B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced ...BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.
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•LIBRA-seq: high-throughput mapping of BCR sequence to antigen specificity•Identified HIV- and influenza-specific B cells in two HIV-infected subjects•Predicted antigen reactivity for thousands of single B cells•Identified a previously unknown broadly neutralizing HIV antibody
LIBRA-seq enables high-throughput mapping of B cell receptor sequence to antigen specificity at the single-cell level.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Severe injury to the lining of the stomach leads to changes in the epithelium (reprogramming) that protect and promote repair of the tissue, including development of spasmolytic ...polypeptide-expressing metaplasia (SPEM) and tuft and foveolar cell hyperplasia. Acute gastric damage elicits a type-2 inflammatory response that includes production of type-2 cytokines and infiltration by eosinophils and alternatively activated macrophages. Stomachs of mice that lack interleukin 33 (IL33) or interleukin 13 (IL13) did not undergo epithelial reprogramming after drug-induced injury. We investigated the role of group 2 innate lymphoid cells (ILC2s) in gastric epithelial repair.
Acute gastric injury was induced in C57BL/6J mice (wild-type and RAG1 knockout) by administration of L635. We isolated ILC2s by flow cytometry from stomachs of mice that were and were not given L635 and performed single-cell RNA sequencing. ILC2s were depleted from wild-type and RAG1-knockout mice by administration of anti-CD90.2. We assessed gastric cell lineages, markers of metaplasia, inflammation, and proliferation. Gastric tissue microarrays from patients with gastric adenocarcinoma were analyzed by immunostaining.
There was a significant increase in the number of GATA3-positive ILC2s in stomach tissues from wild-type mice after L635-induced damage, but not in stomach tissues from IL33-knockout mice. We characterized a marker signature of gastric mucosal ILC2s and identified a transcription profile of metaplasia-associated ILC2s, which included changes in expression of Il5, Il13, Csf2, Pd1, and Ramp3; these changes were validated by quantitative polymerase chain reaction and immunocytochemistry. Depletion of ILC2s from mice blocked development of metaplasia after L635-induced injury in wild-type and RAG1-knockout mice and prevented foveolar and tuft cell hyperplasia and infiltration or activation of macrophages after injury. Numbers of ILC2s were increased in stomach tissues from patients with SPEM compared with patients with normal corpus mucosa.
In analyses of stomach tissues from mice with gastric tissue damage and patients with SPEM, we found evidence of type 2 inflammation and increased numbers of ILC2s. Our results suggest that ILC2s coordinate the metaplastic response to severe gastric injury.
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Characterization of single antibody lineages within infected individuals has provided insights into the development of Env-specific antibodies. However, a systems-level understanding of the humoral ...response against HIV-1 is limited. Here, we interrogated the antibody repertoires of multiple HIV-infected donors from an infection-naive state through acute and chronic infection using next-generation sequencing. This analysis revealed the existence of “public” antibody clonotypes that were shared among multiple HIV-infected individuals. The HIV-1 reactivity for representative antibodies from an identified public clonotype shared by three donors was confirmed. Furthermore, a meta-analysis of publicly available antibody repertoire sequencing datasets revealed antibodies with high sequence identity to known HIV-reactive antibodies, even in repertoires that were reported to be HIV naive. The discovery of public antibody clonotypes in HIV-infected individuals represents an avenue of significant potential for better understanding antibody responses to HIV-1 infection, as well as for clonotype-specific vaccine development.
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•Within-donor longitudinal antibody repertoire to HIV-1 infection was analyzed by NGS•Public antibody clonotypes shared among multiple HIV-infected individuals were uncovered•A public antibody clonotype shared by three donors was confirmed to be HIV reactive•Antibody sequences from HIV-naive repertoires are similar to known HIV antibodies
The overall antibody repertoires of HIV-infected subjects are considered to be unique. Setliff et al. analyze the longitudinal antibody repertoire of HIV-1-infected individuals to uncover the existence of “public” HIV-reactive antibodies in multiple subjects. Antibody sequences with high identity to known HIV-reactive antibodies were identified even in HIV-naive repertoires.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, ...mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a
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pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.
Human immunodeficiency virus (HIV) can adapt to an individual's T cell immune response via genomic mutations that affect antigen recognition and impact disease outcome. These viral adaptations are ...specific to the host's human leucocyte antigen (HLA) alleles, as these molecules determine which peptides are presented to T cells. As HLA molecules are highly polymorphic at the population level, horizontal transmission events are most commonly between HLA-mismatched donor/recipient pairs, representing new immune selection environments for the transmitted virus. In this study, we utilised a deep sequencing approach to determine the HIV quasispecies in 26 mother-to-child transmission pairs where the potential for founder viruses to be pre-adapted is high due to the pairs being haplo-identical at HLA loci. This scenario allowed the assessment of specific HIV adaptations following transmission in either a non-selective immune environment, due to recipient HLA mismatched to original selecting HLA, or a selective immune environment, mediated by matched donor/recipient HLA. We show that the pattern of reversion or fixation of HIV adaptations following transmission provides insight into the replicative cost, and likely compensatory networks, associated with specific adaptations in vivo. Furthermore, although transmitted viruses were commonly heavily pre-adapted to the child's HLA genotype, we found evidence of de novo post-transmission adaptation, representing new epitopes targeted by the child's T cell response. High-resolution analysis of HIV adaptation is relevant when considering vaccine and cure strategies for individuals exposed to adapted viruses via transmission or reactivated from reservoirs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens
. In most mammals, antibodies are composed of heavy and light chains ...that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens
. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes
. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Lower CD4
cell count in people with HIV infection (PWH) is associated with increased cardiovascular disease (CVD) risk. Whether subsets of CD4
T helper cells are linked with CVD is unclear.
