Biology subjects

Alt, F. W.

Publications and source records attributed to Alt, F. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Robust V(D)J Recombination in the Absence of SA1/2-mediated Chromatin Loops

The cohesin ring complex organizes and mediates functional activity of the genome through chromatin loop extrusion1,2. Cohesin comprises a core SMC1-SMC3-RAD21 ring associated with multiple regulatory proteins, including SA1 and SA2 paralogs that bind mutually-exclusively to a RAD21 site3,4. Functional studies of cohesin complexes in vivo often employ inactivation of particular components2,3. However, inactivation of SA1 or SA2 in mice is limited by mouse embryonic lethality5-7 and, for dual SA1/2-inactivation by cycling cell lethality8-10. For these reasons, it remains unknown whether the requirement for SA1/2 in loop extrusion in vitro11,12 applies in vivo1,2, and whether these factors have overlapping or distinct functions in regulating this process3. Here, we employ G0/G1-arrested v-Abl pro-B lines, which are viable in the absence of cohesin-complex factors13,14, to dissect SA1 and SA2 functions in V(D)J recombination, a strictly G0/G1-specific process that harnesses loop extrusion to effect distinct Igh and Ig{kappa} locus recombination mechanisms15,16. Robust long-range Igh or Ig{kappa} V(D)J recombination continued unabated in the absence of SA1 or SA2. However, inactivation of both abrogated most positioned chromatin loops genome-wide and also abolished long-range Igh V(D)J recombination with a phenotype similar to RAD21 (cohesin) inactivation13. Yet, robust Ig{kappa} V(D)J recombination continued upon dual SA1/2-inactivation, which along with Hi-C findings, demonstrates ongoing loop-extrusion in the absence of SA1/2. Correspondingly, replacement of weak Igh V(D)J recombination signal sequences (RSSs) that target V(D)J recombination activity, with strong Ig{kappa} counterparts15 restored long-range Igh V(D)J recombination in SA1/2 inactivated cells. We conclude that SA1/2 together maintain loop extrusion impediments and genome-wide architecture in G0/G1-arrested cells; and, in their absence, loop extrusion proceeds unimpeded. Remarkably, strong RSSs allow Ig{kappa} to function normally in the absence of internal positioned loop structure across the 3.1-mega-base Ig{kappa} locus. We discuss implications of our findings for long-range gene regulation in G0/G1-arrested cells more broadly.

immunology↗

bioRxiv2026

Asymmetric Boost Responses to an HIV-1 Vaccine: Boost-site Controls Secondary B-cell Fates

Induction of broadly neutralizing antibody (bnAb) responses remains a central challenge to designing efficacious HIV vaccines. Lineage design strategies, in which bnAb precursors are guided via iterative immunizations to their mature forms, depend on high levels of somatic mutation and the recall of antigen-specific B cells. Recent studies have identified spatial context as an important determinant of boosting efficacy, but the application of this to HIV vaccines and the underlying mechanisms are incompletely understood. Here, using mice harboring a V3-glycan bnAb lineage precursor knock-in combined with lineage-tracing and single-cell analyses, we show that persistent germinal centers (GCs) support continued affinity maturation of founder clones and ipsilateral boosting preferentially engages these lineages in secondary GCs. In contrast, contralateral boosting predominantly recruits naive B cells and memory B cells not directed towards the immunizing antigen. The few memory cells recruited at this site were biased towards a plasma cell fate. Finally, we identify disfavored mutational trajectories within the V3-glycan bnAb lineage, revealing intrinsic constraints on bnAb lineage evolution.

immunology↗

bioRxiv2026

B Cell Receptor Signaling Sets Boundaries for Selection of Broadly Neutralizing Antibody Functional Mutations

