Frederick W Alt

Influenza viruses escape immunity owing to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. We found that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 bound and disengaged CD19 from its chaperone CD81, permitting CD19 to translocate to the B cell receptor complex to trigger signaling. Moreover, Gb3 regulated major histocompatibility complex class II expression to increase diversity of T follicular helper and GC B cells reactive with subdominant epitopes...

UNLABELLED: Influenza viruses escape immunity due to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. Here, we demonstrate that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 binds and disengages CD19 from its chaperone CD81 for subsequent translocation to the B cell receptor (BCR) complex to trigger signaling...

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 JH s 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...

UNLABELLED: Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from V H , D, and J H gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a J H -based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to J H s to form a DJ H -RC. Igh has a provocative number and organization of CTCF-binding-elements (CBEs) that can impede loop extrusion...

Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities...

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is prevalent and may affect cognitive function. We studied associations of NAFLD with risk of cognitive impairment. Secondarily we evaluated liver biomarkers (alanine aminotransferase (ALT), aspartate aminotransferase (AST), their ratio, and gamma-glutamyl transpeptidase). METHODS: In a prospective cohort study, the REasons for Geographic and Racial Differences in Stroke, among 30,239 black and white adults aged ≥45,495 cases of incident cognitive impairment were identified over 3...

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 nontemplated 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...

The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M, D, G, E or A for antigen recognition1-3 and a disulfide-linked Igα and Igβ heterodimer (Igα/β) that functions as the signalling entity through their intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5 . The organizing principle of the BCR remains elusive. Here we report cryogenic electron microscopy structures of mouse full-length IgM BCR at 8.2 Å resolution and its Fab-deleted form at 3...

After nearly four decades of research, a safe and effective HIV-1 vaccine remains elusive. There are many reasons why the development of a potent and durable HIV-1 vaccine is challenging, including the extraordinary genetic diversity of HIV-1 and its complex mechanisms of immune evasion. HIV-1 envelope glycoproteins are poorly recognized by the immune system, which means that potent broadly neutralizing antibodies (bnAbs) are only infrequently induced in the setting of HIV-1 infection or through vaccination...

SARS-CoV-2 Omicron subvariants have generated a worldwide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage Omicron subvariants and prepare for new ones, additional means of isolating broad and potent humanized SARS-CoV-2 neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human VH 1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC)...

SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF...

Immunoglobulin class switch recombination (CSR) plays an important role in humoral imm\une responses by changing the effector functions of antibodies. CSR occurs between highly repetitive switch (S) sequences located upstream of immunoglobulin constant gene exons. Switch sequences differ in size, the nature of their repeats, and the density of the motifs targeted by the activation-induced cytidine deaminase (AID), the enzyme that initiates CSR. CSR involves double-strand breaks (DSBs) at the universal Sµ donor region and one of the acceptor S regions...

In activated B cells, activation-induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C-terminal peptide. In immunodeficient-patient cells expressing mutant AID lacking its C-terminus, a catalytically active AID-delC protein accumulates in the nucleus but nevertheless fails to support CSR...

Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species1,2 . However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments...

BACKGROUND: We have recently reported a mouse model of PN-associated cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of canalicular bile acid, bilirubin and sterol transporters Abcb11, Abcc2 and Abcg5/8. The aim of this study was to examine the role of TNFα in promoting PNAC in mice. METHODS: First, recombinant TNFα was administered to mice as well as in hepatocyte cell culture...

Although axonal damage induces rapid changes in gene expression in primary sensory neurons, it remains unclear how this process is initiated. The transcription factor ATF3, one of the earliest genes responding to nerve injury, regulates expression of downstream genes that enable axon regeneration. By exploiting ATF3 reporter systems, we identify topoisomerase inhibitors as ATF3 inducers, including camptothecin. Camptothecin increases ATF3 expression and promotes neurite outgrowth in sensory neurons in vitro and enhances axonal regeneration after sciatic nerve crush in vivo...

Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand 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/Lig4 are absolutely required for joining RAG1/2 endonuclease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways...

V(D)J recombination generates mature B cells that express huge repertoires of primary antibodies as diverse immunoglobulin (Ig) heavy chain (IgH) and light chain (IgL) of their B cell antigen receptors (BCRs). Cognate antigen binding to BCR variable region domains activates B cells into the germinal center (GC) reaction in which somatic hypermutation (SHM) modifies primary variable region-encoding sequences, with subsequent selection for mutations that improve antigen-binding affinity, ultimately leading to antibody affinity maturation...

Vaccine elicitation of broadly neutralizing antibodies (bnAbs) is a key HIV-research goal. The VRC01 class of bnAbs targets the CD4-binding site on the HIV-envelope trimer and requires extensive somatic hypermutation (SHM) to neutralize effectively. Despite substantial progress, vaccine-induced VRC01-class antibodies starting from unmutated precursors have exhibited limited neutralization breadth, particularly against viruses bearing glycan on loop D residue N276 (glycan276), present on most circulating strains...

RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a JH -recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs1,2 . The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3 . Scanning of RAG from the DJH -RC-RSS to upstream convergent VH -RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2-5 ...