The aim ...of this study was to explore the association between peripherally circulating CD4
T cell subsets and incident CVD.
Data from 1,860 participants (1,270 PWH) without prevalent CVD from the VACS (Veterans Aging Cohort Study), a prospective, observational cohort of veterans with and without HIV infection, were analyzed. T cell subsets were quantified in baseline samples using flow cytometry. Incident CVD events were identified using International Classification of Diseases-9th Revision and International Classification of Diseases-10th Revision diagnosis and procedure codes. Participants were followed from baseline date (2005-2006) to the first of CVD incidence, death, or September 30, 2016. Cox proportional hazards regression was used to model associations between these T cell subsets and the risk for incident CVD while adjusting for demographics and other CVD risk factors.
The median participant age at baseline was 51.6 years. Most were male (94%) and of Black race (69.1%). There were 344 incident CVD events (219 in PWH) during follow-up (median 9.8 years). In PWH, higher proportions (per SD increment) of T helper type 17 cells (adjusted HR: 1.19; 95% CI: 1.08-1.31), T effector memory cells re-expressing CD45RA (adjusted HR: 1.19; 95% CI: 1.07-1.34), and CD28
cells (adjusted HR: 1.18; 95% CI: 1.03-1.34) were significantly associated with an increased risk for incident CVD. Among those without HIV infection, no T cell subsets were significantly associated with CVD.
Among PWH, T helper type 17 cells, senescent cells, and CD4
T effector memory cells re-expressing CD45RA were significantly associated with incident CVD that was not explained by CVD risk factors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Adipose tissue comprises one of the largest organs in the body and performs diverse functions including energy storage and release, regulation of appetite and other neuroendocrine signaling, and ...modulation of immuity, among others. Adipocytes reside in a complex compartment where antigen, antigen presenting cells, innate immune cells, and adaptive immune cells interact locally and exert systemic effects on inflammation, circulating immune cell profiles, and metabolic homeostasis. T lymphocytes are a major component of the adipose tissue milieu which are altered in disease states such as obesity and human immunodeficiency virus (HIV) infection. While obesity, HIV infection, and simian immunodeficiency virus (SIV; a non-human primate virus similar to HIV) infection are accompanied by enrichment of CD8
T cells in the adipose tissue, major phenotypic differences in CD4
T cells and other immune cell populations distinguish HIV/SIV infection from obesity. Furthermore, DNA and RNA species of HIV and SIV can be detected in the stromal vascular fraction of visceral and subcutaneous adipose tissue, and replication-competent HIV resides in local CD4
T cells. Here, we review studies of adipose tissue CD4
and CD8
T cell populations in HIV and SIV, and contrast the findings with those reported in obesity.
Adipose tissue (AT) CD4
and CD8
T cells contribute to obesity-associated insulin resistance. Prior studies identified conserved T-cell receptor (TCR) chain families in obese AT, but the presence and ...clonal expansion of specific TCR sequences in obesity has not been assessed. We characterized AT and liver CD8
and CD4
TCR repertoires of mice fed a low-fat diet (LFD) and high-fat diet (HFD) using deep sequencing of the TCRβ chain to quantify clonal expansion, gene usage, and CDR3 sequence. In AT CD8
T cells, HFD reduced TCR diversity, increased the prevalence of public TCR clonotypes, and selected for TCR CDR3 regions enriched in positively charged and less polarized amino acids. Although TCR repertoire alone could distinguish between LFD- and HFD-fed mice, these properties of the CDR3 region of AT CD8
T cells from HFD-fed mice led us to examine the role of negatively charged and nonpolar isolevuglandin (isoLG) adduct-containing antigen-presenting cells within AT. IsoLG-adducted protein species were significantly higher in AT macrophages of HFD-fed mice; isoLGs were elevated in M2-polarized macrophages, promoting CD8
T-cell activation. Our findings demonstrate that clonal TCR expansion that favors positively charged CDR3s accompanies HFD-induced obesity, which may be an antigen-driven response to isoLG accumulation in macrophages.
The development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. We screened the circulating B cell repertoires of COVID-19 ...survivors and vaccinees to isolate over 9,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific monoclonal antibodies (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. Among the recovered antibodies was TXG-0078, an N-terminal domain (NTD)-specific neutralizing mAb that recognizes diverse alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy-chain-dominant binding pattern seen in other NTD-supersite-specific neutralizing Abs with much narrower specificity. We also report CC24.2, a pan-sarbecovirus neutralizing antibody that targets a unique receptor-binding domain (RBD) epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 shows protection in vivo, suggesting their potential use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.
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•Next-generation antigen barcoding allows deep mining of antigen-specific antibody repertories•TXG-0078 achieves remarkable breadth by uniquely targeting the SARS-CoV-2 N-terminal domain•A cocktail of complementary coronavirus-specific antibodies shows in vivo protection
Broad and potently neutralizing antibodies aid in identifying conserved sites of viral vulnerability; however, such antibodies are often very rare. Hurtado et al. utilize a next-generation antigen barcoding approach to deeply survey the SARS-CoV-2-specific antibody response in COVID-19 survivors and vaccinees, aiming to identify a cocktail of protective antibodies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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