B cell signaling is required for germinal center (GC) selection and synergizes with T cell-help leading to differentiation into effector cells and a protective antibody response. Here, we studied the relationship between B cell signaling and the selection of functional mutations in a humanized mouse model of a CD4 binding-site specific HIV-1 broadly neutralizing antibody precursor. While BCR signaling increased with antigen affinity, immunization-induced frequency of a functional mutation was inversely related to affinity and favored a gain in association rate. Antigen-specific GC B cells and key mutation frequency were higher in the mid-affinity (0.5 - 5M) than in either the higher or lower affinity group, and were consistent with the significantly higher serum neutralization titers in the mid-range group. Our studies show that BCR signaling imposes boundaries (upper/lower) for selection of antibody functional mutations and support a "Goldilocks Zone" model that defines the favored BCR signaling strength for GC selection.

immunology↗

bioRxiv2026

Human antibody heavy chain variable region exon interacts with 3' regulatory region to form a stable somatic hypermutation center

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) of immunoglobulin heavy chain (HC) variable region exons in germinal center (GC) B cells, allowing selection of affinity-matured B cell receptors. The V(D)J exon location is privileged for SHM targeting of diverse sequences within it. In analogy to the related HC class switch recombination center, we proposed that cohesin-mediated loop extrusion juxtaposes widely separated enhancers, including 3 Igh regulatory region ("3RR"), with the V(D)J exon in GC B cells to establish a SHM-center (SHM-C) "privileged" for AID access. To test this hypothesis, we employed a human RAMOS GC B lymphoma cell SHM model with inducible AID expression in which we targeted an unmutated mouse HC V(D)J exon in place of its human counterpart. Activation of AID expression in this cycling RAMOS line for 10 days led to robust SHM accumulation in the mouse V(D)J exon with a pattern similar to that in mouse GC B cells. In this model, the mouse V(D)J exon was indeed juxtaposed to the two human 3RRs and other elements consistent with SHM-C formation. While deletion of individual 3RRs had little effect, deletion of both greatly reduced SHM, but left V(D)J exon transcription unabated. Moreover, the SHM-C and SHM accumulation remained intact over a 10-day period in viably G1-arrested and viably G1-arrested RAD21 (cohesin)-depleted versions of the model. We discuss implications for SHM-C structure and function, its long-term functional stability in the absence of the loop extrusion process proposed to assemble it, and for 3RR SHM functions beyond transcriptional activation. Significance StatementUpon encountering pathogens, B cells enter specialized lymphoid structures known as germinal centers (GCs). GC B cells express activation-induced cytidine deaminase (AID) that initiates somatic hypermutation (SHM) of the portion of antibody genes that encodes antigen recognition. B cells in which mutations increase antibody affinity for activating pathogen are selected to enhance antibody responses. We proposed SHM occurs in a chromosomal structure in the antibody gene locus termed a SHM-center (SHM-C). By studying SHM in a human B cell lymphoma model, we provide direct evidence for existence of a SHM-C assembled from different functional components normally separated by considerable distances. We also find that the SHM-C is surprisingly stable and continues to function for many days in non-dividing cells.

immunology↗

bioRxiv2026

Mouse models with human antibody repertoires for inducing multiple lineages of HIV-1 broadly neutralizing antibodies

The complementarity determining region (CDR) 1, 2 and 3 of antibodies are the principal antigen contact sites. The heavy chain CDR3 (CDR H3) is highly variable, because it includes random nucleotide additions by the terminal deoxynucleotidyl transferase (TdT). Some broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1) rely heavily on CDR H3 to recognize conserved epitopes on HIV-1 Envelope (Env) protein. Elicitation of comparable bnAbs is a prime goal of HIV-1 vaccine development, but the shortage of precursor antibodies with suitable CDR H3s in human repertoire is a major challenge for immunogen design. To aid this effort, we generated six mouse models for inducing bnAbs against major HIV-1 Env epitopes. In each mouse model, the immunoglobulin heavy and light chain loci were engineered to predominantly rearrange the germline V, D and J segments of a bnAb lineage. Owing to CDR3 diversity, only a small subset of the V(D)J recombination products may encode variable regions that can engage bnAb epitopes with sufficient affinity to initiate immune response. Therefore, these mouse models can be used to test and optimize immunization strategies to induce bnAbs from rare and diverse precursors in complex antibody repertoires.

