Program
The 2025 Immcantation Users Group Meeting will take place on January 30th, 2025.
Note that all times shown here use ET.
Abstracts
Influenza Vaccine Format Mediates Distinct Cellular and Antibody Responses in Human Immune Organoids
Jenna M. Kastenschmidt1, Suhas Sureshchandra2, Aarti Jain2, Jenny E. Hernandez-Davies2, Rafael de Assis2, Zachary W. Wagoner1, Andrew M. Sorn1, Mahina Tabassum Mitul1, Aviv I Benchorin1, Elizabeth Levendosky3, Gurpreet Ahuja4, Qiu Zhong4, Douglas Trask5, Jacob Boeckmann5, Rie Nakajima2, Algimantas Jasinskas2, Naresha Saligrama3, D Huw Davies2, Lisa E. Wagar1*
1Department of Physiology & Biophysics; Institute for Immunology; Center for Virus Research; Vaccine R&D Center, University of California Irvine, Irvine, CA 92617, USA; 2Vaccine R&D Center, University of California Irvine, Irvine, CA 92617, USA; 3Department of Neurology, Washington University School of Medicine in St. Louis, MO 63112, USA; 4Department of Pediatric Otolaryngology, Children’s Hospital of Orange County, Orange, CA 92868, USA; 5Department of Otolaryngology-Head and Neck Surgery, University of California Irvine, Orange, CA 92868, USA. *Corresponding author.
Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying responses to different antigen formats. In this study, we sought to understand how antigen-specific B and T cells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to inactivated (IIV) and live-attenuated influenza vaccines (LAIV) compared to wild-type influenza A virus. Our analyses suggest that LAIV elicited an early and robust type I interferon response, stronger B cell differentiation, higher HA+ B cells, and a more robust neutralizing antibody response than IIV or wild-type virus. We used several Immcantation packages to define how each antigen format elicited distinct B cell responses, including their magnitude, diversity, and breadth. Change-O was used to assign V(D)J annotations and clones were further defined with SCOPer. Size of B cell clonal families were calculated using Alakazam, while mutational frequencies within B cell subsets estimated using SHazaM. We demonstrate that LAIV recruits B cells from both naïve and memory pools, resulting in a diversified response with broader epitope targeting and increased BCR diversity over time. In contrast, antigen-specific responses to IIV primarily relied on pre-existing memory B cells, resulting in early class switching and higher somatic hypermutation rates. These observations emphasize the importance of antigen format and suggest that LAIV may provide superior and diversified protection against influenza.
Integrative Bioinformatics for Characterizing Atypical Memory B Cells Through Single-Cell RNA-seq in COVID-19
Melissa Garcia-Vega1, Mara Anais Llamas-Covarrubias2, Martin Loza3, Monica Resendiz-Sandoval1, Diana Hinojosa-Trujillo1, Edgar Melgoza-Gonzalez1, Olivia Valenzuela4, Veronica Mata-Haro5, Miguel Hernandez-Oñate6, Alan Soto-Gaxiola7, Karina Chavez-Rueda8, Kenta Nakai3*, Jesus Hernandez1*
1Laboratorio de Inmunología, Centro de Investigación en Alimentación y Desarrollo, A.C, Hermosillo, Sonora, Mexico; 2Research Institute for Microbial Diseases, Osaka University, Suita, Japan; 3The Institute of Medical Science, The University of Tokyo, Minato‐ku, Tokyo, Japan; 4Departamento de Ciencias Químico Biológicas, División de Ciencias Biológicas y de la Salud, Universidad de Sonora, Hermosillo, Sonora, Mexico; 5Laboratorio de Microbiología e Inmunología, Centro de Investigación en Alimentación y Desarrollo, A.C, Hermosillo, Sonora, Mexico; 6Laboratorio de Fisiología y Biología Molecular de Plantas, Centro de Investigación en Alimentación y Desarrollo, A.C, Hermosillo, Sonora, Mexico; 7Hospital General del Estado de Sonora “Dr. Ernesto Ramos Bours”, Hermosillo, Sonora, Mexico; 8Unidad de Investigación Médica en Inmunología, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, Mexico. *Corresponding author.
