VH Gene Research Articles

A B cell screen against endogenous retroviruses identifies glycan-reactive IgM that recognizes a broad array of enveloped viruses.

Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. ERVs with near-intact coding potential reactivate in B cell-deficient mice. To study how B cells contribute to host anti-ERV immunity, we used an antigen-baiting strategy to enrich B cells reactive to ERV surface antigens. We identified ERV-reactive B-1 cells expressing germline-encoded natural IgM antibodies in naïve mice, the level of which further increases upon innate immune sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of the Igh VH gene that gives rise to glycan-specific antibodies targeting terminal N-acetylglucosamine moieties on ERV glycoproteins, which further engage the complement pathway to mediate anti-ERV responses. These same antibodies also recognize glycoproteins of other enveloped viruses but not self-proteins. These results reveal an innate antiviral mechanism of germline-encoded antibodies with broad reactivity to enveloped viruses, which constitutes a natural antibody repertoire capable of preventing the emergence of infectious ERVs.

Characterization of anti-EBNA-1 antibodies and exploration of their molecular mimicry potential in an EBV-infected Sjögren's syndrome patient

There is a potential link between autoimmune diseases and Epstein-Barr virus (EBV) infections, with EBV playing a substantial role in the onset of Sjögren's syndrome (SjS). Some EBV proteins could mimic host self-antigens post-infection, leading to molecular mimicry. This similarity may cause the immune system to attack its tissues mistakenly. Among the various proteins associated with EBV, nuclear antigen 1 (EBNA-1) is essential for the latent replication of infected cells and is prevalent in all EBV-related diseases. In the study, single-chain variable fragment (scFv) antibodies targeting EBNA-1 were isolated using phage display technology from a primary SjS patient who also had a chronic active EBV infection. The specific clones were enriched after panning, and the binding activity of selected scFvs targeting EBNA-1 was confirmed. Sequence analysis indicated that the scFvs exhibiting positive signals could be grouped into five clones, all of which used homologous heavy chain V regions derived from germline Vh4-39, and two types of light chain V regions stemming from germline Vλ1-44 and Vλ3-15. These scFvs were found to exhibit a high degree of somatic mutations, likely indicative of antigen selection. Of the scFvs, P1-3 demonstrated the strongest binding affinity to EBNA-1, exhibiting a determined value of 7.3 x 10-8 M, and showed cross-reactivity to the SjS associated La/SSB self-antigen. The experimental results combined with AlphaFold 3 predictions revealed a potential epitope for scFv P1-3 binding to EBNA-1. Additionally, scFv P1-3 could also cross-bind to the modeled structure of La/SSB. We inferred a possible structural correlation between EBNA-1 and La/SSB involving an X2AX6PG epitope motif. This research contributes to our understanding of the structural basis of the interactions between antibodies and EBNA-1, shedding light on the VH and VL gene usage of anti-EBNA-1 antibodies in EBV-infected SjS patients and the potential origins of autoantibodies.

Invariant VD and DJ Motifs Define a Novel Class of Human Antibodies and TCRs Prototyped by antigen receptors of Dual-Expresser Lymphocytes

ABSTRACT Introduction Dual-expressing lymphocytes (DEs) are unique immune cells that express both B cell receptors (BCRs, surface antibody) and T cell receptors (TCRs). In type 1 diabetes, DE antibodies are predominated by one antibody (x-mAb), an IgM monoclonal antibody with a germline-encoded CDR3 that recognizes self-reactive TCRs. We explored if x-mAb and its interacting TCRs have distinct structural features. Methods Using bioinformatics, we compared x-mAb and its most common interacting TCRαβ to billions of antigen receptor sequences to determine if they were unique or randomly generated. Results X-mAb represents a unique class of human antibodies with a conserved CDR3 sequence (CARx1-4DTAMVYYFYDW), consisting of a fixed DJH motif (DTAMVYYFDYW) paired with various VH genes. A public TCRβ clonotype (CASSPGTEAFF) associated with x-mAb on DEs features two invariant segments, VβD (CASSPGT) and DJβ (PGTEAFF), key to two large families of public TCRβ clonotypes—CASSPGT-Jβx and CASSPGT-Jβx—formed by recombining the VβD motif with Jβ genes and the DJβ motif with Vβ genes. B cells also use CASSPGT as a VHD motif for public IGH clonotypes (CASSPGT—Jβx). Discussion DEs, unlike conventional T and B cells, use invariant motifs to create public antibodies and TCRs, a trait previously seen only in cartilaginous fish.

