Biology subjects

Hoehn, K. B.

Publications and source records attributed to Hoehn, K. B..

11 recordsLinked to original sources

bioRxiv2026

Maternal vaccination timing shapes the composition and maturation of antibody clonotypes transferred to the newborn

Newborns rely on maternally transferred antibodies for immune protection, acquired across the placenta and through breast milk. Despite this importance, how closely the inherited antibody repertoire resembles the mothers has not been examined at the clonotypic level. We combined BCR-Seq with proteomic Ig-Seq to track SARS-CoV-2-specific antibody clonotypes across maternal blood, cord blood, and breast milk from six mRNA-immunized pregnant individuals. Vaccination earlier in gestation generated more diverse peak IgG repertoires but greater contraction before delivery, yielding fewer transferred clonotypes. Vaccination later in gestation produced more restricted peak repertoires, but more clonotypes persisted to delivery and transferred to cord. Infant cord was enriched for persistent, highly somatically mutated, and intraclonally diverse IgG clonotypes, consistent with preferential transfer of affinity-matured antibodies. In contrast, breast milk IgA repertoires were largely distinct from systemic repertoires and underwent substantial remodeling despite stable antigen titers. These findings define molecular determinants of passive immunity shaped by vaccination timing relevant to optimizing maternal immunization.

immunology↗

bioRxiv2026

Within-host population structure, migration, and parallel adaptive evolution of Pseudomonas aeruginosa in cystic fibrosis lung disease

Pseudomonas aeruginosa infections in adults with cystic fibrosis (CF) are comprised of heterogeneous populations, most often tracing ancestry back to a single recent common ancestor. What is not clear is the physical spatial structure within the lung infection population, its stability over time and whether this physical structure leads to different evolutionary trajectories in different adaptive environments. To compare the P. aeruginosa populations across a single lung, we performed whole genome sequence analyses of 450 isolates recovered from lavage samples of the three different lobes of the right lung from a person with mild-to-moderate CF lung disease at three time points over the course of [~]1.5 years. We found that isolates fell into five distinct phylogenetic lineages with evidence for repeated translocation of isolates from different lineages across lobes and loss-of-function mutations in lasR and mucA were present in all 450 isolates. The well-resolved phylogenetic analyses revealed a structured population in which we find the coexistence of a slowly evolving lineage and more rapidly evolving lineages. There is also support for numerous migration events. Further, strong evidence for parallel adaptive mutations in multiple genes revealed distinct evolutionary paths affecting mucoid phenotypes and genetic variation in antibiotic resistance-associated pathways across coexisting populations within a single individual over time. These results provide an example of within-host evolution leading to microheterogeneity that may be useful to consider in future study of infection metapopulations dynamics over the course of chronic infection. IMPORTANCEIndividuals with cystic fibrosis (CF) commonly have chronic lung infections that contain clonally derived Pseudomonas aeruginosa populations with genotypic and phenotypic diversity. This study describes a substantial dataset containing 450 isolates from different lobes of the right lung across three timepoints from an individual with mild-to-moderate CF lung disease. Some regional enrichment for specific lineages with parallel mutations among individual lobes of the lung was observed, but longitudinal analysis also demonstrated that compartmentalization is not strictly maintained and that isolates migrate between lobes of the lung over time. Perspectives on within lung evolution will be important for understanding the pathogen populations in chronic respiratory infections in CF and other diseases.

microbiology↗

bioRxiv2025

TyCHE enables time-resolved lineage tracing of heterogeneously-evolving populations

