Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation in a pediatric-dominated cohort.

Introduction: It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of recipient gut lymphocyte populations in immunosuppressed conditions. Methods: Using polychromatic flow cytometry that includes HLA allele group-specific antibodies distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. Results: We confirm the early presence of naïve donor B cells in the circulation (donor age range: 1-14 years, median: 3 years) and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa (recipient age range at the time of transplant: 1-44 years, median: 3 years). Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection (recipient age range at the time of transplant: 1-9 years, median: 2 years) revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in deceased adult donors. In available pan-scope biopsies from pediatric recipients, we observed higher percentages of naïve recipient B cells in colon allograft compared to small bowel allograft and increased BCR overlap between native colon vs colon allograft compared to that between native colon vs ileum allograft in most cases, suggesting differential clonal distribution in large intestine vs small intestine. Discussion: Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of stabilization of the mucosal B cell repertoire in pediatric ITx patients.

Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation.

It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of gut lymphocyte populations. Using polychromatic flow cytometry that includes HLA allele group-specific mAbs distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. We confirm the early presence of naïve donor B cells in the circulation and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa. Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in healthy control adults. Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of establishment of a stable mucosal B cell repertoire.

Induction of bronchus-associated lymphoid tissue is an early life adaptation for promoting human B cell immunity.

Infants and young children are more susceptible to common respiratory pathogens than adults but can fare better against novel pathogens like severe acute respiratory syndrome coronavirus 2. The mechanisms by which infants and young children mount effective immune responses to respiratory pathogens are unknown. Through investigation of lungs and lung-associated lymph nodes from infant and pediatric organ donors aged 0-13 years, we show that bronchus-associated lymphoid tissue (BALT), containing B cell follicles, CD4+ T cells and functionally active germinal centers, develop during infancy. BALT structures are prevalent around lung airways during the first 3 years of life, and their numbers decline through childhood coincident with the accumulation of memory T cells. Single-cell profiling and repertoire analysis reveals that early life lung B cells undergo differentiation, somatic hypermutation and immunoglobulin class switching and exhibit a more activated profile than lymph node B cells. Moreover, B cells in the lung and lung-associated lymph nodes generate biased antibody responses to multiple respiratory pathogens compared to circulating antibodies, which are mostly specific for vaccine antigens in the early years of life. Together, our findings provide evidence for BALT as an early life adaptation for mobilizing localized immune protection to the diverse respiratory challenges during this formative life stage.

Diversification of immunoglobulin genes by gene conversion in the domestic chicken (Gallus gallus domesticus).

Sustainable modern poultry production depends on effective protection against infectious diseases and a diverse range of antibodies is key for an effective immune response. In the domestic chicken, somatic gene conversion is the dominant process in which the antibody immunoglobulin genes are diversified. Affinity maturation by somatic hypermutation (SHM) also occurs, but the relative contribution of gene conversion versus somatic hypermutation to immunoglobulin (Ig) gene diversity is poorly understood. In this study, we use high throughput long-read sequencing to study immunoglobulin diversity in multiple immune-associated tissues in Rhode Island Red chickens. To better understand the impact of genetic diversification in the chicken, a novel gene conversion identification software was developed (BrepConvert). In this study, BrepConvert enabled the identification of over 1 million gene conversion events. Mapping the occurrence of putative somatic gene conversion (SGC) events throughout the variable gene region revealed repetitive and highly restricted patterns of genetic insertions in both the antibody heavy and light chains. These patterns coincided with the locations of genetic variability in available pseudogenes and align with antigen binding sites, predominately the complementary determining regions (CDRs). We found biased usage of pseudogenes during gene conversion, as well as immunoglobulin heavy chain diversity gene (IGHD) preferences during V(D)J gene rearrangement, suggesting that antibody diversification in chickens is more focused than the genetic potential for diversity would suggest.

Chickens, more than humans, focus the diversity of their immunoglobulin genes on the complementarity-determining region but utilise amino acids, indicative of a more cross-reactive antibody repertoire.

The mechanisms of B-cell diversification differ greatly between aves and mammals, but both produce B cells and antibodies capable of supporting an effective immune response. To see how differences in the generation of diversity might affect overall repertoire diversity, we have compared the diversity characteristics of immunoglobulin genes from domestic chickens to those from humans. Both use V(D)J gene rearrangement and somatic hypermutation, but only chickens use somatic gene conversion. A range of diversity analysis tools were used to investigate multiple aspects of amino acid diversity at both the germline and repertoire levels. The effect of differing amino acid usages on antibody characteristics was assessed. At both the germline and repertoire levels, chickens exhibited lower amino acid diversity in comparison to the human immunoglobulin genes, especially outside of the complementarity-determining region (CDR). Chickens were also found to possess much larger and more hydrophilic CDR3s with a higher predicted protein binding potential, suggesting that the antigen-binding site in chicken antibodies is more flexible and more polyreactive than that seen in human antibodies.

