Aberrant B cell repertoire selection associated with HIV neutralizing antibody breadth.

A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.

Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination.

Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody.

Effects of invivo CXCR4 blockade and proteasome inhibition on bone marrow plasma cells in HLA-sensitized kidney transplant candidates.

To date, plasma cell (PC)-targeted therapies have been limited by suboptimal PC depletion and antibody rebound. We hypothesized this is partly because of PC residence in protective bone marrow (BM) microenvironments. The purpose of this proof-of-concept study was to examine the effects of the CXCR4 antagonist, plerixafor, on PC BM residence; its safety profile (alone and in combination with a proteasome inhibitor, bortezomib); and the transcriptional effect on BMPCs in HLA-sensitized kidney transplant candidates. Participants were enrolled into 3 groups: group A (n = 4), plerixafor monotherapy; and groups B (n = 4) and C (n = 4), plerixafor and bortezomib combinations. CD34+ stem cell and PC levels increased in the blood after plerixafor treatment. PC recovery from BM aspirates varied depending on the dose of plerixafor and bortezomib. Single-cell RNA sequencing on BMPCs from 3 group C participants pretreatment and posttreatment revealed multiple populations of PCs, with a posttreatment enrichment of oxidative phosphorylation, proteasome assembly, cytoplasmic translation, and autophagy-related genes. Murine studies demonstrated dually inhibiting the proteasome and autophagy resulted in greater BMPC death than did monotherapies. In conclusion, this pilot study revealed anticipated effects of combined plerixafor and bortezomib on BMPCs, an acceptable safety profile, and suggests the potential for autophagy inhibitors in desensitization regimens.

Multi-omic characterization of pediatric ARDS via nasal brushings.

RATIONALE: While nasal brushing transcriptomics can identify disease subtypes in chronic pulmonary diseases, it is unknown whether this is true in pediatric acute respiratory distress syndrome (PARDS). OBJECTIVES: Determine whether nasal transcriptomics and methylomics can identify clinically meaningful PARDS subgroups that reflect important pathobiological processes. METHODS: Nasal brushings and serum were collected on days 1, 3, 7, and 14 from control and PARDS subjects from two centers. PARDS duration was the primary endpoint. MEASUREMENTS AND MAIN RESULTS: Twenty-four control and 39 PARDS subjects were enrolled. Two nasal methylation patterns were identified. Compared to Methyl Subgroup 1, Subgroup 2 had hypomethylation of inflammatory genes and was enriched for immunocompromised subjects. Four transcriptomic patterns were identified with temporal patterns indicating injury, repair, and regeneration. Over time, both inflammatory (Subgroup B) and cell injury (Subgroup D) patterns transitioned to repair (Subgroup A) and eventually homeostasis (Subgroup C). When control specimens were included, they were largely Subgroup C. In comparison with 17 serum biomarkers, the nasal transcriptome was more predictive of prolonged PARDS. Subjects with initial Transcriptomic Subgroup B or D assignment had median PARDS duration of 8 days compared to 2 in A or C (p = 0.02). For predicting PARDS duration ≥ 3 days, nasal transcriptomics was more sensitive and serum biomarkers more specific. CONCLUSIONS: PARDS nasal transcriptome may reflect distal lung injury, repair, and regeneration. A combined nasal PCR and serum biomarker assay could be useful for predictive and diagnostic enrichment. Trial registration Clinicaltrials.gov NCT03539783 May 29, 2018.

B cell clonal expansion within immune infiltrates in human cardiac allograft vasculopathy.

Cardiac allograft vasculopathy (CAV) is associated with intragraft B cell infiltrates. Here, we studied the clonal composition of B cell infiltrates using 4 graft specimens with CAV. Using deep sequencing, we analyzed the immunoglobulin heavy chain variable region repertoire in both graft and blood. Results showed robust B cell clonal expansion in the graft but not in the blood for all cases. Several expanded B cell clones, characterized by their uniquely rearranged complementarity-determining region 3, were detected in different locations in the graft. Sequences from intragraft B cells also showed elevated levels of mutated rearrangements in the graft compared to blood B cells. The number of somatic mutations per rearrangement was also higher in the graft than in the blood, suggesting that B cells continued maturing in situ. Overall, our studies demonstrated B cell clonal expansion in human cardiac allografts with CAV. This local B cell response may contribute to the pathophysiology of CAV through a mechanism that needs to be identified.

Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.

Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

B-cell repertoire responses to varicella-zoster vaccination in human identical twins.

Adaptive immune responses in humans rely on somatic genetic rearrangements of Ig and T-cell receptor loci to generate diverse antigen receptors. It is unclear to what extent an individual's genetic background affects the characteristics of the antibody repertoire used in responding to vaccination or infection. We studied the B-cell repertoires and clonal expansions in response to attenuated varicella-zoster vaccination in four pairs of adult identical twins and found that the global antibody repertoires of twin pair members showed high similarity in antibody heavy chain V, D, and J gene segment use, and in the length and features of the complementarity-determining region 3, a major determinant of antigen binding. These twin similarities were most pronounced in the IgM-expressing B-cell pools, but were seen to a lesser extent in IgG-expressing B cells. In addition, the degree of antibody somatic mutation accumulated in the B-cell repertoire was highly correlated within twin pair members. Twin pair members had greater numbers of shared convergent antibody sequences, including mutated sequences, suggesting similarity among memory B-cell clonal lineages. Despite these similarities in the memory repertoire, the B-cell clones used in acute responses to ZOSTAVAX vaccination were largely unique to each individual. Taken together, these results suggest that the overall B-cell repertoire is significantly shaped by the underlying germ-line genome, but that stochastic or individual-specific effects dominate the selection of clones in response to an acute antigenic stimulus.

Human B-cell isotype switching origins of IgE.

BACKGROUND: B cells expressing IgE contribute to immunity against parasites and venoms and are the source of antigen specificity in allergic patients, yet the developmental pathways producing these B cells in human subjects remain a subject of debate. Much of our knowledge of IgE lineage development derives from model studies in mice rather than from human subjects. OBJECTIVE: We evaluate models for isotype switching to IgE in human subjects using immunoglobulin heavy chain (IGH) mutational lineage data. METHODS: We analyzed IGH repertoires in 9 allergic and 24 healthy adults using high-throughput DNA sequencing of 15,843,270 IGH rearrangements to identify clonal lineages of B cells containing members expressing IgE. Somatic mutations in IGH inherited from common ancestors within the clonal lineage are used to infer the relationships between B cells. RESULTS: Data from 613,641 multi-isotype B-cell clonal lineages, of which 592 include an IgE member, are consistent with indirect switching to IgE from IgG- or IgA-expressing lineage members in human subjects. We also find that these inferred isotype switching frequencies are similar in healthy and allergic subjects. CONCLUSIONS: We found evidence that secondary isotype switching of mutated IgG1-expressing B cells is the primary source of IgE in human subjects, with lesser contributions from precursors expressing other switched isotypes and rarely IgM or IgD, suggesting that IgE is derived from previously antigen-experienced B cells rather than naive B cells that typically express low-affinity unmutated antibodies. These data provide a basis from which to evaluate allergen-specific human antibody repertoires in healthy and diseased subjects.

Single B-cell deconvolution of peanut-specific antibody responses in allergic patients.

BACKGROUND: The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance. OBJECTIVE: We sought to characterize peanut allergen-specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy. METHODS: B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones. RESULTS: Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG4 members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase. CONCLUSION: Most peanut allergen-binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG4.

Effects of aging, cytomegalovirus infection, and EBV infection on human B cell repertoires.

Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.

Defining the T cell transcriptional landscape in pediatric liver transplant rejection at single cell resolution.

Acute cellular rejection (ACR) affects >80% of pediatric liver transplant recipients within 5 years, and late ACR is associated with graft failure. Traditional anti-rejection therapy for late ACR is ineffective and has remained unchanged for six decades. Although CD8+ T cells promote late ACR, little has been done to define their specificity and gene expression. Here, we used single-cell sequencing and immune repertoire profiling (10X Genomics) on 30 cryopreserved 16G liver biopsies from 14 patients (5 pre-transplant or with no ACR, 9 with ACR). We identified expanded intragraft CD8+ T cell clonotypes (CD8EXP) and their gene expression profiles in response to anti-rejection treatment. Notably, we found that expanded CD8+ clonotypes (CD8EXP) bore markers of effector and CD56hiCD161- 'NK-like' T cells, retaining their clonotype identity and phenotype in subsequent biopsies from the same patients despite histologic ACR resolution. CD8EXP clonotypes localized to portal infiltrates during active ACR, and persisted in the lobule after histologic ACR resolution. CellPhoneDB analysis revealed differential crosstalk between KC and CD8EXP during late ACR, with activation of the LTB-LTBR pathway and downregulation of TGFß signaling. Therefore, persistently-detected intragraft CD8EXP clones remain active despite ACR treatment and may contribute to long-term allograft fibrosis and failure of operational tolerance.