immunology↗

bioRxiv2026

Maturation of HIV-1 neutralizing antibodies in a germinal center conditional expression mouse model

In germinal centers, activated B cells modify their antigen receptors through somatic hypermutation (SHM), followed by antigenic selection that favors expansion of high affinity B cells. The affinity maturation process is critical for development of broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs have been isolated from some people living with HIV-1. Because these antibodies target conserved epitopes of the HIV-1 Envelope (Env) protein, they inhibit a broad spectrum of viruses. Eliciting bnAbs by vaccination is a top priority for HIV-1 prevention, but reproducing the lengthy maturation of bnAbs is a major challenge. The problem is typified by VRC01 class antibodies, which recognize the CD4 binding site of HIV-1 Env protein. To reach the CD4 binding site, antibodies need to navigate through adjacent glycans. Accommodating the glycans requires multiple SHMs in germinal center (GC) B cells, including infrequent events. For this reason, VRC01 vaccine development often stalls at this point. We have generated a mouse model aimed at providing a potential solution for navigating this vaccine design impediment. To this end, we made a mouse model that expresses a stalled VRC01 intermediate conditionally in GC B cells. This system has three advantages: 1) direct expression of the intermediate obviates prior immunization steps, thereby shortening the immunization scheme; 2) the conditional expression system bypasses tolerance control checkpoints that sometimes delete B cells expressing bnAbs; 3) the intermediate responds to immunization in GCs, the physiological site of affinity maturation. With this model, we established an immunization method to mature the VRC01 intermediate into heterologous neutralizing antibodies against viruses with a native glycan shield. Since high mutation load is common among bnAbs, the germinal center conditional expression system could provide a general tool for boost immunogen design to overcome roadblocks in the maturation pathway. Author summaryIn response to antigenic stimulation, cognate B cells become activated and form germinal centers in lymphoid tissues. Germinal center B cells modify their antigen receptors through somatic hypermutation (SHM) of immunoglobulin variable region gene exons, with antigen selecting for high affinity B cells by providing survival advantage. This mechanism accounts for antibody affinity maturaton over the gradual course of an immune response. Affinity maturation is critical for generating potent, neutralizing antibodies against diverse strains of the human immunodeficiency virus-1 (HIV-1). These broadly neutralizing antibodies (bnAbs) are heavily mutated, reflecting lengthy affinity maturation over years of chronic infection. Recapitulating the affinity maturation process is a major challenge for bnAb induction by vaccination. In immunization experiments, bnAb development often stalls at rate limiting steps that involve infrequent, but functionally important, mutational events. Overcoming such obstacles requires boost immunogens that can stimulate the stalled B cells to acquire the requisite mutations. To this end, we recapitulated the maturation arrest of a bnAb lineage by expressing a stalled antibody in mouse germinal center B cells. Using this mouse model, we developed boost immunization conditions that advanced the antibody maturation beyond a roadblock to attain neutralizing activities against heterogenous viruses.

immunology↗

bioRxiv2023

Contribution of the IGCR1 regulatory element and the 3'Igh CBEs 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 3Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Down-regulation 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 3Igh-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 deleting or inverting IGCR1 or 3Igh-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 3Igh-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 down-regulation mechanism in progenitor B cells as opposed to a strict developmental switch. SIGNIFICANCE STATEMENTTo counteract diverse pathogens, vertebrates evolved adaptive immunity to generate diverse antibody repertoires through a B lymphocyte-specific somatic gene rearrangement process termed V(D)J recombination. Tight regulation of the V(D)J recombination process is vital to generating antibody diversity and preventing off-target activities that can predispose the oncogenic translocations. Recent studies have demonstrated V(D)J rearrangement is driven by cohesin-mediated chromatin loop extrusion, a process that establishes genomic loop domains by extruding chromatin, predominantly, between convergently-oriented CTCF looping factor-binding elements (CBEs). By deleting and inverting CBEs within a critical antibody heavy chain gene locus developmental control region and a loop extrusion chromatin-anchor at the downstream end of this locus, we reveal how these elements developmentally contribute to generation of diverse antibody repertoires.