Targeting BMI-1 in B cells restores effective humoral immune responses and controls chronic viral infection.

Ineffective antibody-mediated responses are a key characteristic of chronic viral infection. However, our understanding of the intrinsic mechanisms that drive this dysregulation are unclear. Here, we identify that targeting the epigenetic modifier BMI-1 in mice improves humoral responses to chronic lymphocytic choriomeningitis virus. BMI-1 was upregulated by germinal center B cells in chronic viral infection, correlating with changes to the accessible chromatin landscape, compared to acute infection. B cell-intrinsic deletion of Bmi1 accelerated viral clearance, reduced splenomegaly and restored splenic architecture. Deletion of Bmi1 restored c-Myc expression in B cells, concomitant with improved quality of antibody and coupled with reduced antibody-secreting cell numbers. Specifically, BMI-1-deficiency induced antibody with increased neutralizing capacity and enhanced antibody-dependent effector function. Using a small molecule inhibitor to murine BMI-1, we could deplete antibody-secreting cells and prohibit detrimental immune complex formation in vivo. This study defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection.

Using Domain Based Latent Personal Analysis of B Cell Clone Diversity Patterns to Identify Novel Relationships Between the B Cell Clone Populations in Different Tissues.

The B cell population is highly diverse and very skewed. It is divided into clones (B cells with a common mother cell). It is thought that each clone represents an initial B cell receptor specificity. A few clones are very abundant, comprised of hundreds or thousands of B cells while the majority have only a few cells per clone. We suggest a novel method - domain-based latent personal analysis (LPA), a method for spectral exploration of entities in a domain, which can be used to find the spectral spread of sub repertoires within a person. LPA defines a domain-based spectral signature for each sub repertoire. LPA signatures consist of the elements, in our case - the clones, that most differentiate the sub repertoire from the person's abundance of clones. They include both positive elements, which describe overabundant clones, and negative elements that describe missing clones. The signatures can also be used to compare the sub repertoires they represent to each other. Applying LPA to compare the repertoires found in different tissues, we reiterated previous findings that showed that gut and blood tissues have separate repertoires. We further identify a third branch of clonal patterns typical of the lymphatic organs (Spleen, MLN, and bone marrow) separated from the other two categories. We developed a python version of LPA analysis that can easily be applied to compare clonal distributions - https://github.com/ScanLab-ossi/LPA. It could also be easily adapted to study other skewed sequence populations used in the analysis of B cell receptor populations, for instance, k-mers and V gene usage. These analysis types should allow for inter and intra-repertoire comparisons of diversity, which could revolutionize the way we understand repertoire changes and diversity.

BCR-Induced Ca2+ Signals Dynamically Tune Survival, Metabolic Reprogramming, and Proliferation of Naive B Cells.

B cell receptor (BCR) engagement induces naive B cells to differentiate and perform critical immune-regulatory functions. Acquisition of functional specificity requires that a cell survive, enter the cell cycle, and proliferate. We establish that quantitatively distinct Ca2+ signals triggered by variations in the extent of BCR engagement dynamically regulate these transitions by controlling nuclear factor κB (NF-κB), NFAT, and mTORC1 activity. Weak BCR engagement induces apoptosis by failing to activate NF-κB-driven anti-apoptotic gene expression. Stronger signals that trigger more robust Ca2+ signals promote NF-κB-dependent survival and NFAT-, mTORC1-, and c-Myc-dependent cell-cycle entry and proliferation. Finally, we establish that CD40 or TLR9 costimulation circumvents these Ca2+-regulated checkpoints of B cell activation and proliferation. As altered BCR signaling is linked to autoimmunity and B cell malignancies, these results have important implications for understanding the pathogenesis of aberrant B cell activation and differentiation and therapeutic approaches to target these responses.

Serine substitutions are linked to codon usage and differ for variable and conserved protein regions.