Adult microglial TGFß1 is required for microglia homeostasis via an autocrine mechanism to maintain cognitive function in mice.

While TGF-ß signaling is essential for microglial function, the cellular source of TGF-ß1 ligand and its spatial regulation remains unclear in the adult CNS. Our data supports that microglia but not astrocytes or neurons are the primary producers of TGF-ß1 ligands needed for microglial homeostasis. Microglia-Tgfb1 KO leads to the activation of microglia featuring a dyshomeostatic transcriptome that resembles disease-associated, injury-associated, and aged microglia, suggesting microglial self-produced TGF-ß1 ligands are important in the adult CNS. Astrocytes in MG-Tgfb1 inducible (i)KO mice show a transcriptome profile that is closely aligned with an LPS-associated astrocyte profile. Additionally, using sparse mosaic single-cell microglia KO of TGF-ß1 ligand we established an autocrine mechanism for signaling. Here we show that MG-Tgfb1 iKO mice present cognitive deficits, supporting that precise spatial regulation of TGF-ß1 ligand derived from microglia is required for the maintenance of brain homeostasis and normal cognitive function in the adult brain.

Disease diagnostics using machine learning of immune receptors.

Clinical diagnosis typically incorporates physical examination, patient history, and various laboratory tests and imaging studies, but makes limited use of the human system's own record of antigen exposures encoded by receptors on B cells and T cells. We analyzed immune receptor datasets from 593 individuals to develop MAchine Learning for Immunological Diagnosis (Mal-ID) , an interpretive framework to screen for multiple illnesses simultaneously or precisely test for one condition. This approach detects specific infections, autoimmune disorders, vaccine responses, and disease severity differences. Human-interpretable features of the model recapitulate known immune responses to SARS-CoV-2, Influenza, and HIV, highlight antigen-specific receptors, and reveal distinct characteristics of Systemic Lupus Erythematosus and Type-1 Diabetes autoreactivity. This analysis framework has broad potential for scientific and clinical interpretation of human immune responses.

Comprehensive whole-genome sequencing of an early-stage primary myelofibrosis patient defines low mutational burden and non-recurrent candidate genes.

In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: i) a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; ii) a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and iii) a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).

ENCODE whole-genome data in the UCSC genome browser (2011 update).

The ENCODE project is an international consortium with a goal of cataloguing all the functional elements in the human genome. The ENCODE Data Coordination Center (DCC) at the University of California, Santa Cruz serves as the central repository for ENCODE data. In this role, the DCC offers a collection of high-throughput, genome-wide data generated with technologies such as ChIP-Seq, RNA-Seq, DNA digestion and others. This data helps illuminate transcription factor-binding sites, histone marks, chromatin accessibility, DNA methylation, RNA expression, RNA binding and other cell-state indicators. It includes sequences with quality scores, alignments, signals calculated from the alignments, and in most cases, element or peak calls calculated from the signal data. Each data set is available for visualization and download via the UCSC Genome Browser (http://genome.ucsc.edu/). ENCODE data can also be retrieved using a metadata system that captures the experimental parameters of each assay. The ENCODE web portal at UCSC (http://encodeproject.org/) provides information about the ENCODE data and links for access.

VExD: a curated resource for human gene expression alterations following viral infection.