Here we performed single-cell RNA sequencing of S1 and RBD protein-specific B cells from convalescent COVID-19 patients with different clinical manifestations. This study aimed to evaluate the role and developmental pathways of atypical memory B cells (MBCs) in response to SARS-CoV-2 infection. We employed bioinformatics tools to conduct multiple analyses, including the Immcantation framework particularly Change-O for VDJ alignment, clonal assignment, and germline reconstruction, along with SHazaM for analyzing somatic hypermutations as part of repertoire characterization. Additional analyses included differential gene expression, pathway enrichment, and trajectory analysis to further explore B cell responses. The results revealed a proinflammatory signature across B cell subsets associated with disease severity, as evidenced by the upregulation of genes such as GADD45B, MAP3K8, and NFKBIA critical and severe individuals. The repertoire analysis demonstrated variations in somatic hypermutation rates and gene usage between atypical and conventional MBCs. Furthermore, the analysis of atypical MBCs suggested a developmental pathway similar to that of conventional MBCs through germinal centers, as indicated by the expression of several genes involved in germinal center processes, including CXCR4, CXCR5,BCL2, and MYC. Additionally, the upregulation of genes characteristic of the immune response in COVID-19, such as ZFP36 and DUSP1, suggested that the differentiation and activation of atypical MBCs may be influenced by exposure to SARS-CoV-2 contributing to the immune response necessary for COVID-19 recovery. Our study contributes to a better understanding of atypical MBCs in COVID-19 and the role of other B cell subsets across different clinical manifestations.
Funding Information: Consejo Nacional de Ciencia y Tecnología grant 312677
B3E-Seq: Full-Length BCR Sequencing from 3’-Barcoded scRNA-seq
Y. Jason Zhang1,2, Duncan M. Morgan1,3, Darrell J. Irvine1,2, J. Christopher Love1,3,*
1Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA.; 2Department of Biological Engineering, MIT, Cambridge, MA, USA.; 3Department of Chemical Engineering, MIT, Cambridge, MA, USA. *Corresponding author
Single-cell RNA sequencing (scRNA-seq) can resolve transcriptional features from individual cells, but scRNA-seq techniques capable of resolving the variable regions of B cell receptors (BCRs) remain limited, especially from widely-used 3’-barcoded libraries. Here, we report a method that can recover heavy/light chain paired, full-length variable region sequences of BCRs from 3’-barcoded scRNA-seq libraries. We first verify this method (B3E-seq) can produce accurate, full-length BCR sequences. We then apply this method to profile B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in five infant rhesus macaques. We identify BCR features associated with specificity for the ST3 antigen which are present in multiple vaccinated monkeys, indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and profile antigen-specific responses elicited by vaccination.
Funding Information: The authors declare the following competing interests: J.C.L. has interests in Sunflower Therapeutics PBC, Honeycomb Biotechnologies, OneCyte Biotechnologies, SQZ Biotech, Alloy Therapeutics, QuantumCyte, Amgen, and Repligen (these interests are reviewed and managed under Massachusetts Institute of Technology’s policies for potential conflicts of interest); in addition, he receives sponsored research support at Massachusetts Institute of Technology from Amgen, the Bill and Melinda Gates Foundation, Biogen, Pfizer, Sartorius, Mott Corp, TurtleTree, Takeda, and Sanofi, and his spouse is an employee of Sunflower Therapeutics. Pfizer was involved in the design, analysis, and interpretation of the data in this research study.
Longitudinal Profiling of the BCR Repertoire During Primary DENV1 Infection
T.-W. Verdonckt1*, K.K. Ariën1, F. Van Nieuwerburgh2, A.S. Vermeersch2, C. Struyfs3, O. Lagatie3
1Unit of Virology, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; 2Laboratory for Pharmaceutical Biotechnology/NXTGNT, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium; 3Janssen Pharmaceutica NV, Beerse, Belgium. *Corresponding author.