immunology↗

bioRxiv2022

Mutation-Guided Vaccine Design: A Strategy for Developing Boosting Immunogens for HIV Broadly Neutralizing Antibody Induction

A major goal of HIV-1 vaccine development is induction of broadly neutralizing antibodies (bnAbs). While success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knock-in mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof-of concept and milestone towards development of an HIV vaccine. One-Sentence SummaryA vaccine strategy for selecting key rare antibody mutations is shown to induce HIV broadly neutralizing antibodies in mice.

immunology↗

bioRxiv2022

Humanized V(D)J-rearranging and TdT-expressing Mouse Vaccine Models with Physiological HIV-1 Broadly Neutralizing Antibody Precursors

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through non-templated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs V{kappa}3-20 or V{kappa}1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had non-physiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as V{kappa}1-33 and/or V{kappa}3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated V{kappa}-to-J{kappa} joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center (GC) B cell responses. V{kappa}3-20-based responses were enhanced by N-region addition, which generates V{kappa}3-20-to-J{kappa} junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs. Significance StatementMouse models that express human precursors of HIV-1 broadly neutralizing antibodies (bnAbs) are useful for evaluating vaccination strategies for eliciting such bnAbs in humans. Prior models were handicapped by non-physiological frequency and/or diversity of B lymphocytes that express the bnAb precursors. We describe a new class of mouse models in which the mice express humanized bnAb precursors at a more physiologically relevant level through developmental rearrangement of both antibody heavy and light chain gene segments that encode the precursors. The model also incorporated a human enzyme that diversifies the rearranging gene segments and promotes generation of certain variable region sequences needed for the response. This new class of mouse models should facilitate preclinical evaluation of candidate human HIV-1 vaccination strategies.

immunology↗

bioRxiv2022

Structural principles of B-cell antigen receptor assembly

The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M, D, G, A or E for antigen recognition and a disulfide-linked heterodimer between Ig and Ig{beta} (Ig/{beta}, also known as CD79A and CD79B) that functions as the signalling entity. The organizing principle of BCR assembly remains elusive. Here we report the cryo-electron microscopy structures of the intact IgM class BCR at 8.2 [A] resolution and its Fab-deleted form (IgM BCR{Delta}Fab) at 3.6 [A] resolution. At the ectodomain (ECD), Ig and Ig{beta} position their respective Ig folds roughly in parallel with an approximate 2-fold symmetry, which is distinct from structures of Ig{beta}/{beta} homodimers. Unlike previous predictions, the BCR structure displays an asymmetric arrangement, in which the Ig/{beta} ECD heterodimer mainly uses Ig to associate with C{micro}3-C{micro}4 domains of one heavy chain ({micro}HC) while leaving the other heavy chain ({micro}HC) empty. The transmembrane domain (TMD) helices of the two {micro}HCs also deviate from the 2-fold symmetry of the C{micro}3-C{micro}4 domain dimer and form together with the TMD helices of the Ig/{beta} heterodimer a tight 4-helix bundle. The asymmetry at the TMD helices prevents the recruitment of two Ig/{beta} heterodimers. Surprisingly, the connecting peptides (CPs) between the ECD and TMD are braided together through striking charge complementarity, resulting in intervening of the CP of {micro}HC in between those of Ig and Ig{beta} and crossover of the TMD relative to ECD for the Ig/{beta} heterodimer, to guide the TMD assembly. Interfacial analyses suggest that the IgM BCR structure we present here may represent a general organizational architecture of all BCR classes. Our studies thus provide a structural platform for understanding B-cell signalling and for designing rational therapies against BCR-mediated diseases.

immunology↗

bioRxiv2022

Humanized antibody potently neutralizes all SARS-CoV-2 variants by a novel mechanism