Serine is the only amino acid that is encoded by two disjoint codon sets (TCN & AGY) so that a tandem substitution of two nucleotides is required to switch between the two sets. We show that these codon sets underlie distinct substitution patterns at positions subject to purifying and diversifying selections. We found that in humans, positions that are conserved among ~100 vertebrates, and thus subjected to purifying selection, are enriched for substitutions involving serine (TCN, denoted S'), proline, and alanine, (S'PA). In contrast, the less conserved positions are enriched for serine encoded with AGY codons (denoted S″), glycine and asparagine, (GS″N). We tested this phenomenon in the HIV envelope glycoprotein (gp120), and the V-gene that encodes B-cell receptors/antibodies. These fast evolving proteins both have hypervariable positions, which are under diversifying selection, closely adjacent to highly conserved structural regions. In both instances, we identified an opposite abundance of two groups of serine substitutions, with enrichment of S'PA in the conserved positions, and GS″N in the hypervariable regions. Finally, we analyzed the substitutions across 60,000 individual human exomes to show that, when serine has a specific functional constraint of phosphorylation capability, S' codons are 32-folds less prone than S″ to substitutions to Threonine or Tyrosine that could potentially retain the phosphorylation site capacity. Combined, our results, that cover evolutionary signals at different temporal scales, demonstrate that through its encoding by two codon sets, serine allows for the existence of alternating substitution patterns within positions of functional maintenance versus sites of rapid diversification.

Liver Is a Generative Site for the B Cell Response to Ehrlichia muris.

The B cell response to Ehrlichia muris is dominated by plasmablasts (PBs), with few-if any-germinal centers (GCs), yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs) that express the transcription factor T-bet and harbor V-region mutations. Because Ehrlichia prominently infects the liver, we investigated the nature of liver B cell response and that of the spleen. B cells within infected livers proliferated and underwent somatic hypermutation (SHM). Vh-region sequencing revealed trafficking of clones between the spleen and liver and often subsequent local clonal expansion and intraparenchymal localization of T-bet+ MBCs. T-bet+ MBCs expressed MBC subset markers CD80 and PD-L2. Many T-bet+ MBCs lacked CD11b or CD11c expression but had marginal zone (MZ) B cell phenotypes and colonized the splenic MZ, revealing T-bet+ MBC plasticity. Hence, liver and spleen are generative sites of B cell responses, and they include V-region mutation and result in liver MBC localization.

Caspase-8 promotes c-Rel-dependent inflammatory cytokine expression and resistance against Toxoplasma gondii.

Caspase-8 is a key integrator of cell survival and cell death decisions during infection and inflammation. Following engagement of tumor necrosis factor superfamily receptors or certain Toll-like receptors (TLRs), caspase-8 initiates cell-extrinsic apoptosis while inhibiting RIPK3-dependent programmed necrosis. In addition, caspase-8 has an important, albeit less well understood, role in cell-intrinsic inflammatory gene expression. Macrophages lacking caspase-8 or the adaptor FADD have defective inflammatory cytokine expression and inflammasome priming in response to bacterial infection or TLR stimulation. How caspase-8 regulates cytokine gene expression, and whether caspase-8-mediated gene regulation has a physiological role during infection, remain poorly defined. Here we demonstrate that both caspase-8 enzymatic activity and scaffolding functions contribute to inflammatory cytokine gene expression. Caspase-8 enzymatic activity was necessary for maximal expression of Il1b and Il12b, but caspase-8 deficient cells exhibited a further decrease in expression of these genes. Furthermore, the ability of TLR stimuli to induce optimal IκB kinase phosphorylation and nuclear translocation of the nuclear factor kappa light chain enhancer of activated B cells family member c-Rel required caspase activity. Interestingly, overexpression of c-Rel was sufficient to restore expression of IL-12 and IL-1ß in caspase-8-deficient cells. Moreover, Ripk3-/-Casp8-/- mice were unable to control infection by the intracellular parasite Toxoplasma gondii, which corresponded to defects in monocyte recruitment to the peritoneal cavity, and exogenous IL-12 restored monocyte recruitment and protection of caspase-8-deficient mice during acute toxoplasmosis. These findings provide insight into how caspase-8 controls inflammatory gene expression and identify a critical role for caspase-8 in host defense against eukaryotic pathogens.

ImmuneDB, a Novel Tool for the Analysis, Storage, and Dissemination of Immune Repertoire Sequencing Data.

ImmuneDB is a system for storing and analyzing high-throughput immune receptor sequencing data. Unlike most existing tools, which utilize flat-files, ImmuneDB stores data in a well-structured MySQL database, enabling efficient data queries. It can take raw sequencing data as input and annotate receptor gene usage, infer clonotypes, aggregate results, and run common downstream analyses such as calculating selection pressure and constructing clonal lineages. Alternatively, pre-annotated data can be imported and analyzed data can be exported in a variety of common Adaptive Immune Receptor Repertoire (AIRR) file formats. To validate ImmuneDB, we compare its results to those of another pipeline, MiXCR. We show that the biological conclusions drawn would be similar with either tool, while ImmuneDB provides the additional benefits of integrating other common tools and storing data in a database. ImmuneDB is freely available on GitHub at https://github.com/arosenfeld/immunedb, on PyPi at https://pypi.org/project/ImmuneDB, and a Docker container is provided at https://hub.docker.com/r/arosenfeld/immunedb. Full documentation is available at http://immunedb.com.