Approximately 400 million people contract dengue virus (DENV) annually, typically resulting in mild or asymptomatic infections. However, secondary infections with a different DENV serotype increases the risk of severe dengue, often due to immune mechanisms like antigenic imprinting and antibody-dependent enhancement (ADE). Understanding immune responses during primary infection is therefore essential.
We conducted a retrospective analysis on nine healthy volunteers experimentally infected with dengue virus serotype 1 (DENV1) under controlled conditions. Blood samples were collected on days 0, 8, 10, 14, and 28 post-infection, and bulk B-cell receptor sequencing was performed with the NEBNext Immune Sequencing kit at each time point to track adaptive immune responses.
BCR sequences were assembled through the nf-core/airrflow analysis pipeline version 4.1.0 using the nebnext_umi protocol. Downstream analysis was carried out on the airrflow-generated datasets in R, aided by the Immcantation tool suite. In particular, public clones were determined through scoper, diversity metrics were calculated through alakazam, and clonal trees analyzed and plotted with dowser.
Our findings indicate that primary DENV1 infection initiates two distinct immune responses. In the acute phase, public IgM junctions emerge with low somatic hypermutation (SHM) rates, suggesting an origin from naïve B cells. As the infection progresses, these IgM clones undergo class switch recombination to IgG, retaining low SHM rates, which implies maturation outside germinal centers. In parallel, we observed IgA clone expansion with high SHM rates, indicative of cross-reactive memory B cells.
By characterizing these processes, we aim to uncover biomarkers predictive of severe dengue
Consistency and Reproducibility Across Germline Databases in Immune Repertoire Analysis
Ariel Erijman1, Chen Song1, Evan Janzen1, Bradley W. Langhorst1*
1New England Biolabs. *Corresponding author.
The immune repertoire, comprising the diverse range of T and B cell antigen receptor transcripts, is key to understanding immune responses in various diseases. High-throughput sequencing has transformed immune repertoire analysis, enabling detailed profiling of clonal diversity and evolution. Although bulk sequencing cannot link receptor sequences to individual cells, its large-scale throughput provides valuable, population-wide insights into clonal diversity, frequency, and immune dynamics.
Despite its advantages, bulk immune repertoire analysis presents notable bioinformatic challenges. Since each individual’s immune transcripts are unique, it’s not possible to align to a reference as in typical RNA-seq analysis methods. These vast datasets require robust pipelines for quality assessment, assembly, clustering, and annotation with known alleles. Furthermore, clonal assignment is complicated by somatic hypermutation and sequence similarity, leading to potential ambiguity in clonotype definitions. Standardized workflows like Immcantation help to address some of these technical challenges. Availability of curated V, D, and J alleles from B and T cells across diverse populations make it possible to annotate assembled clones, providing confidence in the biological relevance of each sequence. In this study, we analyze high quality B-Cell Receptor (BCR) data produced from cell line and clinical samples using the New England Biolabs Immune Sequencing kit. We evaluate clonal assignment differences between the widely used IMGT germline database and the new open-source OGRDB, highlighting the impact of reference database selection on immune repertoire analysis.
Funding Information: New England Biolabs supported this work
Pathogenic IgE-Fated B Cell Memory Retains Functional Plasticity
Kelly Bruton1,2†*, Allyssa Phelps1†, Atai Ariaz1, Niels Peter H Knudsen3, Peter S Andersen3, Manel Jordana1
1Department of Medicine, McMaster University, Hamilton, ON, CA; 2Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; 3 ALK-Abelló A/S, Hørsholm, DK. †Equal contribution. *Corresponding author.