Phylogenetic methods for cell lineage tracing have driven significant insights into organismal development, immune responses, and tumor evolution. While most methods estimate mutation trees, time-resolved lineage trees are more interpretable and could relate events like cellular migration and differentiation to perturbations like vaccines and drug treatments. However, somatic mutation rates vary dramatically by cell type, significantly biasing existing methods. We introduce TyCHE (Type-linked Clocks for Heterogeneous Evolution), a Bayesian phylogenetics package that infers time-resolved phylogenies of populations with distinct evolutionary rates. We demonstrate that TyCHE improves tree accuracy using a new simulation package SimBLE (Simulator of B cell Lineage Evolution). We use TyCHE to infer patterns of memory B cell differentiation during HIV infection, dynamics of recall germinal centers following influenza vaccination, evolution of a glioma tumor lineage, and progression of a bacterial lung infection. TyCHE and SimBLE are available as open-source software packages compatible with the BEAST2 and Immcantation ecosystems.

immunology↗

bioRxiv2025

Pre-Omicron Immunity Generates IgG⁺ but Not IgA⁺ Memory B Cells Reactive to Omicron Spike Protein

The emergence of the Omicron variant marked a major antigenic shift from previous SARS-CoV-2 variants. While the emergence of new variants of concern was associated with loss of protection from infection, protection from serious disease was maintained. What was less clear was whether this protection was associated with expansion, affinity maturation of pre-existing cross-reactive B cells, or de novo generation of variant-specific B cells. To define the cross-reactivity of memory B cell populations generated through infection and/or vaccination with pre-Omicron variant spike, we utilised archived samples from healthcare workers enrolled in the extensively monitored Panther study cohort. Using samples collected from Omicron-naive individuals approximately three months (106 days) after a third BNT162b2 vaccine dose, we analyzed the spike-specific B cell repertoire via multiparameter flow cytometry and B cell receptor (BCR) sequencing. Reactivity to both Lineage A and Omicron spike proteins was higher for serum IgG than IgA, and serum neutralisation potency was significantly reduced against pseudoviruses bearing the Omicron spike compared to those with Lineage A spike. The frequency of Omicron-reactive IgA+ B cells was markedly lower than that of Lineage A-reactive cells, whereas Omicron-reactive IgG+ memory B cells were more frequent. These responses were predominantly localized to classical memory and double-negative (DN) B cell subsets. BCR sequencing of donors unexposed to Omicron confirmed that Omicron-binding clones were class-switched and somatically mutated, indicating they originated from pre-existing memory B cells primed by vaccination or infection. Our findings demonstrate that memory B cells generated through infection and/or immunisation with pre-Omicron variants were primed for broad antigenic recognition despite Omicrons extensive genetic divergence. However, the diminished IgA+ B cell frequency suggests a weakened mucosal antibody defence, potentially explaining the continued susceptibility to infection despite preserved protection from severe disease.

immunology↗

bioRxiv2023

Inferring B cell phylogenies from paired heavy and light chain BCR sequences with Dowser

Antibodies are vital to human immune responses and are composed of genetically variable heavy and light chains. These structures are initially expressed as B cell receptors (BCRs). BCR diversity is shaped through somatic hypermutation and selection during immune responses. This evolutionary process produces B cell clones, cells that descend from a common ancestor but differ by mutations. Phylogenetic trees inferred from BCR sequences can reconstruct the history of mutations within a clone. Until recently, BCR sequencing technologies separated heavy and light chains, but advancements in single cell sequencing now pair heavy and light chains from individual cells. However, it is unclear how these separate genes should be combined to infer B cell phylogenies. In this study, we investigated strategies for using paired heavy and light chain sequences to build phylogenetic trees. We found incorporating light chains significantly improved tree accuracy and reproducibility across all methods tested. This improvement was greater than the difference between tree building methods and persisted even when mixing bulk and single cell sequencing data. However, we also found that many phylogenetic methods estimated significantly biased branch lengths when some light chains were missing, such as when mixing single cell and bulk BCR data. This bias was eliminated using maximum likelihood methods with separate branch lengths for heavy and light chain gene partitions. Thus, we recommend using maximum likelihood methods with separate heavy and light chain partitions, especially when mixing data types. We implemented these methods in the R package Dowser: https://dowser.readthedocs.io.

