Biology subjects

Rosenberg, A. F.

Publications and source records attributed to Rosenberg, A. F..

12 recordsLinked to original sources

bioRxiv2026

Comprehensive characterization of V(D)J recombination from long-read transcriptomic data with VDJcraft

V(D)J recombination generates antigen receptor diversity in developing B and T cells. Long-read transcriptome technologies (e.g., PacBio Iso-Seq, Nanopore RNA/cDNA) capture full-length transcripts and thus resolve V(D)J events more accurately than short-read platforms. However, existing short-read tools are not applicable to or optimized for long-read data. We developed VDJcraft, the first integrated pipeline designed for V(D)J recombination analysis using long-read transcriptome sequencing data. The workflow uses a two-pass alignment strategy: global alignment to the GENCODE reference with minimap2, followed by local realignment and annotation using the international ImMunoGeneTics information system (IMGT). A customized module enhances D-gene detection sensitivity and positional precision. Sequencing errors are reduced through consensus-based correction toward the predominant subclass. Antigen-binding regions are annotated using IMGT-defined motifs to characterize CDRs and binding site composition. VDJcraft was validated on simulated and Human Genome Structural Variation Consortium (HGSVC) datasets and applied to disease datasets. It accurately recovered full-length V(D)J-C sequences and outperformed existing methods in gene detection and recombination accuracy. Long-read calls also showed significantly higher concordance with high-confidence short-read calls (Mann-Whitney U test, p = 1.55 x 10-4). Additionally, we identified 31 putative novel gene subclasses absent from the IMGT database from HGSVC datasets. Analyses of longitudinal blood samples from a COVID-19 patient revealed distinct V(D)J recombination patterns and segment enrichment, characterized by increased IGHV1-2 usage, enrichment of the IGHV3-7/IGHD6-9/IGHJ5_02 rearranged clonotype, and a transient peak in IgG2 levels at day 4 followed by a gradual return to baseline. In conclusion, VDJcraft provides a robust framework for long-read V(D)J characterization and enables the discovery of disease-associated immune signatures.

bioinformatics↗

bioRxiv2026

Terminally Differentiated Influenza-Specific Effector Memory B Cells Circulate after Live Attenuated Influenza Vaccination

Live attenuated influenza vaccination (LAIV) is the only FDA approved mucosal vaccine. Easily assayed, circulating correlates of protection after LAIV are lacking. Using fluorochrome labeled hemagglutinin (HA) antigen, we previously identified a subset of HA-specific (HA+) IgDneg memory B cells that circulate after inactivated intramuscular vaccination (IIV), express the master transcriptional regulator, T-bet, as well as effector memory genes and predict durable antibody (Ab) responses to IIV. Here we profile the circulating HA+ IgDneg memory B cell response in a cohort of immunized patients who seroconvert after LAIV to identify which, if any, circulating HA+ B cells predict antibody responses after LAIV. Although we report LAIV elicits circulating T-bet+ HA+ IgDneg B cells that are phenotypically similar to those we described after IIV, we find no correlation between the magnitude of these cells and systemic HA-IgG responses after LAIV. Supervised and unsupervised analyses demonstrate that unlike IIV, LAIV preferentially elicits circulating HA+ IgDneg B cells that co-express TBX21 and the terminal effector cell gene, Zeb2. Consistent with their terminal differentiation status, LAIV-elicited T-bet+ cells cannot be recalled as short-lived antibody-secreting cells (ASCs) after systemic or mucosal antigen re-challenge. We conclude that the transcriptional profiles and functions of HA+ IgDneg B cells vary by influenza vaccine platform.

immunology↗

bioRxiv2025

Diversity of motility gene signatures distinguishes commensal Clostridiathat elicit divergent host immune responses

Adaptive immune responses to commensal flagellins are hallmarks of Crohns disease, but it is unclear whether flagellins themselves promote inflammation or whether flagellated commensals can also be colitogenic. Here, we show that the arrangement of motility loci and the diversity of encoded flagellins can separate flagellated gut-derived Clostridia into at least 2 functionally distinct groups. In gnotobiotic mice, both groups induce tolerogenic responses but only one group promoted tissue inflammation following barrier disruption. Accordingly, specific flagellins expressed by members of this pro-inflammatory group displayed a heightened capacity for TLR5 activation which could be altered by modification of a defined region of the flagellin D0 domain. Finally, bacteria belonging to the pro-inflammatory group were found to be elevated in Crohns disease biopsies. Collectively, our study identified key features of specific commensal bacteria that possess colitogenic potential and revealed one mechanism whereby these organisms can potentially initiate intestinal inflammation.

immunology↗

bioRxiv2024

Human Anti-Glycan Reactivity is Driven by the Selection of B cells Utilizing Private Antibody Gene Rearrangements that are Affinity Maturated in Germinal Centers

