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Three-dimensional genome organization orchestrates recombination and transcription of immunoglobulin heavy chain (Igh) genes. The structure of wild-type (WT) alleles includes a prominent architectural stripe that extends from a cluster of CTCF binding elements at the 3' end of the locus (3'CBE), suggesting interactions of this end with sequences throughout the 2 Mb Igh TAD. Here we elucidate interplay between regulatory elements located in the 3'Igh domain (260 kb) that impact the stripe. The CTCF-lacking intronic enhancer, Eµ, promotes stripe formation and tethers sub-TADs between flanking CTCF-bound 3'CBE and IGCR1. Substituting Eµ with an EF1α promoter in different orientations partially recapitulates epigenetic features of WT Igh alleles, including active histone modifications, sub-TAD formation and interactions with the 3'CBE, but does not restore VDJ recombination. Loss of IGCR1 increases the stripe while inverting the 3'CBE redirects the stripe away from the Igh locus. However, inverted 3'CBE continue to serve as a boundary against aberrant activation of genes outside the Igh domain by Eµ. Our observations provide insights into mechanisms by which regulatory elements modulate chromatin structure and stripe formation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

V(D)J recombination secures the production of functional immunoglobulin (Ig) genes and antibody diversity during the early stages of B-cell development through long-distance interactions mediated by cis-regulatory elements and trans-acting factors. O-GlcNAcylation is a dynamic and reversible posttranslational modification of nuclear and cytoplasmic proteins that regulates various protein functions, including DNA-binding affinity and protein-protein interactions. However, the effects of O-GlcNAcylation on proteins involved in V(D)J recombination remain largely unknown. To elucidate this relationship, we downregulated O-GlcNAcylation in a mouse model by administering an O-GlcNAc inhibitor or restricting the consumption of a regular diet. Interestingly, the inhibition of O-GlcNAcylation in mice severely impaired Ig heavy-chain (IgH) gene rearrangement. We identified several factors crucial for V(D)J recombination, including YY1, CTCF, SMC1, and SMC3, as direct targets of O-GlcNAc modification. Importantly, O-GlcNAcylation regulates the physical interaction between SMC1 and SMC3 and the DNA-binding patterns of YY1 at the IgH gene locus. Moreover, O-GlcNAc inhibition downregulated DDX5 protein expression, affecting the functional association of CTCF with its DNA-binding sites at the IgH locus. Our results showed that locus contraction and long-range interactions throughout the IgH locus are disrupted in a manner dependent on the cellular O-GlcNAc level. In this study, we established that V(D)J recombination relies on the O-GlcNAc status of stage-specific proteins during early B-cell development and identified O-GlcNAc-dependent mechanisms as new regulatory components for the development of a diverse antibody repertoire., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to CSI and USTC.)

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

The rearrangement and expression of the immunoglobulin μ heavy chain (Igh) gene require communication of the intragenic Eμ and 3' regulatory region (RR) enhancers with the variable (V H ) gene promoter. Eμ binding of the transcription factor YY1 has been implicated in enhancer-promoter communication, but the YY1 protein network remains obscure. By analyzing the comprehensive proteome of the 1-kb Eμ wild-type enhancer and that of Eμ lacking the YY1 binding site, we identified the male-specific lethal (MSL)/MOF complex as a component of the YY1 protein network. We found that MSL2 recruitment depends on YY1 and that gene knockout of Msl2 in primary pre-B cells reduces μ gene expression and chromatin looping of Eμ to the 3' RR enhancer and V H promoter. Moreover, Mof heterozygosity in mice impaired μ expression and early B cell differentiation. Together, these data suggest that the MSL/MOF complex regulates Igh gene expression by augmenting YY1-mediated enhancer-promoter communication., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, most importantly in nonsense-mediated mRNA decay (NMD). Here, we show that UPF1 is upregulated during the early stages of B cell development and is important for early B cell development in the bone marrow. B-cell-specific Upf1 deletion in mice severely impedes the early to late LPre-B cell transition, in which V H -D H J H recombination occurs at the Igh gene. Furthermore, UPF1 is indispensable for V H -D H J H recombination, without affecting D H -J H recombination. Intriguingly, the genetic pre-arrangement of the Igh gene rescues the differentiation defect in early LPre-B cells under Upf1 deficient conditions. However, differentiation is blocked again following Ig light chain recombination, leading to a failure in development into immature B cells. Notably, UPF1 interacts with and regulates the expression of genes involved in immune responses, cell cycle control, NMD, and the unfolded protein response in B cells. Collectively, our findings underscore the critical roles of UPF1 during the early LPre-B cell stage and beyond, thus orchestrating B cell development., (© 2024. The Author(s).)

Background: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive., Results: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus., Conclusion: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability., (© 2024. The Author(s).)

