline genes. We found somatic gene conversion events in the VDJ genes from AID+ B cells, but not in those from AID- B cells. These results demonstrate that diversification of the rabbit primary antibody repertoire begins around 1 week of age, in B cells residing at the basolateral region of appendix follicles. This work was supported by NIH Grant 1 RO1 AI49458-01A1.