The Igh distal enhancers modulate locus topology and repertoire diversity in pro-B cells

Abstract The Igh locus undergoes large-scale locus contraction prior to V(D)J recombination in pro B cells. Our Hi-C analyses have identified chromatin loops anchored by a series of distal VH enhancers, EVH1-4 and Pax5-activated intergenic repeat 4 (PAIR4) implying that these interactions contribute to Igh locus contraction. Indeed, EVH1 modulates Igh locus spatial organization and VH gene usage in VDJ recombination linking Igh locus conformation with function (Nat Comm. 14:1225). Loop extrusion is a process that forms chromatin loops by extruding chromatin through cohesin’s ring structure and is a critical regulator of distal V(D)J recombination. However, the relationship between chromatin looping and loop extrusion remains incompletely understood. We posit that EVHs and PAIR4 provide structure independent of loop extrusion which then interacts with loop extrusion to shape higher order locus architecture. Wings apart-like protein (Wapl) is a cohesin release factor. Acute WAPL-depletion increases loop extrusion, enabling us to directly test the relationship between Igh regulatory elements and loop extrusion in Abelson transformed (Abl-t) pro-B cell lines. We show that EVH4 and PAIR4 deletion reduce distal Igh locus interactions despite increased loop extrusion. Our studies support a model in which cis regulatory elements modulate loop extrusion to shape Igh locus architecture and consequently direct VH gene usage.

VH14-2 mediated nuclear speckles association contributes to radial positioning of the Igh locus and regulates VDJ recombination

Abstract The Igh locus undergoes large-scale contraction and repositions to the nuclear center prior to V(D)J recombination. However, little is known about the mechanisms that mediate these processes. We recently identified a chromatin loop anchor which corresponds to a highly transcribed VH gene promoter (VH14-2 Pr) (Nat Commun 14:1225, 2023). Here we report that VH14-2 deletion increased locus contraction which was expected to increase VDJ recombination. However, deletion of the VH14-2 gene severely reduced VDJ recombination. This implied that another mechanism may contribute to VDJ recombination. Strikingly, Igh loci accumulated toward nuclear periphery in Rag2-/-VH14-2-/- pro-B cells indicating the failure of radial repositioning. Thus, efficient VDJ recombination is linked to repositioning of the Igh locus and implies that the VH14-2 gene anchors the locus in the nuclear center. Highly transcribed genes often associate with nuclear speckles that tend to locate to the nuclear center. As VH14-2 is highly transcribed we asked whether it associates with nuclear speckles. We found that the Igh locus overlaps with nuclear speckles and this association was significantly reduced in Rag2-/-VH14-2-/- pro B cells as visualized using a combination of DNA and immuno-fluorescence in situ hybridization (FISH). This study identified the VH14-2 gene as a tether that anchors the locus to nuclear speckles and in turn contributes to radial positioning and VDJ recombination.