The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes young and old bone marrow progenitor (pro-) B cells. These changes result in increased interactions at the compartment level and reduced interactions within topologically associated domains (TADs). The gene encoding Ebf1, a key B cell regulator, switches from compartment A to B with age. Genetically reducing Ebf1 recapitulates some features of old pro-B cells. TADs that are most reduced with age contain genes important for B cell development, including the immunoglobulin heavy chain (Igh) locus. Weaker intra-TAD interactions at Igh correlate with altered variable (V), diversity (D) and joining (J) gene recombination. Our observations implicate three-dimensional chromatin reorganization as a major driver of pro-B cell phenotypes that impair B lymphopoiesis with age., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Since the initial report of V (D) J "allelic exclusion/inclusion" (allelic exclusion rearrangement or allelic inclusion rearrangement) and the concept of the "dual B cell receptor (BCR)" in 1961, despite ongoing discoveries, the precise proportion and source mechanism of dual BCR under physiological conditions have been puzzling immuologists. This study takes advantage of the single cell B cell receptor sequencing (scBCR-seq) technology, which can perfectly match the heavy and light chains of BCR at the level of a single B cell, and obtain the full length mRNA sequence of the complementary determining region 3 (CDR3). Through analyzing the pairing of functional IGH (immunoglobulin heavy chain) and IGL (immunoglobulin light chain) in single B cell from both human and mouse bone marrow and peripheral blood, it was observed that dual BCR B cells exhibit stable and high levels of expression. Among them, the human bone marrow and peripheral blood contain about 10% dual (or multiple) BCR B cells, while in mouse peripheral blood and bone marrow memory B cells, this proportion reaches around 20%. At the same time, we innovatively found that in each research sample of humans and mice, there are three (or more) functional rearrangements (mRNA level) of a single chain in a single B cell. By analyzing the position, direction and other compositional characteristics of the V(D)J gene family, we found that at least two (or more) of them are derived from over two (or more) specific allelic inclusion rearrangements of a single chromosome (mRNA molecular level evidence), our findings also highlighted the necessity of classified single cell sequencing data based on single, dual (or multiple) and cannot be assembled into BCR when analyzing the B cell repertoire. The results of this article provides new methods and modeling references for evaluating the proportion and source mechanisms of dual BCR B cells, as well as potential significance of allelic inclusion (exclusion escape) of V(D)J rearrangement., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain ( IgH ) locus under control of its 3' cis -regulatory region ( 3'RR ). The 3'RR is itself transcribed and can undergo "locus suicide recombination" (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined., Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3'RR ., Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies., Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro , this study suggests that LSR may contribute to the elimination of self-reactive B cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Denis-Lagache, Oblet, Marchiol, Baylet, Têteau, Dalloul, Dalloul, Zawil, Dézé, Cook-Moreau, Saintamand, Boutouil, Khamlichi, Carrion, Péron, Le Noir, Laffleur and Cogné.)

Genome methylation profiles define naïve-like (n-CLL), memory-like (m-CLL), and intermediate (i-CLL) subsets of chronic lymphocytic leukaemia (CLL). The profiles can be easily determined by the analysis of the five-CpG signature. m-CLL, i-CLL, and n-CLL with the good, intermediate, and poor prognoses, respectively, differ by the somatic hypermutation status of the immunoglobulin heavy chain variable gene (IGHV), a widely used prognostic predictor in CLL. We have previously shown that the expression of WNT5A , encoding a ROR1 ligand, distinguishes patients with the worse outcome within the prognostically favourable IGHV-mutated subgroup. To analyse the mechanisms controlling WNT5A expression, we investigated the methylation status of 54 CpG sites within the WNT5A promoter and its relation to the WNT5A gene expression. In a cohort of 59 CLL patients balanced for combinations of IGHV and WNT5A statuses, we identified three promoter CpG sites whose methylation level correlated with the WNT5A expression within the IGHV-mutated subgroup. Further, we complemented our data with the methylation status of the five-CpG signature. IGHV-mutated/ WNT5A -negative and IGHV-mutated/ WNT5A -positive cases overlapped with m‑CLL and i‑CLL methylation subgroups, respectively, while most IGHV‑unmutated samples were assigned to n-CLL. Median methylation levels of all the three CpG sites in the WNT5A promoter were lowest in i-CLL. Finally, a detailed analysis of m-CLL and i-CLL showed that undetectable WNT5A expression predicts longer treatment-free survival with higher statistical significance than the classification according to the five-CpG signature. To conclude, a favourable m-CLL subgroup is associated with mutated IGHV and undetectable WNT5A expression due to its promoter methylation.

The transcriptional coactivator Med12 regulates gene expression through its kinase module. Here, we show a kinase module-independent function of Med12 in CSR. Med12 is essential for super-enhancer activation by collaborating with p300-Jmjd6/Carm1 coactivator complexes. Med12 loss decreases H3K27 acetylation and eRNA transcription with concomitant impairment of AID-induced DNA breaks, S-S synapse formation, and 3'RR-Eμ interaction. CRISPR-dCas9-mediated enhancer activation reestablishes the epigenomic and transcriptional hallmarks of the super-enhancer and fully restores the Med12 depletion defects. Moreover, 3'RR-derived eRNAs are critical for promoting S region epigenetic regulation, synapse formation, and recruitment of Med12 and AID to the IgH locus. We find that XLID syndrome-associated Med12 mutations are defective in both 3'RR eRNA transcription and CSR, suggesting that B and neuronal cells may have cell-specific super-enhancer dysfunctions. We conclude that Med12 is essential for IgH 3'RR activation/eRNA transcription and plays a central role in AID-induced antibody gene diversification and genomic instability in B cells.

Cohesin mediates chromatin loop formation across the genome by extruding chromatin between convergently oriented CTCF-binding elements. Recent studies indicate that cohesin-mediated loop extrusion in developing B cells presents immunoglobulin heavy chain (Igh) variable (V), diversity (D) and joining (J) gene segments to RAG endonuclease through a process referred to as RAG chromatin scanning. RAG initiates V(D)J recombinational joining of these gene segments to generate the large number of different Igh variable region exons that are required for immune responses to diverse pathogens. Antigen-activated mature B cells also use chromatin loop extrusion to mediate the synapsis, breakage and end joining of switch regions flanking Igh constant region exons during class-switch recombination, which allows for the expression of different antibody constant region isotypes that optimize the functions of antigen-specific antibodies to eliminate pathogens. Here, we review recent advances in our understanding of chromatin loop extrusion during V(D)J recombination and class-switch recombination at the Igh locus., (© 2022. Springer Nature Limited.)