The human antibody repertoire is broadly reactive with carbohydrate antigens represented in the universe of all living things, including both the host/self-as well as the commensal microflora-derived glycomes. Here we have used BCR receptor cloning and expression together with single-cell transcriptomics to analyze the B cell repertoire to the ubiquitous N-acetyl-D-glucosamine (GlcNAc) epitope in human cohorts and dissect the immune phylogeny of this predominant class of antibodies. We find that circulating anti-GlcNAc B cells exhibiting canonical BMem phenotypes emerge rapidly after birth and couple this observation with evidence for germinal center-dependent affinity maturation of carbohydrate-specific B cell receptors in situ during early childhood. Direct analysis of individual B cell clonotypes reveals they exhibit strikingly distinct fine-specificity profiles for palettes of GlcNAc containing moieties. These results suggest that a generalized exposure to complex environmental glycans drives the steady state anti-glycan repertoire.

immunology↗

bioRxiv2023

Crohn's patients and healthy infants share immunodominant B cell response to commensal flagellin peptide epitopes

About half of patients with Crohns disease (CD) develop selective serum IgG response to flagellin proteins of the Lachnospiraceae family. Here, we identified a dominant B cell peptide epitope in CD, locating in the highly conserved "hinge region" between the D0 and D1 domains at the amino-terminus of Lachnospiraceae flagellins. Serum IgG reactive to this epitope is present at an elevated level in adult CD patients and in pediatric CD patients at diagnosis. Most importantly, high levels of serum IgG to the hinge epitope were found in most infants from 3 different geographic regions (Uganda, Sweden, and the USA) at one year of age. This vigorous homeostatic response decrements with age as it is not present in healthy adults. These data identify a distinct subset of CD patients, united by a shared reactivity to this dominant flagellin epitope that may represent failure of a homeostatic response beginning in infancy.

immunology↗

bioRxiv2023

Alloreactivity and autoreactivity converge to support B cell epitope targeting in transplant rejection

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. The rules governing the immunogenicity of non-self donor HLA are poorly understood. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific B cell response in the kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We observed an immunodominant B cell antibody response focused on topographically exposed, solvent-accessible mismatched HLA residues along the peptide-binding groove - a subregion comprising only 20% of the HLA molecule. We further demonstrated that, even within a diverse cohort of transplant recipients, the B cell alloresponse consistently converges on this same immunodominant subregion on the crown of the HLA molecule. Based on these findings, we propose that B cell immunodominance in transplant rejection relies on antigenic topography, and we suggest that this link could be exploited for organ matching and therapeutics.

immunology↗

bioRxiv2023

A high-throughput multiplex array for antigen-specific serology with automated analysis.

High-throughput customizable CBA automated script-based analysisThe utility of high-throughput systems to evaluate antigen-specific antibody (Ab) has been highlighted by the SARS-CoV-2 pandemic. Pathogen-specific Ab levels are often used to assess protection following vaccination and, in the case of novel pathogens, an indication of prior exposure. Several platforms exist to visualize antigen-specific Ab, however most are not quantitative and are difficult to scale for population level studies. Additionally, the sensitivity across platforms differs making direct comparisons between studies difficult. Cytometric bead arrays (CBA) are an attractive platform for antigen-specific Ab measurements as they can be used to assess Ab reactivity against several antigens and of several isotypes to be performed simultaneously. Additionally, CBAs exhibit high sensitivity and can be designed to provide quantitative measurements. Using commercially available particles, a biotin-Streptavidin bead loading strategy, and the inclusion of indirect standards, we describe a flexible system that can be modified to include a variety of antigens. Here we describe two arrays, focused on antigens derived from either {beta}-coronaviruses or influenza virus. To support the high-throughput capacity of this system, we developed a suit of automated software tools, the CBA Toolbox, to process raw data into antigen-reactive IgM, IgA, and IgG concentrations. We describe quality control requirements, assay performance, and normalizations to accurately quantitate antigen-specific Ig.

immunology↗

bioRxiv2022

IL-6 prevents Th2 cell polarization by promoting SOCS3-dependent suppression of IL-2 signaling

Defective interleukin-6 (IL-6) signaling has been associated with Th2 bias and elevated IgE. However, the underlying mechanism by which IL-6 prevents the development of Th2-driven diseases remains unknown. Using a model of house-dust-mite (HDM)-induced Th2 differentiation and allergic airway inflammation, we show that IL-6 signaling in allergen-specific T cells was required to prevent Th2 development and subsequent IgE response and allergic inflammation. Th2 cell lineage commitment required strong sustained IL-2 signaling. Importantly, we found that IL-6 turned off IL-2 signaling during early T cell activation and thus inhibited Th2 priming. Mechanistically, we found that IL-6-driven inhibition of IL-2 signaling in responding T cells was mediated by upregulation of Suppressor Of Cytokine Signaling 3 (SOCS3). This mechanism could be mimicked by pharmacological Janus Kinase-1 (JAK1) inhibition. Collectively, our results identify an unrecognized mechanism that prevents the development of unwanted Th2 cell responses and associated diseases and outline potential preventive interventions.

immunology↗

bioRxiv2022

Chitinase-3-like 1 regulates TH2 cells, TFH cells and IgE following helminth infection