immunology↗

bioRxiv2021

The Physiological Landscape and Specificity of Antibody Repertoires

Diverse antibody repertoires spanning multiple lymphoid organs (e.g., bone marrow, spleen, lymph nodes) form the foundation of protective humoral immunity. Changes in their composition across lymphoid organs are a consequence of B-cell selection and migration events leading to a highly dynamic and unique physiological landscape of antibody repertoires upon antigenic challenge (e.g., vaccination). However, to what extent B cells encoding identical or similar antibody sequences (clones) are distributed across multiple lymphoid organs and how this is shaped by the strength of a humoral response, remains largely unexplored. Here, we performed an in-depth systems analysis of antibody repertoires across multiple distinct lymphoid organs of immunized mice, and discovered that organ-specific antibody repertoire features (e.g., germline V-gene usage and clonal expansion profiles) equilibrated upon a strong humoral response (multiple immunizations and high serum titers). This resulted in a surprisingly high degree of repertoire consolidation, characterized by highly connected and overlapping B-cell clones across multiple lymphoid organs. Finally, we revealed distinct physiological axes indicating clonal migrations and showed that antibody repertoire consolidation directly correlated with antigen-specificity. Our study uncovered how a strong humoral response resulted in a more uniform but redundant physiological landscape of antibody repertoires, indicating that increases in antibody serum titers were a result of synergistic contributions from antigen-specific B-cell clones distributed across multiple lymphoid organs. Our findings provide valuable insights for the assessment and design of vaccine strategies.

immunology↗

bioRxiv2021

Elevated N-glycosylation of immunoglobulin G variable regions in myasthenia gravis highlights a commonality across autoantibody-associated diseases

Elevated N-linked glycosylation of immunoglobulin G variable regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design included adaptive immune receptor repertoire sequencing to quantify and characterize N-glycosylation sites in the global B cell receptor repertoire, proteomics to examine glycosylation patterns of the circulating IgG, and production of human-derived recombinant autoantibodies, which were studied with mass spectrometry and antigen binding assays to confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the B cell repertoire of MG patients when compared to healthy donors. Motifs were introduced by both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the circulating IgG in a subset of MG patients. Autoantigen binding, by patient-derived MG autoantigen-specific monoclonal antibodies with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of variable region N-linked glycosylation in autoimmunity to MG subtypes. Although occupied IgG-VN-Glyc motifs are found on MG autoantigen-specific monoclonal antibodies, they are not required for binding to the autoantigen in this disease.

immunology↗

bioRxiv2021

Human B cell lineages engaged by germinal centers following influenza vaccination are measurably evolving

Poor efficacy of seasonal influenza virus vaccines is often attributed to pre-existing immunity interfering with the persistence and maturation of vaccine-induced B cell responses.1 Consistent with this notion, no significant increase in somatic hypermutation (SHM) among circulating influenza-binding lineages was detected following seasonal vaccination in humans.2 A more recent study showed that at least a subset of vaccine-induced B cell lineages are recruited into germinal centers (GCs) following vaccination, suggesting that affinity maturation of these lineages can occur.3 Crucially, however, it has not been demonstrated whether these GC-engaged lineages actually accumulate additional SHM. Here, we address this point using a phylogenetic test of measurable evolution. We first validate this test through simulations and demonstrate measurable B cell evolution in known examples of affinity maturation such as the response to HIV infection. We then show that lineages in the blood are rarely measurably evolving following influenza vaccination, but that GC-engaged lineages - likely derived from memory B cells - are frequently measurably evolving. These findings confirm that seasonal influenza virus vaccination can stimulate additional SHM among responding B cell lineages.

immunology↗

bioRxiv2020

Phylogenetic analysis of migration, differentiation, and class switching in B cells