SARS-CoV-2 Omicron variants have generated a world-wide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of antibodies induced by vaccination. Here, we describe the SARS-CoV-2 neutralizing SP1-77 antibody that was generated from a humanized mouse model with a single human VH1-2 and V{kappa}1-33-associated with immensely diverse complementarity-determining-region-3 (CDR3) sequences. SP1-77 potently and broadly neutralizes SARS-CoV-2 variants of concern and binds the SARS-CoV-2 spike protein receptor-binding-domain (RBD) via a novel-CDR3-based mode. SP1-77 does not block RBD-binding to the ACE2-receptor or endocytosis step of viral entry, but rather blocks membrane fusion. Our findings provide the first mechanistic insight into how a non-ACE2 blocking antibody potently neutralizes SARS-CoV-2, which may inform strategies for designing vaccines that robustly neutralize current and future SARS-CoV-2 variants.

immunology↗

bioRxiv2022

TrieDedup: A fast trie-based deduplication algorithm to handle ambiguous bases in high-throughput sequencing

BackgroundHigh-throughput sequencing is a powerful tool that is extensively applied in biological studies. However, sequencers may produce low-quality bases, leading to ambiguous bases, Ns. PCR duplicates introduced in library preparation should usually be removed in genomics studies, and several deduplication tools have been developed for this purpose. However, two identical reads may appear different due to ambiguous bases and the existing tools cannot address Ns correctly or efficiently. ResultsHere we proposed and implemented TrieDedup, which uses trie (prefix tree) data structure to compare and store sequences. TrieDedup can handle ambiguous base Ns, and efficiently deduplicate at the level of raw sequences. We also reduced its memory usage by approximately 20% by implementing restrictedDict in Python. We benchmarked the performance of the algorithm and showed that TrieDedup can deduplicate reads up to 270-fold faster than pairwise comparison at a cost of 32-fold higher memory usage. ConclusionsTrieDedup algorithm may facilitate PCR deduplication, barcode or UMI assignment and repertoire diversity analysis of large scale high-throughput sequencing datasets with its ultra-fast algorithm that can account for ambiguous bases due to sequencing errors. AvailabilityTrieDedup is available at https://github.com/lolrenceH/TrieDedup

bioinformatics↗

bioRxiv2021

Immunoglobulin enhancers increase RNA polymerase stalling at somatic hypermutation target sequences

Somatic hypermutation (SHM) drives the genetic diversity of immunoglobulin (Ig) genes in activated B cells and supports the generation of antibodies with increased affinity for antigen. SHM is targeted to Ig genes by their enhancers (DIVACs; diversification activators), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase 2 (Pol2) and Pol2 occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol2 or production of full-length transcripts, indicating accumulation of stalled Pol2. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of single-stranded DNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol2 stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

immunology↗

bioRxiv2021

Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells

Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but predominates in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Ligase IV are absolutely required for joining RAG1/2-endonucease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Ligase IV also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Ligase IV-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially, in association with G1-phase down-regulation of Ligase1. Finally, we suggest that differential impacts of Ku-deficiency versus Lig4-deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-defcient developing lymphocyte and neuronal cells. Significance StatementAlternative end-joining (A-EJ) is implicated in oncogenic translocations and mediating DNA double-strand break (DSB) repair in cycling cells when classical nonhomologous endjoining (C-NHEJ) factors of the C-NHEJ Ligase complex are absent. However, V(D)J recombination-associated DSBs that occur in G1 cell cycle-phase progenitor lymphocytes are joined exclusively by the C-NHEJ pathway. Until now, however, the overall mechanisms that join general DSBs in G1-phase progenitor B cells had not been fully elucidated. Here, we report that Ku, a core C-NHEJ double-strand break recognition complex, directs repair of a variety of different targeted DSBs towards C-NHEJ and suppresses A-EJ in G1-phase cells. We suggest this Ku activity explains how Ku-deficiency can rescue the neuronal development and embryonic lethality phenotype of Ligase 4-deficient mice.