An 85-year-old man with a history of aortic dissection suddenly fainted, underwent cardiac heart arrest, and died. An autopsy was performed, but the cause of death was not grossly identified. Congo red staining detected amyloid deposits in systemic organs, including the heart, lungs, liver, and kidneys. Immunohistochemical (IHC) analysis revealed immunoglobulin (Ig) λ light chain (-λ) in systemic blood vessels and transthyretin (TTR) in the heart and lungs. Ig-λ was predominantly positive in the blood vessels of the lungs, while TTR was detected in the alveolar septum. In the heart, Ig-λ was positive in the endocardium and blood vessels, and TTR was positive in nodular deposits between cardiomyocytes. The concurrent deposition of Ig-λ and TTR in the heart was further substantiated by laser microdissection (LMD)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) at each deposition site. Despite systemic deposition of Ig-λ, bone marrow biopsy findings were not diagnostic for multiple myeloma. In summary, we present an autopsy case of concurrent Ig-λ and TTR deposition as revealed by IHC and LC-MS/MS. When Congo red staining and IHC results are indeterminate due to the deposition of multiple amyloid proteins, LMD-LC-MS/MS is useful for determining the precursor protein., (© 2021 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Simplified monoclonal antibodies can be produced by fusing a VHH or nanobody, derived from camelid heavy-chain-only antibodies to the Fc domain of either IgG (VHH-IgG), IgA (VHH-IgA), or IgY (VHH-IgY). These recombinant antibodies are encoded by a single gene and their production can be easily scaled up in plants. This chapter contains methods for Gateway cloning of VHH-Fc fusions into the binary T-DNA vectors pEAQ-HT-DEST1 and pPhasGW, electroporation of Agrobacterium with the resulting constructs, transient antibody expression in Nicotiana benthamiana leaves, and stable antibody expression in Arabidopsis thaliana seeds. The properties of chimeric VHH-based antibodies produced in plants enable novel passive immunization treatments, such as in-feed oral delivery or intravenous injection., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Different HIV vaccine regimens elicit distinct plasma antibody responses in both human and nonhuman primate models. Previous studies in human and non-human primate infants showed that adjuvants influenced the quality of plasma antibody responses induced by pediatric HIV envelope vaccine regimens. We recently reported that use of the 3M052-SE adjuvant and longer intervals between vaccinations are associated with higher magnitude of antibody responses in infant rhesus macaques. However, the impact of different adjuvants in HIV vaccine regimens on the developing infant B cell receptor (BCR) repertoire has not been studied. This study evaluated whether pediatric HIV envelope vaccine regimens with different adjuvants induced distinct antigen-specific memory B cell repertoires and whether specific immunoglobulin (Ig) immunogenetic characteristics are associated with higher magnitude of plasma antibody responses in vaccinated infant rhesus macaques. We utilized archived preclinical pediatric HIV vaccine studies PBMCs and tissue samples from 19 infant rhesus macaques immunized either with (i) HIV Env protein with a squalene adjuvant, (ii) MVA-HIV and Env protein co-administered using a 3-week interval, (iii) MVA-HIV prime/ protein boost with an extended 6-week interval between immunizations, or (iv) with HIV Env administered with 3M-052-SE adjuvant. Frequencies of vaccine-elicited HIV Env-specific memory B cells from PBMCs and tissues were similar across vaccination groups (frequency range of 0.06-1.72%). There was no association between vaccine-elicited antigen-specific memory B cell frequencies and plasma antibody titer or avidity. Moreover, the epitope specificity and Ig immunogenetic features of vaccine-elicited monoclonal antibodies did not differ between the different vaccine regimens. These data suggest that pediatric HIV envelope vaccine candidates with different adjuvants that previously induced higher magnitude and quality of plasma antibody responses in infant rhesus macaques were not driven by distinct antigen-specific memory BCR repertoires., Competing Interests: The authors have declared that no competing interests exist.

Leukopenia is a common manifestation of many diseases, including global outbreak SAS-CoV-2 infection. Granulocyte-macrophage colony-stimulating factor (GM -CSF) has been proved to be effective in promoting lymphocyte regeneration, but adverse immunological effects have also emerged. This study aim to investigate the effect of GM -CSF on BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration. Cyclophosphamide (CTX) and GM -CSF were used to inhibit and stimulate bone marrow hematopoiesis, respectively. High throughput sequencing was applied to detect the characteristics of BCR CDR3 repertoire in controls, CTX group and GM -CSF group. The white blood cells (WBCs) were quickly reduced (P < 0.05) with lymphocytes decreasing causing by CTX, and the WBCs and lymphocytes returned to the level of controls after GM -CSF treatment. The diversity of BCR heavy chain CDR3 repertoire was also significantly decreased in CTX group. Although there is still a big gap from the controls, the diversity was picked up after GM -CSF treatment. The expression of IGHD01-01, IGHD02-14 and IGHJ04-01 with high-frequency usage regularly and significantly changed in three groups, and many genes with low-frequency usage lost in CTX group and did not reappear in GM -CSF group. Moreover, two shared sequences and accounted for the highest proportion in GM -CSF group have been detected in animal model of chronic lymphocytic leukemia. These results revealed that GM -CSF can partially restore changes in the BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration, but it may also lead to rearrangement, proliferation and activation of abnormal B cells, which can provide a basis for further study on the adverse immunological effects and mechanism of GM -CSF treatment., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Autonomous heavy-chain variable (V H ) domains are the smallest functional antibody fragments, and they possess unique features, including small size and convex paratopes, which provide enhanced targeting of concave epitopes that are difficult to access with larger conventional antibodies. However, human V H domains have evolved to fold and function with a light chain partner, and alone, they typically suffer from low stability and high aggregation propensity. Development of autonomous human V H domains, in which aggregation propensity is reduced without compromising antigen recognition, has proven challenging. Here, we used an autonomous human V H domain as a scaffold to construct phage-displayed synthetic libraries in which aspartate was systematically incorporated at different paratope positions. In selections, the library yielded many anti-EphA1 receptor V H domains, which were characterized in detail. Structural analyses of a parental anti-EphA1 V H domain and an improved variant provided insights into the effects of aspartate and other substitutions on preventing aggregation while retaining function. Our naïve libraries and in vitro selection procedures offer a systematic approach to generating highly functional autonomous human V H domains that resist aggregation and could be used for basic research and biomedical applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SμGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N 6 -methyladenosine (m 6 A) RNA modification drives recognition and 3' end processing of SμGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m 6 A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SμGLT. Direct suppression of m 6 A modification of SμGLT or of m 6 A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m 6 A modification, and m 6 A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The orange-spotted grouper (Epinephelus coioides) is an important marine farmed fish in China. It is affected by the bacterial pathogen Vibrio alginolyticus, which causes high mortality and substantial economic losses. We studied the transcriptional changes of the IgZ gene in E. coioides following V. alginolyticus stimulation and investigated the distribution of IgZ in different tissues. The highest expression level of IgZ occurred in the head kidney. When fish were stimulated with live and inactivated V. alginolyticus, the expression levels of IgZ in the head kidney, spleen, intestine, gills and blood cells were significantly upregulated. In an in situ hybridization study, IgZ mRNA-positive cells were detected in the head kidney, spleen and gill, but positive signals were not detected in the liver and intestine. IgZ-labelled cells increased in the head kidney, spleen and gills post-infection with V. alginolyticus for 21 days. The present study provides additional evidence that IgZ is involved in mucosal immune responses and helps explain the role of IgZ in E. coioides defence against V. alginolyticus infection., (© 2021 John Wiley & Sons Ltd.)

Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus , are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens., Competing Interests: The authors declare no competing interest.

Immunoglobulin class switch recombination (CSR) plays a crucial role in adaptive immune responses through a change of the effector functions of antibodies and is triggered by T-cell-dependent as well as T-cell-independent antigens. Signals generated following encounter with each type of antigen direct CSR to different isotypes. At the genomic level, CSR occurs between highly repetitive switch sequences located upstream of the constant gene exons of the immunoglobulin heavy chain locus. Transcription of switch sequences is mandatory for CSR and is induced in a stimulation-dependent manner. Switch transcription takes place within dynamic chromatin domains and is regulated by long-range regulatory elements which promote alignment of partner switch regions in CSR centers. Here, we review recent work and models that account for the function of long-range transcriptional regulatory elements and the chromatin-based mechanisms involved in the control of CSR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dauba and Khamlichi.)

Background: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of β2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers., Methods: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling., Results: The solution structure of isolated Nb23 nanobody was determined., Conclusions: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ + B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ + B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Nanobodies (VHHs) are the single variable immunoglobulin domains of heavy chain antibodies (hcAbs) that naturally occur in alpacas and other camelids. The two variable domains of conventional antibodies typically interact via a hydrophobic interface. In contrast, the corresponding surface area of nanobodies is hydrophilic, rendering these single immunoglobulin domains highly soluble, robust to harsh environments, and exceptionally easy to format into bispecific reagents. In homage to Geoffrey Burnstock, the pioneer of purinergic signaling, we provide a brief history of nanobody-mediated modulation of purinergic signaling, using our nanobodies targeting P2X7 and the NAD + -metabolizing ecto-enzymes CD38 and ARTC2.2 as examples., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from V H 10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (V H 10) or not (V H 4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. V H 10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the V H 4 segment did not. The CDR2 loop shuffling hampered V H 10 reactivity while displaying a gain-of-function in the nonbinding V H 4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the V H 10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.

Background: Immunoglobulins (Igs) distributed among systemic immune tissues and mucosal immune tissues play important roles in protecting teleosts from infections in the pathogen-rich aquatic environment. Teleost IgZ/IgT subclasses with different tissue expression patterns may have different immune functions., Results: In the present study, a novel secreted IgZ heavy chain gene was cloned and characterized in common carp (Cyprinus carpio). This gene exhibited a different tissue-specific expression profile than the reported genes IgZ1 and IgZ2. The obtained IgZ-like subclass gene designated CcIgZ3, had a complete open reading frame contained 1650 bp encoding a protein of 549 amino acid residues. Phylogenetic analysis revealed that CcIgZ3 was grouped with carp IgZ2 and was in the same branch as IgZ/IgT genes of other teleosts. Basal expression detection of the immunoglobulin heavy chain (IgH) in healthy adult common carp showed that CcIgZ3 transcripts were widely expressed in systemic immune tissues and mucosal-associated lymphoid tissues. CcIgZ3 was expressed at the highest levels in the head kidneys, gills, and gonads, followed by the spleen, hindgut, oral epithelium, liver, brain, muscle, foregut, and blood; it was expressed at a very low level in the skin. The transcript expression of CcIgZ3 in leukocytes isolated from peripheral blood cells was significantly higher than that in leukocytes isolated from the spleen. Different groups of common carp were infected with Aeromonas hydrophila via intraperitoneal injection or immersion. RT-qPCR analysis demonstrated that significant differences in CcIgZ3 mRNA levels existed between the immersion and injection groups in all the examined tissues, including the head kidney, spleen, liver, and hindgut; in particular, the CcIgZ3 mRNA level in the hindgut was higher in the immersion group than in the injection group. The different routes of A. hydrophila exposure in common carp had milder effects on the IgM response than on the CcIgZ3 response. Further study of the relative expression of the IgH gene during the development of common carp showed that the tissue-specific expression profile of CcIgZ3 was very different from those of other genes. RT-qPCR analysis demonstrated that the CcIgZ3 mRNA level increased gradually in common carp during the early larval development stage from 1 day post fertilization (dpf) to 31 dpf with a dynamic tendency similar to those of IgZ1 and IgZ2, and IgM was the dominant Ig with obviously elevated abundance. Analyses of the tissue-specific expression of IgHs in common carp at 65 dpf showed that CcIgZ3 was expressed at mucosal sites, including both the hindgut and gill; in contrast, IgZ1 was preferentially expressed in the hindgut, and IgZ2 was preferentially expressed in the gill. In addition to RT-qPCR analysis, in situ hybridization was performed to detect CcIgZ3-expressing cells and IgM-expressing cells. The results showed that CcIgZ3 and IgM transcripts were detectable in the spleens, gills, and hindguts of common carp at 65 dpf., Conclusions: These results reveal that CcIgZ3 gene transcripts are expressed in common carp during developmental stage not only in systemic tissues but also in mucosal tissues. CcIgZ3 expression can be induced in immune tissues by A. hydrophila challenge via immersion and intraperitoneal injection with significantly different expression profiles, which indicates that CcIgZ3 is involved in the antimicrobial immune response and might play an important role in gut mucosal immunity.