Much of the host antiviral response is mediated through changes to host gene expression levels. Likewise, viruses induce changes to host gene expression levels in order to promote the viral life cycle and evade the host immune system. However, there is no resource that specifically collects human gene expression levels pre- and post-virus infection. Further, public gene expression repositories do not contain enough specialized metadata to easily find relevant experiments. Here, we present the Virus Expression Database (VExD), a freely available website and database, that collects human gene expression datasets in response to viral infection. VExD contains ∼8,000 uniformly processed samples obtained from 289 studies examining 51 distinct human viruses. We show that the VExD processing pipeline captures known antiviral responses in the form of interferon-stimulated genes. We further show that the datasets collected in VExD can be used to quickly identify supporting data for experiments performed in human cells or model organisms. VExD is freely available at https://vexd.cchmc.org/.

B cell repertoire in children with skin barrier dysfunction supports altered IgE maturation associated with allergic food sensitization.

The skin is a major immune organ and skin barrier dysfunction is a major risk factor for the development of the inappropriate immune response seen in allergic disease. Skin barrier disruption alters the landscape of antigens experienced by the immune system and the downstream impacts on the antibody repertoire remain poorly characterized, particularly for the IgE isotype responsible for allergic specificity and in early life, when allergic disease is developing. In this study, we sequenced antibody gene repertoires from a large and well-characterized cohort of children with atopic dermatitis and found that food sensitization was associated with lower mutation frequencies in the IgE compartment. This trend was abrogated in children living with pets during the first year of life. These results elucidate potential molecular mechanisms underlying the protective effects of pet ownership and non-antiseptic environs reported for allergic disease, and the hygiene hypothesis more broadly. We also observed increased IgE diversity and increased isotype-switching to the IgE isotype, suggesting that B cell development, particularly isotype-switching, is heavily altered in the those with food allergen sensitizations relative to those without food allergen sensitizations. Unlike for food antigens, aeroallergen sensitization exhibited no effect on IgE mutation or diversity. Consistent patterns of antibody rearrangement were associated with food allergen sensitization in subjects with atopic dermatitis. Thus, we propose the Immune Repertoire in Atopic Disease (IRAD) score, to quantify this repertoire shift and to aid clinically in patient diagnosis and risk stratification.

Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/ß sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/ß cDNAs from CD8EXP into Jurkat 76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

Meta-alignment with crumble and prune: partitioning very large alignment problems for performance and parallelization.

BACKGROUND: Continuing research into the global multiple sequence alignment problem has resulted in more sophisticated and principled alignment methods. Unfortunately these new algorithms often require large amounts of time and memory to run, making it nearly impossible to run these algorithms on large datasets. As a solution, we present two general methods, Crumble and Prune, for breaking a phylogenetic alignment problem into smaller, more tractable sub-problems. We call Crumble and Prune meta-alignment methods because they use existing alignment algorithms and can be used with many current alignment programs. Crumble breaks long alignment problems into shorter sub-problems. Prune divides the phylogenetic tree into a collection of smaller trees to reduce the number of sequences in each alignment problem. These methods are orthogonal: they can be applied together to provide better scaling in terms of sequence length and in sequence depth. Both methods partition the problem such that many of the sub-problems can be solved independently. The results are then combined to form a solution to the full alignment problem. RESULTS: Crumble and Prune each provide a significant performance improvement with little loss of accuracy. In some cases, a gain in accuracy was observed. Crumble and Prune were tested on real and simulated data. Furthermore, we have implemented a system called Job-tree that allows hierarchical sub-problems to be solved in parallel on a compute cluster, significantly shortening the run-time. CONCLUSIONS: These methods enabled us to solve gigabase alignment problems. These methods could enable a new generation of biologically realistic alignment algorithms to be applied to real world, large scale alignment problems.

Shared B cell memory to coronaviruses and other pathogens varies in human age groups and tissues.

Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells, which encode humoral immune memory. We evaluated pediatric and adult blood and deceased adult organ donor tissues to determine convergent antigen-specific antibody genes of similar sequences shared between individuals. B cell memory varied for different pathogens. Polysaccharide antigen-specific clones were not exclusive to the spleen. Adults had higher clone frequencies and greater class switching in lymphoid tissues than blood, while pediatric blood had abundant class-switched convergent clones. Consistent with reported serology, prepandemic children had class-switched convergent clones to severe acute respiratory syndrome coronavirus 2 with weak cross-reactivity to other coronaviruses, while adult blood or tissues showed few such clones. These results highlight the prominence of early childhood B cell clonal expansions and cross-reactivity for future responses to novel pathogens.