genetics↗

bioRxiv2020

An In Vivo Method for Diversifying the Functions of Therapeutic Antibodies

V(D)J recombination generates mature B cells that express huge repertoires of primary antibodies as diverse immunoglobulin heavy (IgH) and light chains (IgL) of their B cell antigen receptors (BCRs). Cognate antigen binding to BCR variable region domains activates B cells into the germinal center (GC) reaction in which somatic hypermutation (SHM) modifies primary variable region-encoding sequences, with subsequent selection for mutations that improve antigen-binding affinity, ultimately leading to antibody affinity maturation. Based on these principles, we developed a humanized mouse model approach to diversify an anti-PD1 therapeutic antibody and allow isolation of variants with novel properties. In this approach, component Ig gene segments of the anti-PD1 antibody underwent de novo V(D)J recombination to diversify the anti-PD1 antibody in the primary antibody repertoire in the mouse models. Immunization of these mouse models further modifies the anti-PD1 antibodies through SHM. Known anti-PD1 antibodies block interaction of PD1 with its ligands to alleviate PD1-mediated T cell suppression, thereby boosting anti-tumor T cell responses. By diversifying one such anti-PD1 antibody, we derived many new anti-PD1 antibodies, including anti-PD1 antibodies with the opposite activity of enhancing PD1/ligand interaction. Such antibodies theoretically might suppress deleterious T cell activities in autoimmune diseases. The approach we describe should be generally applicable for diversifying other therapeutic antibodies. Significance StatementThe immune system is capable of producing enormous varieties of antibodies to counter pathogenic infections. In this study, we devised a method to harness the power of the immune system to produce potentially therapeutic antibodies. As a test, we used this method to generate antibodies against the human PD1 molecule, a negative regulator of T cell activity. Available anti-PD1 therapeutic antibodies inhibit the function of PD1, thereby boosting T cell activity against tumor cells. Through our approach, we diversified one such anti-PD1 antibody in our mouse models. From this work, we obtained multiple new anti-PD1 antibodies, some of which can stimulate, rather than inhibit, PD1 function in vitro. Such PD1 stimulatory antibodies could potentially be used to suppress pathogenic T cell activities in autoimmune diseases. This method could be used to diversify the functions of other therapeutic antibodies.

immunology↗

bioRxiv2020

Physiological role of the 3'IgH CBEs super-anchor in antibody class switching

IgH class switch recombination (CSR) replaces C constant region (CH) exons with one of six downstream CHS by joining transcription-targeted DSBs in the C switch (S) region to DSBs in a downstream S region. Chromatin loop extrusion underlies fundamental CSR mechanisms including 3IgH regulatory region (3IgHRR)-mediated S region transcription, CSR center formation, and deletional CSR joining. There are ten consecutive CTCF binding elements (CBEs) downstream of the 3IgHRR, termed the "3IgH CBEs". Prior studies showed that deletion of eight 3IgH CBEs did not detectably affect CSR. Here, we report that deletion of all 3IgH CBEs impacts, to varying degrees, germline transcription and CSR of upstream S regions, except S{gamma}1. Moreover, deletion of all 3IgH CBEs rendered the 6kb region just downstream highly transcribed and caused sequences within to be aligned with S, broken, and joined to form aberrant CSR rearrangements. These findings implicate the 3IgH CBEs as a critical insulator for focusing loop extrusion-mediated 3IgHRR transcriptional and CSR activities on upstream CH locus targets. SignificanceB lymphocytes change antibody heavy chain (IgH) isotypes by a recombination/deletion process called IgH class switch recombination (CSR). CSR involves introduction of DNA breaks into a donor switch (S) region and also into one of six downstream S regions, with joining of the breaks changing antibody isotype. A chromatin super-anchor, of unknown function, is located just downstream of the IgH locus. We show that complete deletion of this super-anchor variably decreases CSR to most S regions and creates an ectopic S region downstream of IgH locus that undergoes aberrant CSR-driven chromosomal rearrangements. Based on these and other findings, we conclude that the super-anchor downstream of IgH is a critical insulator for focusing potentially dangerous CSR rearrangements to the IgH locus.