A translocation involving the cyclin-dependent kinase 6 (CDK6) gene [t(CDK6)] is a rare but recurrent abnormality in B-cell neoplasms. To further characterise this aberration, we studied 57 cases; the largest series reported to date. Fluorescence in situ hybridisation analysis confirmed the involvement of CDK6 in all cases, including t(2;7)(p11;q21) immunoglobulin kappa locus (IGK)/CDK6 (n = 51), t(7;14)(q21;q32) CDK6/immunoglobulin heavy locus (IGH) (n = 2) and the previously undescribed t(7;14)(q21;q11) CDK6/T-cell receptor alpha locus (TRA)/T-cell receptor delta locus (TRD) (n = 4). In total, 10 patients were diagnosed with chronic lymphocytic leukaemia, monoclonal B-cell lymphocytosis or small lymphocytic lymphoma, and 47 had small B-cell lymphoma (SmBL) including 36 cases of marginal zone lymphoma (MZL; 34 splenic MZLs, one nodal MZL and one bronchus-associated lymphoid tissue lymphoma). In all, 18 of the 26 cytologically reviewed cases of MZL (69%) had an atypical aspect with prolymphocytic cells. Among the 47 patients with MZL/SmBL, CD5 expression was found in 26 (55%) and the tumour protein p53 (TP53) deletion in 22 (47%). The TP53 gene was mutated in 10/30 (33%); the 7q deletion was detected in only one case, and no Notch receptor 2 (NOTCH2) mutations were found. Immunoglobulin heavy-chain variable-region (IGHV) locus sequencing revealed that none harboured an IGHV1-02*04 gene. Overall survival was 82% at 10 years and not influenced by TP53 aberration. Our present findings suggest that most t(CDK6)+ neoplasms correspond to a particular subgroup of indolent marginal zone B-cell lymphomas with distinctive features., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Aims: Elevated serum immunoglobulins have been associated with experimental and human hypertension for decades but whether immunoglobulins and B cells play a causal role in hypertension pathology is unclear. In this study, we sought to determine the role of B cells and high-affinity class-switched immunoglobulins on hypertension and hypertensive end-organ damage to determine if they might represent viable therapeutic targets for this disease., Methods and Results: We purified serum immunoglobulin G (IgG) from mice exposed to vehicle or angiotensin (Ang) II to induce hypertension and adoptively transferred these to wild type (WT) recipient mice receiving a subpressor dose of Ang II. We found that transfer of IgG from hypertensive animals does not affect blood pressure, endothelial function, renal inflammation, albuminuria, or T cell-derived cytokine production compared with transfer of IgG from vehicle infused animals. As an alternative approach to investigate the role of high-affinity, class-switched immunoglobulins, we studied mice with genetic deletion of activation-induced deaminase (Aicda-/-). These mice have elevated levels of IgM but virtual absence of class-switched immunoglobulins such as IgG subclasses and IgA. Neither male nor female Aicda-/- mice were protected from Ang II-induced hypertension and renal/vascular damage. To determine if IgM or non-immunoglobulin-dependent innate functions of B cells play a role in hypertension, we studied mice with severe global B-cell deficiency due to deletion of the membrane exon of the IgM heavy chain (µMT-/-). µMT-/- mice were also not protected from hypertension or end-organ damage induced by Ang II infusion or deoxycorticosterone acetate-salt treatment., Conclusions: These results suggest that B cells and serum immunoglobulins do not play a causal role in hypertension pathology., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

A distinct B cell population marked by elevated CD11c expression is found in patients with systemic lupus erythematosus (SLE). Cells with a similar phenotype have been described during chronic infection, but variable gating strategies and nomenclature have led to uncertainty of their relationship to each other. We isolated CD11c hi cells from peripheral blood and characterized them using transcriptome and IgH repertoire analyses. Gene expression data revealed the CD11c hi IgD + and IgD - subsets were highly similar to each other, but distinct from naive, memory, and plasma cell subsets. Although CD11c hi B cells were enriched in some germinal center (GC) transcripts and expressed numerous negative regulators of B cell receptor (BCR) activation, they were distinct from GC B cells. Gene expression patterns from SLE CD11c hi B cells were shared with other human diseases, but not with mouse age-associated B cells. IgH V-gene sequencing analysis showed IgD + and IgD - CD11c hi B cells had somatic hypermutation and were clonally related to each other and to conventional memory and plasma cells. However, the IgH repertoires expressed by the different subsets suggested that defects in negative selection during GC transit could contribute to autoimmunity. The results portray a pervasive B cell population that accumulates during autoimmunity and chronic infection and is refractory to BCR signaling., Competing Interests: MDC, PB, ACG, and PEL are employed by AMPEL BioSolutions, LLC. VK is a full-time employee and shareholder of AstraZeneca. RE and SWare full time employees and shareholders of Viela Bio, and shareholders of AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Maul, Catalina, Kumar, Bachali, Grammer, Wang, Yang, Hasni, Ettinger, Lipsky and Gearhart.)

Cancer immunotherapy using T cells redirected with chimeric antigen receptor (CAR) has shown a lot of promise. We have established a single-chain antibody (scFv) generation system in which scFv library-expressing CAR-T cells can be screened appropriately based on their antitumor functions. A variable region library containing the variable and J regions of the human immunoglobulin light or heavy chain was fused with the variable region of a heavy or light chain encoded by an existing tumor-specific antibody to generate a new scFv library. Then, scFv library-expressing CAR-T cells were generated and stimulated with target cells to concentrate the antigen-specific population. Using this system, target-specific recognition of CAR-T cells appeared to be finely tuned by selecting a new variable region. Importantly, we have demonstrated that the newly optimized scFv-expressing CAR-T cells had better proliferation capacity and durable phenotypes, enabling superior reactivity against advanced tumors in vivo in comparison with the original CAR-T cells. Therefore, the optimization of an scFv is needed to maximize the in vivo antitumor functions of CAR-T cells. This system may allow us to adjust an immunological synapse formed by an scFv expressed by CAR-T cells and a target antigen, representing an ideal form of CAR-T-cell immunotherapy.