B cells undergo rapid mutation and selection for antibody binding affinity when producing antibodies capable of neutralizing pathogens. This evolutionary process can be intermixed with migration between tissues, differentiation between cellular subsets, and switching between functional isotypes. B cell receptor (BCR) sequence data has the potential to elucidate important information about these processes. However, there is currently no robust, generalizable framework for making such inferences from BCR sequence data. To address this, we develop three parsimony-based summary statistics to characterize migration, differentiation, and isotype switching along B cell phylogenetic trees. We use simulations to demonstrate the effectiveness of this approach. We then use this framework to infer patterns of cellular differentiation and isotype switching from high throughput BCR sequence datasets obtained from patients in a study of HIV infection and a study of food allergy. These methods are implemented in the R package dowser, available at https://bitbucket.org/kleinstein/dowser. Author summaryB cells produce high affinity antibodies through an evolutionary process of mutation and selection during adaptive immune responses. Migration between tissues, differentiation to cellular subtypes, and switching between different antibody isotypes can be important factors in shaping the role B cells play in response to infection, autoimmune disease, and allergies. B cell receptor (BCR) sequence data has the potential to elucidate important information about these processes. However, there is currently no robust, generalizable framework for making such inferences from BCR sequence data. Here, we develop three parsimony-based summary statistics to characterize migration, differentiation, and isotype switching along B cell phylogenetic trees. Using simulations, we confirm the effectiveness of our approach, as well as identify some caveats. We further use these summary statistics to investigate patterns of cellular differentiation in three HIV patients, and patterns of isotype switching in an individual with food allergies. Our methods are released in the R package dowser: https://bitbucket.org/kleinstein/dowser.

immunology↗

bioRxiv2019

Single-cell immune repertoire tracing identifies rituximab refractory B cells that emerge during relapse

Rituximab, a B cell-depleting therapy, is indicated for treating a growing number of autoantibody-mediated autoimmune disorders. However, relapses can occur after treatment and autoantibody-producing B cell subsets may be found during relapses. It is not understood if these autoantibody-producing B cell subsets emerge from the failed depletion of pre-existing B cells or are re-generated de novo. To further define the mechanisms that cause post-rituximab relapse, we studied patients with autoantibody-mediated muscle-specific kinase (MuSK) myasthenia gravis (MG) who relapsed after treatment. We carried out single-cell transcriptional and B cell receptor (BCR) profiling on longitudinal B cell samples. We identified clones present prior to therapy that continued to persist during relapse. Persistent B cell clones included both antibody-secreting cells and memory B cells characterized by gene expression signatures associated with B cell survival. A subset of persistent antibody-secreting cells and memory B cells were specific for the MuSK autoantigen. These results demonstrate that rituximab is not fully effective at eliminating autoantibody-producing B cells and provide a mechanistic understanding of post-rituximab relapse in MuSK MG.

immunology↗

bioRxiv2019

Repertoire-wide phylogenetic models of B cell molecular evolution reveal evolutionary signatures of aging and vaccination

In order to produce effective antibodies, B cells undergo rapid somatic hypermutation (SHM) and selection for binding affinity to antigen via a process called affinity maturation. The similarities between this process and evolution by natural selection have led many groups to use evolutionary and phylogenetic methods to characterize the development of immunological memory, vaccination, and other processes that depend on affinity maturation. However, these applications are limited by several features of affinity maturation that violate assumptions in standard phylogenetic models. Further, most phylogenetic models are designed to be applied to individual lineages comprising genetically diverse sequences, while B cell repertoires often consist of hundreds to thousands of separate low-diversity lineages. Here, we introduce a hierarchical phylogenetic framework that incorporates the unique features of SHM, and integrates information from all lineages in a repertoire to more precisely estimate model parameters. We demonstrate the power of this approach by characterizing previously un-described phenomena in affinity maturation. First, we find evidence consistent with age related changes in SHM hot-and cold-spot motifs. Second, we identify a consistent relationship between increased tree length and signs of increased negative selection, apparent in the repertoires of both healthy subjects and those undergoing active immune responses. This suggests that B cell lineages shift towards negative selection over time as a general feature of affinity maturation. Our study provides a framework for undertaking repertoire-wide phylogenetic testing of SHM hypotheses, and provides a new means of charactering the process of mutation and selection during affinity maturation.

immunology↗