Long-lived immunoglobulin (Ig) E responses against innocuous environmental and dietary antigens (Ag) are maintained by an IgG1-dominant memory B cell (MBC) compartment primed for IL-4 responsiveness. The plasticity of the MBC compartment destined for IgE class switch recombination, however, remains poorly understood. In this work, we report a critical IL-4/IL-13 dependency for the pathogenic IgE fate of type 2-polarized MBCs. Initiating a recall response in the absence of IL-4/IL-13 signaling ameliorated the type 2 MBC phenotype in mice and humans and permitted the emergence of long-lived Ag-specific IgG2c+ MBCs. Bulk VDJ sequencing of Ag-specific B cells was performed to investigate how the perturbed recall response modulated the B cell repertoire. Implementing the Immcantation analysis framework, we found that loss of IL-4/IL-13 signaling gave rise to more highly mutated IgG1 and IgG2 clones (SHazaM). Reconstruction of B cell lineage trees (Dowser) revealed that mutated IgG2 B cells were present in trees containing expanded IgM, IgG3, and IgG1 clones, demonstrating that both naïve and class-switched cells can give rise to IgG2. This divergence to a type 1-like response was dependent on IFN-\(\gamma\) signaling and was sustained even beyond therapeutic intervention, revealing fundamental insight into the plasticity of allergen-specific MBCs.
Funding Information: KB was supported by Canadian Institutes of Health Research Doctoral Award and Postdoctoral Fellowship. This research was supported by funding from the Walter & Maria Schroder Foundation, Food Allergy Canada, Canadian Allergy, Asthma, and Immunology Foundation, and ALK-Abelló.
Gut Germinal Centers Support Sequential Class Switching for Antigen-Specific IgA Production
Emily R. Siniscalco1,2, Hailong Meng3, Gisela Gabernet3, Gaspar. A. Pacheco4,5,6,7, Shahab Saghaei4,5,6,7, Shuting Chen1, Xiangyun Yin1, Christine Dien3, Laura R. Hoyt1, Elise G. Liu8, Neima Briggs9, Adam Williams2,10, Vipul Shukla2,11, Duane R. Wesemann4,5,6,7, Steven Kleinstein1,3,12, Joseph Craft1,8*, Stephanie Eisenbarth1,2,10,13*
1 Dept. of Immunobiology, Yale Sch. of Med.; 2 Center for Human Immunology, Northwestern Univ. Feinberg Sch. of Med.; 3 Dept. of Pathology, Yale Sch. of Med.; 4 Dept of Medicine, Div. of Allergy and Clinical Immunology, Div. of Genetics, Brigham and Women’s Hospital; 5 Harvard Med. Sch.; 6 The Broad Institute of MIT and Harvard; 7 The Ragon Institute of MGH, MIT, and Harvard; 8 Section of Rheumatology, Allergy and Immunology, Yale Sch. of Med.; 9 Section of Infectious Diseases, Yale Sch. of Med.; 10 Dept. of Medicine (Allergy and Immunology); Northwestern Univ. Feinberg Sch. of Med.; 11 Dept. of Cell and Developmental Biology, Northwestern Univ. Feinberg Sch. of Med.; 12 Program in Computational Biology and Bioinformatics; 13 Dept. of Laboratory Medicine, Yale Sch. of Med. *Corresponding Author.
The intestine is a barrier site where foreign stimuli interface with the immune system, causing protective and tolerogenic immune responses. IgA is a hallmark of intestinal immunity and is the predominant gut antibody isotype, neutralizing toxins, binding pathogens and foods, and maintaining microbial commensals. Despite IgA’s prevalence, the rules for making gut IgA are unknown. To investigate gut antibody responses, we used fate mapping to track antigen-specific B cells’ GC participation along with single-cell BCR sequencing and analysis with Immcantation to define relationships between antigen-specific B cells in multiple gut tissues. We found that antigen-specific B cells entered gut GCs soon after immunization but did not form GC-derived plasma cells until weeks later. Additionally, we identified an early wave of GC-independent, antigen-specific IgA plasma cells in the gut that accumulated somatic mutations and underwent clonal selection. Through analysis of BCRs in mice and humans using the Dowser package, we observed that IgA and IgG1 B cells were closely related in expanded clones. Further analysis of IgG1 and IgA B cells revealed that gut IgG1 B cells class switched to IgA to contribute to the gut IgA response. These results define several noncanonical pathways of antibody production in the gut, including GC-independent production of affinity matured plasma cells and sequential class switching through IgG1 to IgA, thus illuminating how the unique environment of the gut shapes B cell responses.