Purpose: Approximately 30% of patients with chronic lymphocytic leukemia (CLL) can be grouped into subsets with stereotyped B-cell receptor immunoglobulin (BcR IG) displaying remarkable similarity in the heavy complementarity-determining region 3 (VH CDR3). Here, we investigated whether the consensus VH CDR3 sequences from CLL stereotyped subsets can be exploited for immunotherapy approaches., Experimental Design: Immunogenic epitopes from the consensus VH CDR3 sequence of the clinically aggressive subsets #1 and #2 and from Eμ-TCL1 mice, which spontaneously develop CLL with BcR IG stereotypy, were identified and used to generate specific HLA class I- and II-restricted T cells in vitro . T-cell reactivity was assayed in vitro as IFNγ production. Bone marrow-derived dendritic cells loaded with the peptides were used as vaccination strategy to restrain leukemia development in the Eμ-TCL1 mouse model., Results: These stereotyped epitopes were naturally processed and presented by CLL cells to the VH CDR3-specific T cells. Furthermore, we validated the efficacy of VH CDR3 peptide-based immunotherapy in the Eμ-TCL1 transplantable mouse model. Immunization of mice against defined VH CDR3 peptide epitopes, prior to the challenge with the corresponding leukemia cells, resulted in the control of CLL development in a significant fraction of mice, and increased overall survival., Conclusions: Our data highlight the immunogenicity of stereotyped VH CDR3 sequences and support the feasibility and efficacy of their use for novel cancer vaccine in CLL. Such approach has the advantage to generate "off-the-shelf" therapeutic vaccines for relevant groups of patients belonging to stereotyped subsets. See related commentary by Seiffert, p. 659 ., (©2020 American Association for Cancer Research.)

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab., (© 2021 The Author(s).)

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

Key Message: Present review summarizes the current applications of nanobodies in plant science and biotechnology, including plant expression of nanobodies, plant biotechnological applications, nanobody-based immunodetection, and nanobody-mediated resistance against plant pathogens. Nanobodies (Nbs) are variable domains of heavy chain-only antibodies (HCAbs) isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals. In this review, we summarize the current applications of nanobodies in plant science and biotechnology, focusing on nanobody expression in plants, plant biotechnological applications, determination of plant toxins and pathogens, and nanobody-mediated resistance against plant pathogens. Prospects and challenges of nanobody applications in plants are also discussed.

Treatment for patients with chronic lymphocytic leukemia (CLL) is becoming more individualized due to the recent introduction of novel molecularly targeted therapies into the therapeutic armamentarium. Genomic and molecular risk factors in CLL patients determine the individual risk for disease progression and response to therapy, and can impact survival. In this review article, we discuss current treatment strategies for CLL patients in Japan, where the novel targeted agents, the BTK inhibitor ibrutinib and BCL2 antagonist venetoclax, now are available and increasingly used in clinical practice. We also discuss the importance of CLL risk factors for making therapy decisions, focusing on immunoglobulin variable region heavy chain (IGHV) mutation status, 11q deletion, and 17p deletion. Treatment approaches for CLL have rapidly changed in the past few years because of these new targeted agents. They are highly effective, well tolerated, and have been demonstrated in a series of large randomized clinical trials to improve survival when compared with conventional chemotherapy-based treatment. Therefore, for most patients, especially high-risk CLL patients, BTK inhibitor and BCL2 antagonist therapies are preferred over chemo-immunotherapy. Currently ongoing studies seek to determine the best sequence for these new agents and whether a combination therapy approach is beneficial. With these developments, a new era of chemotherapy-free treatment for CLL patients is expected.

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) ( IgHR ) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the Igh Ter5H∆TM mouse model from Igh Ter5H mice having a premature termination codon at position +5 in leader exon of Igh Ter5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of Igh Ter5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the Igh Ter5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Lymphoma-related amyloidosis is a rare entity. Systemic AL amyloidosis is generally caused by an underlying plasma cell clone in the bone marrow with an intact monoclonal immunoglobulin G (IgG) or IgA protein. The rarity of the lymphoma-related amyloidosis makes the generation of data in randomized trials and the determination of the optimal treatment almost impossible. Therefore, treatment recommendations discussed here are based on either retrospective or small prospective trials of single centers., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

B cell and plasma cell fates are controlled by different transcriptional networks, as exemplified by the mutually exclusive expression and cross-antagonism of the B cell identity factor Pax5 and the plasma cell regulator Blimp1. It has been postulated that repression of Pax5 by Blimp1 is essential for plasma cell development. Here, we challenged this hypothesis by analyzing the IghPax5/+ mouse, which expressed a Pax5 minigene from the immunoglobulin heavy-chain locus. Despite high Pax5 expression, plasma cells efficiently developed in young IghPax5/+ mice at steady state and upon immunization, while their number moderately declined in older mice. Although Pax5 significantly deregulated the plasma cell expression program, key plasma cell regulators were normally expressed in IghPax5/+ plasma cells. While IgM and IgA secretion by IghPax5/+ plasma cells was normal, IgG secretion was modestly decreased. Hence, Pax5 repression is not essential for robust plasma cell development and antibody secretion, although it is required for optimal IgG production and accumulation of long-lived plasma cells., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Liu et al.)

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR., Competing Interests: The authors declare no competing interest.

Immunoglobulin M (IgM) is the major isotype among teleost immunoglobulins. The present study was aimed to explore IgM heavy chain gene and its expression profile in rohu. Full-length IgM heavy chain cDNA of rohu consisted of 1994 bp encoding a polypeptide of 576 amino acid residues including a leader peptide, variable (VH) and constant (CH1-CH2-CH3-CH4) domains confirming the secretory form of IgM. The sequence carries conserved residues such as cysteine, tryptophan and amino acid motifs like 'YYCAR' and 'FDYWGKGT-VTV-S'. The predicted 3 D model confirmed various domains of rohu IgM heavy chain. Phylogenetic tree analysis revealed that IgM heavy chain gene of rohu shared the same cluster with that of other cyprinid fishes. Tissue distribution analysis showed the predominant level of IgM heavy chain gene expression in kidney, spleen and intestine. IgM heavy chain gene expression in rohu kidney was found to be up-regulated and reached a maximum at 7 days post-challenge with Aeromonas hydrophila . These findings demonstrate the first report of full-length secretory IgM heavy chain gene in rohu. Besides, IgM heavy chain gene was highly expressed in major lymphoid tissues and bacterial challenge influenced its expression which further confirmed its role in the adaptive humoral immune response.