Abstract 6317: Developing a fully human heavy chain only Claudin-18.2-specific CAR-T cell therapy

Abstract Camelid VHH/nanobodies have been widely applied as the antigen binding domain of CAR-T cell therapies due to a smaller size and simpler structure compared to the traditionally used single chain variable fragment (scFv). Additionally, camelid VHH/nanobodies provide greater flexibility for dual-targeting CAR-T cell therapies. However, camelid VHH/nanobodies require laborious and costly humanization and still have a significant risk of inducing anti-drug antibodies when administered to patients. Transgenic mice exclusively harboring the most stable human VH genes (HCAb Harbour Mice®) provide one of the best solutions to get fully human heavy chain only antibodies (HCAbs). To efficiently identify lead HCAb VHs for Claudin-18.2-targeting CAR-T cells, we developed an innovative direct CAR-function based HCAb library screening platform. From Claudin-18.2-immunized HCAb Harbour Mice®, we constructed a total VH library in a CAR lentiviral vector that we developed in-house. After producing the lentiviral particle library, we transduced a Jurkat-NFAT-GFP reporter cell line and stimulated with Claudin-18.2 overexpression cell line. Subsequently, GFP positive reporter cells, representing Claudin-18.2-specific CARs, were isolated through FACS sorting, expanded, and further stimulated with Claudin-18.2-positive NUGC-4 tumor cells. Then, Next Generation Sequencing and abundance ranking was performed on VHs recovered from sorted CAR-reporter cells. Lead VH candidates were cloned into the CAR-P2A-RFP lentiviral vector, and many of these VH clones exhibited superior Claudin-18.2-specific functionality compared to a clinically-validated reference scFv in both reporter and primary CAR-T cells. Many lead VH clones also exhibited excellent developability as soluble HCAbs. These data suggest that this innovative method of combining our fully human HCAb and CAR-based library screening platform will provide new avenues for CAR-T cell therapies. Citation Format: Jason Noon, Yao Lu, Itishree Kaushik, Oyku Ay, Adam Luk, Bradley Delaney, David DeMeritt, Musheng Bao. Developing a fully human heavy chain only Claudin-18.2-specific CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6317.

Convergent evolution and targeting of diverse E2 epitopes by human broadly neutralizing antibodies are associated with HCV clearance

The early appearance of broadly neutralizing antibodies (bNAbs) in serum is associated with spontaneous hepatitis C virus (HCV) clearance, but to date, the majority of bNAbs have been isolated from chronically infected donors. Most of these bNAbs use the VH1-69 gene segment and target the envelope glycoprotein E2 front layer. Here, we performed longitudinal B cell receptor (BCR) repertoire analysis on an elite neutralizer who spontaneously cleared multiple HCV infections. We isolated 10,680 E2-reactive B cells, performed BCR sequencing, characterized monoclonal B cell cultures, and isolated bNAbs. In contrast to what has been seen in chronically infected donors, the bNAbs used a variety of VH genes and targeted at least three distinct E2 antigenic sites, including sites previously thought to be non-neutralizing. Diverse front-layer-reactive bNAb lineages evolved convergently, acquiring breadth-enhancing somatic mutations. These findings demonstrate that HCV clearance-associated bNAbs are genetically diverse and bind distinct antigenic sites that should be the target of vaccine-induced bNAbs.

An Engineered Mouse Model That Generates a Diverse Repertoire of Endogenous, High-Affinity Common Light Chain Antibodies.

Bispecific antibodies have gained increasing popularity as therapeutics as they enable novel activities that cannot be achieved with monospecific antibodies. Some of the most popular bispecific formats are molecules in which two Fab arms with different antigen specificities are combined into one IgG-like molecule. One way to produce these bispecific molecules requires the discovery of antibodies against the two antigens of interest that share a common light chain. Here, we present the generation and characterization of a common light chain mouse model, in which the endogenous IGKJ cluster is replaced with a prearranged, modified murine IGKV10-96/IGKJ1 segment. We demonstrate that genetic modification does not impact B-cell development. Upon immunization with ovalbumin, the animals generate an antibody repertoire with VH gene segment usage of a similar diversity to wildtype mice, while the light chain diversity is restricted to antibodies derived from the prearranged IGKV10-96/IGKJ1 germline. We further show that the clonotype diversity of the common light chain immune repertoire matches the diversity of immune repertoire isolated from wildtype mice. Finally, the common light chain anti-ovalbumin antibodies have only slightly lower affinities than antibodies isolated from wildtype mice, demonstrating the suitability of these animals for antibody discovery for bispecific antibody generation.

