scBCR-seq revealed a special and novel IG H&L V(D)J allelic inclusion rearrangement and the high proportion dual BCR expressing B cells.

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.

Attempts to evaluate locus suicide recombination and its potential role in B cell negative selection in the mouse.

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.

XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation.

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.

Memory B-cell like chronic lymphocytic leukaemia is associated with specific methylation profile of WNT5A promoter and undetectable expression of WNT5A gene.

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 m­CLL and i­CLL methylation subgroups, respectively, while most IGHV­unmutated 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 role of chromatin loop extrusion in antibody diversification.

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.

Production of Designer VHH-Based Antibodies in Plants.

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.

Autopsy case with concurrent transthyretin and immunoglobulin amyloidosis.

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.

Different adjuvanted pediatric HIV envelope vaccines induced distinct plasma antibody responses despite similar B cell receptor repertoires in infant rhesus macaques.

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.

Long-Range Control of Class Switch Recombination by Transcriptional Regulatory Elements.

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.

Characterizing the BCR repertoire during lymphocyte reduction and recovery mediated by cyclophosphamide and granulocyte-macrophage colony-stimulating factor.

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.

The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe.

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.

Systematic Engineering of Optimized Autonomous Heavy-Chain Variable Domains.

Autonomous heavy-chain variable (VH) 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 VH 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 VH domains, in which aggregation propensity is reduced without compromising antigen recognition, has proven challenging. Here, we used an autonomous human VH 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 VH domains, which were characterized in detail. Structural analyses of a parental anti-EphA1 VH 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 VH domains that resist aggregation and could be used for basic research and biomedical applications.

Mechanism of noncoding RNA-associated N6-methyladenosine recognition by an RNA processing complex during IgH DNA recombination.

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 N6-methyladenosine (m6A) 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 m6A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SµGLT. Direct suppression of m6A modification of SµGLT or of m6A 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, m6A modification, and m6A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells.

Structure of Nanobody Nb23.

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 ß2-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.

Expression analysis and tissue localization of IgZ in the grouper Epinephelus coioides after Vibrio alginolyticus infection and vaccination.

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.

A Germline-Encoded Structural Arginine Trap Underlies the Anti-DNA Reactivity of a Murine V Gene Segment.

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 VH10 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 (VH10) or not (VH4) 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. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 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.

New Insights into IgZ as a Maternal Transfer Ig Contributing to the Early Defense of Fish against Pathogen Infection.

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.

Molecular characterization of a new IgZ3 subclass in common carp (Cyprinus carpio) and comparative expression analysis of IgH transcripts during larvae development.

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.

Transcriptome and IgH Repertoire Analyses Show That CD11chi B Cells Are a Distinct Population With Similarity to B Cells Arising in Autoimmunity and Infection.

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 CD11chi cells from peripheral blood and characterized them using transcriptome and IgH repertoire analyses. Gene expression data revealed the CD11chi IgD+ and IgD- subsets were highly similar to each other, but distinct from naive, memory, and plasma cell subsets. Although CD11chi 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 CD11chi 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- CD11chi 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.