Data from patient cohorts and mouse models of atopic dermatitis, food allergy and asthma strongly support a role for the chitinase-like protein ChI3L1 in allergic disease. To address whether CHI3L1 also contributes to TH2 responses following nematode infection, we infected Chi3l1-/- mice with Heligmosomoides polygyrus (Hp) and analyzed T cell responses. Not surprisingly, we observed impaired TH2 responses in Hp-infected Chi3l1-/- mice. However, we also found that T cell intrinsic expression of Chi3l1 was required for ICOS upregulation following activation of naive CD4 T cells and was necessary for the development of the IL-4+ TFH subset, which supports germinal center (GC) B cell reactions and IgE responses. The requirement for Chi3l1 in TFH and IgE responses was also seen following alum-adjuvanted vaccination. While Chi3l1 was critical for IgE humoral responses it was not required for vaccine or infection induced IgG1 responses. These results suggest that Chi3l1 specifically modulates IgE responses that are highly dependent on help from IL-4-producing TFH cells.

immunology↗

bioRxiv2021

Mixed Origins: HIV gp120-specific memory develops from pre-existing memory and naive B cells following vaccination in humans

The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naive B cell receptor, fail to bind Env suggesting that the initial responding B cell population is not exclusively comprised of a naive population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants in HVTN 105, an HIV vaccine trial. Using deep sequencing VH-lineage tracking we identified several of these mAb lineages in pre-immune peripheral blood. Several of these pre-immune lineages also persisted in the bone marrow, including CD138+ long-lived plasma cell compartment, [~]7 months after the final vaccination. The majority of the pre-immune lineage members included IgM, however IgG and IgA members were also prevalent and exhibited somatic hypermutation. These results suggest that vaccine-induced gp120-specific antibody lineages originate from both naive and cross-reactive memory B cells.

immunology↗

bioRxiv2021

A Non-redundant Role for T cell-derived IL-22 in Antibacterial Defense of Colonic Crypts

IL-22 is a key cytokine in immune defense against pathogens at barrier sites. In response to enteric attaching and effacing bacteria, IL-22 produced by type 3 innate lymphoid cells (ILC3s) is thought to be important early for induction of antimicrobial peptides (AMPs) that protect intestinal epithelial cells (IECs) in advance of T cell-derived IL-22 that arises later. Yet, the basis for a requirement for both innate and adaptive IL-22-producing immune cells in protecting the intestinal mucosa is unknown. Here, using novel mice that both report IL-22 expression and can be targeted for its lineage-specific deletion, we show that mice with deficiency of IL-22 targeted to innate immune cells, including ILC3s, have impaired STAT3 activation of surface colonic IECs colonized by bacteria early in infection. In contrast, mice with IL-22 deficiency limited to T cells have complete loss of STAT3 activation in IECs lining colonic crypts and fail to protect the crypts from bacterial invasion late despite ongoing production of IL-22 from ILC3s. T cell-derived IL-22 is required for upregulation of many host-protective genes by crypt IECs, including those encoding AMPs, neutrophil-recruiting chemokines, and mucins and mucin-related molecules, while also restricting pro-inflammatory genes downstream of IFN{gamma} and TNF signals. Thus, T cell-derived IL-22 is indispensable for antibacterial defense and damage control of intestinal crypts.

immunology↗

bioRxiv2019

Complex responses to inflammatory oxidants by the probiotic bacterium Lactobacillus reuteri

Inflammatory diseases of the gut are associated with increased intestinal oxygen concentrations and high levels of inflammatory oxidants, including hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), which are antimicrobial compounds produced by the innate immune system. This contributes to dysbiotic changes in the gut microbiome, including increased populations of pro-inflammatory enterobacteria (Escherichia coli and related species) and decreased levels of health-associated anaerobic Firmicutes and Bacteroidetes. The pathways for H2O2 and HOCl resistance in E. coli have been well-studied, but little is known about how commensal and probiotic bacteria respond to inflammatory oxidants. In this work, we have characterized the transcriptomic response of the anti-inflammatory, gut-colonizing Gram-positive probiotic Lactobacillus reuteri to both H2O2 and HOCl. L. reuteri mounts distinct responses to each of these stressors, and both gene expression and survival were strongly affected by the presence or absence of oxygen. Oxidative stress response in L. reuteri required several factors not found in enterobacteria, including the small heat shock protein Lo18, polyphosphate kinase 2, and RsiR, an L. reuteri-specific regulator of anti-inflammatory mechanisms. These results raise the intriguing possibility of developing treatments for inflammatory gut diseases that could sensitize pro-inflammatory enterobacteria to killing by the immune system while sparing anti-inflammatory, health-associated species.\n\nIMPORTANCEIt is becoming increasingly clear that effective treatment of inflammatory gut diseases will require modulation of the gut microbiota. Preventing pro-inflammatory bacteria from blooming while also preserving anti-inflammatory and commensal species is a considerable challenge, but our results suggest that it may be possible to take advantage of differences in the way different species of gut bacteria resist inflammatory oxidants to accomplish this goal.

microbiology↗