AIRR Community Standardized Representations for Annotated Immune Repertoires.

Increased interest in the immune system's involvement in pathophysiological phenomena coupled with decreased DNA sequencing costs have led to an explosion of antibody and T cell receptor sequencing data collectively termed "adaptive immune receptor repertoire sequencing" (AIRR-seq or Rep-Seq). The AIRR Community has been actively working to standardize protocols, metadata, formats, APIs, and other guidelines to promote open and reproducible studies of the immune repertoire. In this paper, we describe the work of the AIRR Community's Data Representation Working Group to develop standardized data representations for storing and sharing annotated antibody and T cell receptor data. Our file format emphasizes ease-of-use, accessibility, scalability to large data sets, and a commitment to open and transparent science. It is composed of a tab-delimited format with a specific schema. Several popular repertoire analysis tools and data repositories already utilize this AIRR-seq data format. We hope that others will follow suit in the interest of promoting interoperable standards.

Human Lymph Nodes Maintain TCF-1hi Memory T Cells with High Functional Potential and Clonal Diversity throughout Life.

Translating studies on T cell function and modulation from mouse models to humans requires extrapolating in vivo results on mouse T cell responses in lymphoid organs (spleen and lymph nodes [LN]) to human peripheral blood T cells. However, our understanding of T cell responses in human lymphoid sites and their relation to peripheral blood remains sparse. In this study, we used a unique human tissue resource to study human T cells in different anatomical compartments within individual donors and identify a subset of memory CD8+ T cells in LN, which maintain a distinct differentiation and functional profile compared with memory CD8+ T cells in blood, spleen, bone marrow, and lungs. Whole-transcriptome and high-dimensional cytometry by time-of-flight profiling reveals that LN memory CD8+ T cells express signatures of quiescence and self-renewal compared with corresponding populations in blood, spleen, bone marrow, and lung. LN memory T cells exhibit a distinct transcriptional signature, including expression of stem cell-associated transcription factors TCF-1 and LEF-1, T follicular helper cell markers CXCR5 and CXCR4, and reduced expression of effector molecules. LN memory T cells display high homology to a subset of mouse CD8+ T cells identified in chronic infection models that respond to checkpoint blockade immunotherapy. Functionally, human LN memory T cells exhibit increased proliferation to TCR-mediated stimulation and maintain higher TCR clonal diversity compared with memory T cells from blood and other sites. These findings establish human LN as reservoirs for memory T cells with high capacities for expansion and diverse recognition and important targets for immunotherapies.

Computational Evaluation of B-Cell Clone Sizes in Bulk Populations.

B cell clones expand and contract during adaptive immune responses and can persist or grow uncontrollably in lymphoproliferative disorders. One way to monitor and track B cell clones is to perform large-scale sampling of bulk cell populations, amplifying, and sequencing antibody gene rearrangements by next-generation sequencing (NGS). Here, we describe a series of computational approaches for estimating B cell clone size in NGS immune repertoire profiling data of antibody heavy chain gene rearrangements. We define three different measures of B cell clone size-copy numbers, instances, and unique sequences-and show how these measures can be used to rank clones, analyze their diversity, and study their distribution within and between individuals. We provide a detailed, step-by-step procedure for performing these analyses using two different data sets of spleen samples from human organ donors. In the first data set, 19 independently generated biological replicates from a single individual are analyzed for B cell clone size, diversity and sampling sufficiency for clonal overlap analysis. In the second data set, B cell clones are compared in eight different organ donors. We comment upon frequently encountered pitfalls and offer practical advice with alternative approaches. Overall, we provide a series of pragmatic analytical approaches and show how different clone size measures can be used to study the clonal landscape in bulk B cell immune repertoire profiling data.

An atlas of B-cell clonal distribution in the human body.

B-cell responses result in clonal expansion, and can occur in a variety of tissues. To define how B-cell clones are distributed in the body, we sequenced 933,427 B-cell clonal lineages and mapped them to eight different anatomic compartments in six human organ donors. We show that large B-cell clones partition into two broad networks-one spans the blood, bone marrow, spleen and lung, while the other is restricted to tissues within the gastrointestinal (GI) tract (jejunum, ileum and colon). Notably, GI tract clones display extensive sharing of sequence variants among different portions of the tract and have higher frequencies of somatic hypermutation, suggesting extensive and serial rounds of clonal expansion and selection. Our findings provide an anatomic atlas of B-cell clonal lineages, their properties and tissue connections. This resource serves as a foundation for studies of tissue-based immunity, including vaccine responses, infections, autoimmunity and cancer.

Applying systems-level spectral imaging and analysis to reveal the organelle interactome.

The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for ß-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.