immunology↗

bioRxiv2020

Loop Extrusion Mediates Physiological Locus Contraction for V(D)J Recombination

Immunoglobulin heavy chain locus (Igh) VH, D, and JH gene segments are developmentally assembled into V(D)J exons. RAG endonuclease initiates V(D)J recombination by binding a JH-recombination signal sequence (RSS) within a chromatin-based recombination center (RC) and then, in an orientation-dependent process, scans upstream D-containing chromatin presented by cohesin-mediated loop extrusion for convergent D-RSSs to initiate DJH-RC formation1,2. In primary pro-B cells, 100s of upstream VH-associated RSSs, embedded in convergent orientation to the DJH-RC-RSS, gain proximity to the DJH-RC for VH-to-DJH joining via a mechanistically-undefined VH-locus contraction process3-7. Here, we report that a 2.4 mega-base VH locus inversion in primary pro-B cells nearly abrogates rearrangements of normally convergent VH-RSSs and cryptic RSSs, even though locus contraction per se is maintained. Moreover, this inversion activated rearrangement of both cryptic VH-locus RSSs normally in the opposite orientation and, unexpectedly, of normally-oriented cryptic RSSs within multiple, sequential upstream convergent-CBE domains. Primary pro-B cells had significantly reduced transcription of Wapl8, a cohesin-unloading factor, versus levels in v-Abl pro-B lines that lack marked locus contraction or distal VH rearrangements2,9-11. Correspondingly, Wapl depletion in v-Abl lines activated VH-locus contraction and orientation-specific RAG-scanning across the VH-locus. Our findings indicate that locus contraction and physiological VH-to-DJH joining both are regulated via circumvention of CBE scanning impediments.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Conditional Antibody Expression to Avoid Central B Cell Deletion in a Humanized HIV-1 Vaccine Mouse Models

HIV-1 vaccine development aims to elicit broadly neutralizing antibodies (bnAbs) against diverse viral strains. In some HIV-1 infected individuals, bnAbs evolve from precursor antibodies through affinity maturation. To induce bnAbs, a vaccine must mediate a similar process of antibody maturation. One way to test vaccination strategies is to immunize mouse models that express human bnAb precursors. Such immunization experiments can assess whether the vaccine can convert precursor antibody into bnAb. A major problem with such mouse models is that bnAb expression often hinders B cell development in the bone marrow. Such developmental blocks may be attributed to unusual properties of bnAb variable regions, such as poly-reactivity and long antigen-binding loops, which are often under negative selection during primary B cell development. To address this problem, we devised a method to circumvent B cell developmental block by expressing bnAbs conditionally in mature B cells. We validated this method by expressing the unmutated common ancestor (UCA) of the human VRC26 bnAb in transgenic mice. Constitutive expression of combined immunoglobulin heavy and light chains of VRC26UCA led to developmental arrest of B cell progenitors in the bone marrow; poly-reactivity of VRC26UCA and poor pairing of VRC26UCA IgH chain with mouse surrogate light chain may contribute to the phenotype. The conditional expression strategy circumvented this developmental impediment, allowing the VRC26UCA to be expressed in mature peripheral B cells. This method should be generally applicable for expressing other antibodies that are under negative selection during B cell development. Significance statementMouse models can provide fast and cost-effective systems to test HIV-1 vaccine candidates at the pre-clinical stage. To serve this purpose, mouse models are engineered to express the precursors of human bnAbs against diverse HIV-1 strains. Immunization of such mouse models can evaluate the ability of vaccines to mature the precursor antibodies into bnAbs. However, due to unusual properties of bnAbs, mouse models expressing their precursors often have B cell developmental defects. In this study, we devised and validated a strategy to address this problem. This method could also facilitate the expression of other clinically relevant antibodies in mature B cells in transgenic mice; immunization of such mice could be used to generate novel antibodies with desirable properties.

immunology↗