Intra- and interchromosomal contact mapping reveals the Igh locus has extensive conformational heterogeneity and interacts with B-lineage genes

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

Signatures of VH1-69-derived hepatitis C virus neutralizing antibody precursors defined by binding to envelope glycoproteins

An effective preventive vaccine for hepatitis C virus (HCV) remains a major unmet need. Antigenic region 3 (AR3) on the E1E2 envelope glycoprotein complex overlaps with the CD81 receptor binding site and represents an important epitope for broadly neutralizing antibodies (bNAbs) and is therefore important for HCV vaccine design. Most AR3 bNAbs utilize the VH1-69 gene and share structural features that define the AR3C-class of HCV bNAbs. In this work, we identify recombinant HCV glycoproteins based on a permuted E2E1 trimer design that bind to the inferred VH1-69 germline precursors of AR3C-class bNAbs. When presented on nanoparticles, these recombinant E2E1 glycoproteins efficiently activate B cells expressing inferred germline AR3C-class bNAb precursors as B cell receptors. Furthermore, we identify critical signatures in three AR3C-class bNAbs that represent two subclasses of AR3C-class bNAbs that will allow refined protein design. These results provide a framework for germline-targeting vaccine design strategies against HCV.

Characteristics of rabbit hapten-specific and germline-based BCR repertoires following repeated immunization

The rabbit is well known for producing diverse antibodies against various antigens including small molecules such as drugs and toxins, due to a robust immune response. Elucidating how hapten repeated immunization shapes the rabbit B cell receptor (BCR) repertoire is crucial to understanding rabbit immune response to small molecules and assisting rare antibody discovery/engineering. In this study, we enriched and sequenced chloramphenicol (CAP)-specific rabbit B cells following repeated immunization, and analyzed both CAP-specific repertoires combined with the structure and affinity features of V1S69/V1S37 germline-based BCRs. The length of rabbit complementarity-determining region 3 of heavy chain (CDRH3) increased after hapten immunization. Repeated immunization significantly reduced the diversity of CAP-specific rabbit BCR clonotypes, and changed the frequency of VDJ usage and the type of V(D)J recombination. The average number of mutations among VL is notably higher than that of VH genes in rabbits, however, they are both not changed along with repeated immunization. Moreover, repeated immunization resulted in an increase surface charge and a decrease in solvent accessible surface area, leading to improvement in the stability of the most abundant V1S69/V1S37 germline-based BCR, along with an affinity increase from an IC50 of 898.2 ng mL−1 at the 1st immunization to 4.16 ng mL−1 at the 6th immunization. The study provides a benchmark for rabbit repertoire-scale analyses and offers a method for antibody discovery of small molecules.

Contribution of the IGCR1 regulatory element and the 3′Igh CTCF-binding elements to regulation of Igh V(D)J recombination

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3'Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3'Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.

Enhancers of the PAIR4 regulatory module promote distal VH gene recombination at the Igh locus

While extended loop extrusion across the entire Igh locus controls VH‐DJH recombination, local regulatory sequences, such as the PAIR elements, may also activate VH gene recombination in pro‐B‐cells. Here, we show that PAIR‐associated VH8 genes contain a conserved putative regulatory element (V8E) in their downstream sequences. To investigate the function of PAIR4 and its V8.7E, we deleted 890 kb containing all 14 PAIRs in the Igh 5′ region, which reduced distal VH gene recombination over a 100‐kb distance on either side of the deletion. Reconstitution by insertion of PAIR4‐V8.7E strongly activated distal VH gene recombination. PAIR4 alone resulted in lower induction of recombination, indicating that PAIR4 and V8.7E function as one regulatory unit. The pro‐B‐cell‐specific activity of PAIR4 depends on CTCF, as mutation of its CTCF‐binding site led to sustained PAIR4 activity in pre‐B and immature B‐cells and to PAIR4 activation in T‐cells. Notably, insertion of V8.8E was sufficient to activate VH gene recombination. Hence, enhancers of the PAIR4‐V8.7E module and V8.8E element activate distal VH gene recombination and thus contribute to the diversification of the BCR repertoire in the context of loop extrusion.