Site-directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity, which is advantageous for many applications. Here, we report a facile, specific, and efficient strategy based on the SpyTag-SpyCatcher chemistry. Two SpyTag-fused model proteins, that is, the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl-7-aminocephalosporanic acid acylase, were easily immobilized onto a SpyCatcher-modified resin directly from cell lysates, with activity recoveries in the range of 85-91%. This strategy was further adapted to protein purification, which proceeded through the selective capture of the SpyCatcher-fused target proteins by a SpyTag-modified resin, with the aid of an intein to generate authentic N-termini. For two model proteins, that is, RFP and a variable domain of a heavy chain antibody, the yields were ∼3-7 mg/L culture with >90% purities. This approach could provide a versatile tool for producing high-performance immobilized protein devices and proteins for industrial and therapeutic uses., (© 2020 Wiley Periodicals LLC.)

Enrichment for tyrosine in immunoglobulin CDR-H3 is due in large part to natural selection of germline immunoglobulin D H sequence. We have previously shown that when D H sequence is modified to reduce the contribution of tyrosine codons, epitope recognition is altered and B cell development, antibody production, autoantibody production, and morbidity and mortality following pathogen challenge are adversely affected. TCRβ diversity (Dβ) gene segment sequences are even more highly conserved than D H , with trout Dβ1 identical to human and mouse Dβ1. We hypothesized that natural selection of Dβ sequence also shapes CDR-B3 diversity and influences T cell development and T cell function. To test this, we used a mouse strain that lacked Dβ2 and contained a novel Dβ1 allele ( DβYTL ) that replaces Dβ1 with an immunoglobulin D H , DSP2.3. Unlike Dβ1, wherein glycine predominates in all three reading frames (RFs), in DSP2.3 there is enrichment for tyrosine in RF1, threonine in RF2, and leucine in RF3. Mature T cells using DβYTL expressed TCRs enriched at particular CDR-B3 positions for tyrosine but depleted of leucine. Changing Dβ sequence altered thymocyte and peripheral T cell numbers and the T cell response to an ovalbumin immunodominant epitope. The differences in tyrosine content might explain, at least in part, why TCRs are more polyspecific and of lower affinity for their cognate antigens than their immunoglobulin counterparts., (Copyright © 2020 Levinson, Khass, Burrows and Schroeder.)

Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; n = 12), other neurologic diseases (ONDs; n = 1), and healthy controls (HCs; n = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS ( n = 4), clinically isolated syndrome ( n = 2), and OND ( n = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; n = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-κB) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-β1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood-brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein-Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype., Competing Interests: Competing interest statement: R.D.S. served on an advisory board for Sanofi Genzyme and is currently an employee of Asceneuron SA. A.J.G. reports personal fees from Inception Sciences and Mylan Pharmaceuticals and has reported serving on an end point adjudication committee for Biogen and Medimmune. He has served on trial steering committees for Novartis and serves on the Scientific Advisory Board for Bionure. R.M.B. has received personal compensation for medical legal consulting and for consulting or serving on the advisory boards of F. Hoffmann-La Roche Ltd., Sanofi-Genzyme, and Novartis. J.M.G. has received research support to University of California, San Francisco from Genentech, personal compensation for consulting for Biogen and Alexion, and personal compensation for medical legal consulting; he has also received honoraria from Dynamed Plus for editorial work. B.A.C.C. receives personal compensation for consulting from Abbvie, Biogen, EMD Serono, GeNeuro, Novartis, and Sanofi Genzyme. S.S.Z. is Deputy Editor of Neurology, Neuroimmunology and Neuroinflammation and is an Associate Editor for Frontiers in Immunology and Frontiers in Neurology. He serves on the Advisory Committee for the American Congress on Treatment and Research in Multiple Sclerosis and on the grant review committee for the National Multiple Sclerosis Society (NMSS). Previously, he has served on the Editorial Board of the Journal of Clinical Investigation, The Journal of Immunology, and The Journal of Neurological Sciences and has been a charter member of the grant review committee for the NIH Clinical Neuroimmunology and Brain Tumors. He has served as a consultant and received honoraria from Biogen-Idec, EMD-Serono, Genzyme, Novartis, Roche/Genentech, and Teva Pharmaceuticals, Inc. and has served on Data Safety Monitoring Boards for Lilly, BioMS, Teva, and Opexa Therapeutics. Currently, S.S.Z. receives research grant support from the NIH, NMSS, Weill Institute, Race to Erase MS, and the Maisin Foundation. S.L.H. currently serves on the Scientific Advisory Board of Alector, Annexon, Bionure, and Molecular Stethoscope and on the Board of Trustees of Neurona. S.L.H. also has received travel reimbursement and writing assistance from F. Hoffmann-La Roche Ltd. and Novartis for CD20-related meetings and presentations. M.R.W. received research funding from Roche/Genentech., (Copyright © 2020 the Author(s). Published by PNAS.)

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B cell system with age is poorly studied. We extensively investigated age-related alterations of naïve and antigen-experienced immunoglobulin heavy chain (IgH) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream IgH constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing IgH repertoires and stress the importance of using well-selected, age-appropriate controls in IgH studies., (Copyright © 2020 Ghraichy, Galson, Kovaltsuk, von Niederhäusern, Pachlopnik Schmid, Recher, Jauch, Miho, Kelly, Deane and Trück.)