Phylogenetic Reconstruction of HIV B Cell Lineages with Dowser: Applications to Mutation-Guided Vaccine Design
James San1, Sravani Venkatayogi1, Elizabeth Van Itallie1, Kevin Wiehe1,2
1Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710; 2Department of Medicine, Duke University Medical Center, Durham, NC, 27710, USA.
A protective HIV vaccine must elicit broadly neutralizing antibodies (bnAbs). HIV bnAbs are challenging to elict because they are the result of long maturation pathways which include mutations that are not routinely made by the somatic hypermutation machinery. To address this challenge, we recently developed the mutation-guided vaccine design strategy in which developmentally rate-limiting bnAb mutations are targeted for selection by specifically designed immunogens. This vaccine strategy relies on accurate reconstruction of the maturation history of target bnAb clones including inferring the unmutated common ancestor and the ancestral sequences in the B cell clonal genealogy. Although recent advancements in sequencing have enabled paired heavy and light chain sequencing, there remains a shortage of tools capable of analyzing paired chain data and accurately reconstructing B cell maturation histories. Dowser is a newly developed lineage reconstruction tool in the Immcantation framework. Using data from highly curated HIV B cell lineages, we compared ancestral states and clonal genealogy trees reconstructed by Dowser to previous methods. Our results demonstrated that Dowser reconstructed highly similar trees to the previously inferred HIV bnAb clonal genealogies. Furthermore, analysis of the inferred ancestral sequences showed that Dowser placed most of the critical, improbable mutations responsible for the breadth and binding affinity of the lineages along similar branches as previous methods. We also used Dowser to reconstruct a large clone elicited in a rhesus macaque demonstrating that Dowser can be applied to paired sequence data from a non-human primate animal model widely used in vaccine research. These findings establish Dowser as a robust and reliable tool for B cell lineage reconstruction, providing a valuable asset for advancing mutation-guided vaccine design strategies.
Linking affinity maturation and B cell differentiation in the germinal center after Plasmodium immunization
Henry J. Sutton1,*, Xin Gao1,*, Hannah G. Kelly1, Brian J. Parker2,3, Mariah Lofgren4, Cherrelle Dacon5, Deepyan Chatterjee1, Robert A. Seder4, Joshua Tan5, Azza H. Idris4, Teresa Neeman2 and Ian A. Cockburn1,†
1Department of Immunology and Infectious Disease, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, 2601, Australia; 2Biological Data Science Institute, The Australian National University, Canberra, ACT, 2601, Australia; 3School of Computing, ANU College of Engineering, Computing & Cybernetics, The Australian National University, Canberra, ACT, 2601, Australia; 4Malaria Unit, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; 5Antibody Biology Unit, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
*These authors contributed equally to this work.
Long-lived plasma cells (PCs) secrete antibodies that can provide sustained immunity against infection. It has been proposed that high affinity cells are preferentially selected into this compartment, potentiating the immune response. We used single cell RNA-seq to track the germinal center (GC) development of Ighg2A10 cells, specific for the Plasmodium falciparum circumsporozoite protein (PfCSP). Following immunization with Plasmodium sporozoites we identified 3 populations of cells in the GC light zone. One population expressed a gene signature associated with the initiation of PC differentiation and had an enhanced propensity to form PCs in vitro. Unexpectedly, the estimated affinity of this putative pre-PC population was indistinguishable from cells in the GC generally. This was also true when high- or low-avidity recombinant PfCSP proteins were used as immunogens. Immunization with low-avidity PfCSP did, however, induce increased affinity maturation. Collectively these findings suggest that the initiation of PC development in the GC occurs via an affinity independent process.