CD62L expression marks SARS-CoV-2 memory B cell subset with preference for neutralizing epitopes

Severe acute respiratory syndrome coronavirus 2–neutralizing antibodies primarily target the spike receptor binding domain (RBD). However, B cell antigen receptors (BCRs) on RBD-binding memory B (B mem ) cells have variation in the neutralizing activities. Here, by combining single B mem cell profiling with antibody functional assessment, we dissected the phenotype of B mem cell harboring the potently neutralizing antibodies in coronavirus disease 2019 (COVID-19)–convalescent individuals. The neutralizing subset was marked by an elevated CD62L expression and characterized by distinct epitope preference and usage of convergent V H (variable region of immunoglobulin heavy chain) genes, accounting for the neutralizing activities. Concordantly, the correlation was observed between neutralizing antibody titers in blood and CD62L + subset, despite the equivalent RBD binding of CD62L + and CD62L − subset. Furthermore, the kinetics of CD62L + subset differed between the patients who recovered from different COVID-19 severities. Our B mem cell profiling reveals the unique phenotype of B mem cell subset that harbors potently neutralizing BCRs, advancing our understanding of humoral protection.

Igh and Igk loci use different folding principles for V gene recombination due to distinct chromosomal architectures of pro-B and pre-B cells

Extended loop extrusion across the immunoglobulin heavy-chain (Igh) locus facilitates VH-DJH recombination following downregulation of the cohesin-release factor Wapl by Pax5, resulting in global changes in the chromosomal architecture of pro-B cells. Here, we demonstrate that chromatin looping and VK-JK recombination at the Igk locus were insensitive to Wapl upregulation in pre-B cells. Notably, the Wapl protein was expressed at a 2.2-fold higher level in pre-B cells compared with pro-B cells, which resulted in a distinct chromosomal architecture with normal loop sizes in pre-B cells. High-resolution chromosomal contact analysis of the Igk locus identified multiple internal loops, which likely juxtapose VK and JK elements to facilitate VK-JK recombination. The higher Wapl expression in Igμ-transgenic pre-B cells prevented extended loop extrusion at the Igh locus, leading to recombination of only the 6 most 3’ proximal VH genes and likely to allelic exclusion of all other VH genes in pre-B cells. These results suggest that pro-B and pre-B cells with their distinct chromosomal architectures use different chromatin folding principles for V gene recombination, thereby enabling allelic exclusion at the Igh locus, when the Igk locus is recombined.

Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known. To identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects. We found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing. Together, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

Affinity maturation generates pathogenic antibodies with dual reactivity to DNase1L3 and dsDNA in systemic lupus erythematosus

Anti-dsDNA antibodies are pathogenically heterogeneous, implying distinct origins and antigenic properties. Unexpectedly, during the clinical and molecular characterization of autoantibodies to the endonuclease DNase1L3 in patients with systemic lupus erythematosus (SLE), we identified a subset of neutralizing anti-DNase1L3 antibodies previously catalogued as anti-dsDNA. Based on their variable heavy-chain (VH) gene usage, these antibodies can be divided in two groups. One group is encoded by the inherently autoreactive VH4-34 gene segment, derives from anti-DNase1L3 germline-encoded precursors, and gains cross-reactivity to dsDNA – and some additionally to cardiolipin – following somatic hypermutation. The second group, originally defined as nephritogenic anti-dsDNA antibodies, is encoded by diverse VH gene segments. Although affinity maturation results in dual reactivity to DNase1L3 and dsDNA, their binding efficiencies favor DNase1L3 as the primary antigen. Clinical, transcriptional and monoclonal antibody data support that cross-reactive anti-DNase1L3/dsDNA antibodies are more pathogenic than single reactive anti-dsDNA antibodies. These findings point to DNase1L3 as the primary target of a subset of antibodies classified as anti-dsDNA, shedding light on the origin and pathogenic heterogeneity of antibodies reactive to dsDNA in SLE.