Erythropoietin (EPO), a hematopoietic growth factor and a promising therapy for Alzheimer's disease, has low permeability across the blood-brain barrier. The transferrin receptor antibody fused to EPO (TfRMAb-EPO) is a chimeric monoclonal antibody that ferries EPO into the brain via the transvascular route. However, TfRMAbs have Fc-effector function-related adverse effects including reticulocyte suppression. To overcome this, we recently developed an effectorless TfRMAb-EPO fusion protein, designated TfRMAb-N292G-EPO, by eliminating the Fc N-linked glycosylation site at position 292 of the antibody heavy chain. The mutant fusion protein showed enhanced plasma clearance and dramatically reduced plasma concentrations compared with the wild-type (WT) nonmutant fusion protein. This increased clearance of the aglycosylated TfRMAb is expected to increase the injection dose of the mutant fusion protein. To provide a basis for future therapeutic uses of this IgG-neurotrophin fusion protein, the current study aimed to characterize the pharmacokinetic profile of this effectorless TfRMAb-N292G-EPO at different doses following different routes of administration in the mouse. Adult C57BL/6J male mice were injected with a single dose (3, 6, 9, or 20 mg/kg; n = 3-6 per dose) of TfRMAb-N292G-EPO through either the subcutaneous (SQ) or intraperitoneal (IP) route. TfRMAb-N292G-EPO plasma concentrations were determined using an enzyme-linked immunosorbent assay. Mice were sacrificed 24 h after injection, and terminal blood was used for a complete blood count. Brain concentrations in the WT- and mutant fusion protein-treated mice were compared. We observed stark differences in the plasma pharmacokinetics of TfRMAb-N292G-EPO between the IP and SQ routes of administration. Dose escalation from 3 to 20 mg/kg increased the plasma C max only 3.5-fold for the SQ route, compared with a 35-fold increase for the IP route. The plasma C max was 15.0 ± 2.0, 21.3 ± 4.1, 21.3 ± 6.4, and 52.8 ± 27.9 ng/mL following SQ injection and 288 ± 47, 389 ± 154, 633 ± 194, and 10,066 ± 7059 ng/mL following IP injection for 3, 6, 9, and 20 mg/kg doses, respectively. The plasma C max following the SQ route was therefore 19- to 190-fold lower than that following the IP route. This finding is consistent with a 31-fold higher apparent clearance following the SQ route compared with the IP route at the highest dose administered. The brain concentrations in the mice treated with a 3 mg/kg dose of the mutant fusion protein were lower than those in the nonmutant WT-treated mice. No reticulocyte suppression was observed at the 3 mg/kg SQ dose of TfRMAb-N292G-EPO. However, reticulocyte suppression increased with an increase in dose and area under the plasma concentration-time curve (AUC) for both the IP and SQ routes. Overall, elimination of Fc N-linked glycosylation, to mitigate TfRMAb effector function side effects, has a profound effect on the plasma exposure of TfRMAb-N292G-EPO at therapeutic as well as high doses (3-20 mg/kg). This effect is more pronounced following SQ injection. The low plasma concentrations of the mutant fusion protein following a 3 mg/kg dose resulted in negligible brain uptake. The beneficial rescue of reticulocyte reduction by the N292G mutation is a function of AUC and is negated at high doses of the N292G mutant.

Bispecific antibodies have received wide attention as promising immunotherapeutic agents because of their high specificity and the ability to target immune cells to tumors. However, analysis of bispecific antibodies is challenging because multiple forms of antibodies are potentially generated during production in cell culture. Most analyses of bispecific antibodies rely on liquid chromatography with mass spectrometry (LC-MS), which could miss detection or becomes less quantitative if those forms are not physically separated. Here, we report a novel and sensitive mixed mode size exclusion chromatography (MM SEC) coupled with multiangle light scattering (MALS) to analyze different forms of bispecific IgG molecules under native conditions. The method displayed great ability to separate various antibody forms with peak resolutions unmatched by other methods we tested, isolating desired bispecific molecules, parental homodimers, half molecules, and antibodies with mispaired light and heavy chains. Each peak was analyzed by online MALS and then identified and confirmed by intact and reduced LC-MS of isolated forms. MM SEC in this study performs by a novel mechanism through the interactions of resin with protein surface hydrophobic clusters distributed across CDRs of light chains. This novel MM SEC allows quantitative detection of even low abundance forms and provides a new tool for screening expression profiles of cell culture clones, monitoring purification, and evaluating drug substance purity.

The relative orientation of the two variable domains, V H and V L , influences the shape of the antigen binding site, that is, the paratope, and is essential to understand antigen specificity. ABangle characterizes the V H -V L orientation by using five angles and a distance and compares it to other known structures. Molecular dynamics simulations of antibody variable domains (Fvs) reveal fluctuations in the relative domain orientations. The observed dynamics between these domains are confirmed by NMR experiments on a single-chain variable fragment antibody (scFv) in complex with IL-1β and an antigen-binding fragment (Fab). The variability of these relative domain orientations can be interpreted as a structural feature of antibodies, which increases the antibody repertoire significantly and can enlarge the number of possible binding partners substantially. The movements of the V H and V L domains are well sampled with molecular dynamics simulations and are in agreement with the NMR ensemble. Fast Fourier transformation of the ABangle metrics allows to assign timescales of 0.1-10 GHz to the fastest collective interdomain movements. The results clearly show the necessity of dynamics to understand and characterize the favorable orientations of the V H and V L domains implying a considerable binding interface flexibility and reveal in all antibody fragments (Fab, scFv, and Fv) very similar V H -V L interdomain variations comparable to the distributions observed for known X-ray structures of antibodies. SIGNIFICANCE STATEMENT: Antibodies have become key players as therapeutic agents. The binding ability of antibodies is determined by the antigen-binding fragment (Fab), in particular the variable fragment region (Fv). Antigen-binding is mediated by the complementarity-determining regions consisting of six loops, each three of the heavy and light chain variable domain V H and V L . The relative orientation of the V H and V L domains influences the shape of the antigen-binding site and is a major objective in antibody design. In agreement with NMR experiments and molecular dynamics simulations, we show a considerable binding site flexibility in the low nanosecond timescale. Thus we suggest that this flexibility and its implications for binding and specificity should be considered when designing and optimizing therapeutic antibodies., (© 2020 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

Protection and neutralization of a vast array of pathogens is accomplished by the tremendous diversity of the B cell receptor (BCR) repertoire. For jawed vertebrates, this diversity is initiated via the somatic recombination of immunoglobulin (Ig) germline elements. While it is clear that the number of these germline segments differs from species to species, the extent of cross-species sequence diversity remains largely uncharacterized. Here we use extensive computational and statistical methods to investigate the sequence diversity and evolutionary relationship between Ig variable (V), diversity (D), and joining (J) germline segments across nine commonly studied species ranging from zebrafish to human. Metrics such as guanine-cytosine (GC) content showed low redundancy across Ig germline genes within a given species. Other comparisons, including amino acid motifs, evolutionary selection, and sequence diversity, revealed species-specific properties. Additionally, we showed that the germline-encoded diversity differs across antibody (recombined V-D-J) repertoires of various B cell subsets. To facilitate future comparative immunogenomics analysis, we created VDJgermlines, an R package that contains the germline sequences from multiple species. Our study informs strategies for the humanization and engineering of therapeutic antibodies.