An Igh distal enhancer modulates antigen receptor diversity by determining locus conformation

The mouse Igh locus is organized into a developmentally regulated topologically associated domain (TAD) that is divided into subTADs. Here we identify a series of distal VH enhancers (EVHs) that collaborate to configure the locus. EVHs engage in a network of long-range interactions that interconnect the subTADs and the recombination center at the DHJH gene cluster. Deletion of EVH1 reduces V gene rearrangement in its vicinity and alters discrete chromatin loops and higher order locus conformation. Reduction in the rearrangement of the VH11 gene used in anti-PtC responses is a likely cause of the observed reduced splenic B1 B cell compartment. EVH1 appears to block long-range loop extrusion that in turn contributes to locus contraction and determines the proximity of distant VH genes to the recombination center. EVH1 is a critical architectural and regulatory element that coordinates chromatin conformational states that favor V(D)J rearrangement.

Nontraditional Roles of DNA Polymerase Eta Support Genome Duplication and Stability.

DNA polymerase eta (Pol η) is a Y-family polymerase and the product of the POLH gene. Autosomal recessive inheritance of POLH mutations is the cause of the xeroderma pigmentosum variant, a cancer predisposition syndrome. This review summarizes mounting evidence for expanded Pol η cellular functions in addition to DNA lesion bypass that are critical for maintaining genome stability. In vitro, Pol η displays efficient DNA synthesis through difficult-to-replicate sequences, catalyzes D-loop extensions, and utilizes RNA-DNA hybrid templates. Human Pol η is constitutively present at the replication fork. In response to replication stress, Pol η is upregulated at the transcriptional and protein levels, and post-translational modifications regulate its localization to chromatin. Numerous studies show that Pol η is required for efficient common fragile site replication and stability. Additionally, Pol η can be recruited to stalled replication forks through protein-protein interactions, suggesting a broader role in replication fork recovery. During somatic hypermutations, Pol η is recruited by mismatch repair proteins and is essential for VH gene A:T basepair mutagenesis. Within the global context of repeat-dense genomes, the recruitment of Pol η to perform specialized functions during replication could promote genome stability by interrupting pure repeat arrays with base substitutions. Alternatively, not engaging Pol η in genome duplication is costly, as the absence of Pol η leads to incomplete replication and increased chromosomal instability.

Biological Features of CD5+ CD19+ B1 Cell and Natural IgM (VH4-34) in Chronic Lymphocytic Leukemia vs. Multiple Sclerosis.

Chronic lymphocytic leukemia (CLL) is the clonal expansion of mature CD5+ B cells and the most common lymphoproliferative disease in adults (B1-CLL). B1 cells' anti-inflammatory effects include the production of natural IgM (nIgM) by the spleen and bone marrow, decreased inflammatory cytokines as a primary response to maintaining tissue homeostasis, and enhanced release of transforming growth factor β (TGFβ). We used the flow cytometry technique in peripheral blood from patients with CLL and multiple sclerosis (MS) to immunophenotype B cells and their subpopulations. Whole blood from CLL and MS patients, as well as healthy controls, was used to detect nIgM using the VH4-34 gene copy number and real-time RT-PCR. We found that the proportion of CD5+ B cells was significantly lower in MS patients than in the control group and that CD5+ B lymphocytes were significantly higher in CLL patients than in the control group. Compared to the control group, CLL patients had significantly higher levels of the VH4-34 gene copy number. On the contrary, MS patients had significantly lower VH4-34 gene copy number levels compared to the control group. As the number of antibodies in CLL patients increases due to the high number of B1 cells, we propose a new way to treat MS by extracting this natural antibody from the sera of CLL patients and injecting it into MS patients.