Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection.

Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

Type I interferons induce an epigenetically distinct memory B cell subset in chronic viral infection.

Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.

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.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ T cell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ T cell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subset also expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ T cells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ T cell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Broadening Horizons: New Antibodies Against Influenza.

Seasonal influenza vaccine formulation efforts struggle to keep up with viral antigenic variation. Two studies now report engineered or naturally occurring human antibodies targeting the influenza hemagglutinin (HA) stem, with exceptional neutralizing breadth (Joyce et al., 2016; Kallewaard et al., 2016). Antibodies with similar structural features are elicited in multiple subjects, suggesting that modified vaccine regimens could provide broad protection.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

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.

Genetic and structural basis of the human anti-α-galactosyl antibody response.

Humans lack the capacity to produce the Galα1-3Galß1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome.

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

On being the right size: antibody repertoire formation in the mouse and human.

The immune systems of all mammals include populations of B cells producing antibodies with incredibly diverse specificities. Repertoire diversity has been described as the "miracle of immunology," and it was long thought to be the result of essentially stochastic processes. Recently, however, analysis of high throughput gene sequencing data has shown that hard-wired biases in these processes result in antibody repertoires that are broadly predictable. The repertoires of mice and humans are both predictable, but they are strikingly different. In this review, features of the naïve antibody repertoires of the two species are contrasted. We show that the mouse repertoire includes a conspicuous population of public clonotypes that are shared by different individuals of an inbred strain. These clonotypes are the result of gene rearrangements that involve little gene processing. By skewing repertoire formation toward such sequences, which probably target commonly encountered pathogens, it may be that the relatively small mouse repertoire is appropriate and effective despite its size. Species like the mouse face challenges that are a direct consequence of their small body sizes and the limitations this places on the antibody arsenal-particularly early in ontogeny. We propose that it is the differences in the naïve repertoires of mice and humans, and the differences in the ways these repertoires are used, which ensure that the very different biological needs of the two species are met. The processes that contribute to repertoire formation may appear to be stochastic, but in both species, evolution has left little to chance.

DJ Pairing during VDJ Recombination Shows Positional Biases That Vary among Individuals with Differing IGHD Locus Immunogenotypes.

Human IgH diversity is influenced by biases in the pairing of IGHD and IGHJ genes, but these biases have not been described in detail. We used high-throughput sequencing of VDJ rearrangements to explore DJ pairing biases in 29 individuals. It was possible to infer three contrasting IGHD-IGHJ haplotypes in nine of these individuals, and two of these haplotypes include deletion polymorphisms involving multiple contiguous IGHD genes. Therefore, we were able to explore how the underlying genetic makeup of the H chain locus influences the formation of particular DJ pairs. Analysis of nonproductive rearrangements demonstrates that 3' IGHD genes tend to pair preferentially with 5' IGHJ genes, whereas 5' IGHD genes pair preferentially with 3' IGHJ genes; the relationship between IGHD gene pairing frequencies and IGHD gene position is a near linear one for each IGHJ gene. However, striking differences are seen in individuals who carry deletion polymorphisms in the D locus. The absence of different blocks of IGHD genes leads to increases in the utilization frequencies of just a handful of genes, and these genes have no clear positional relationships to the deleted genes. This suggests that pairing frequencies may be influenced by additional complex positional relationships that perhaps arise from chromatin structure. In contrast to IGHD gene usage, IGHJ gene usage is unaffected by the IGHD gene-deletion polymorphisms. Such an outcome would be expected if the recombinase complex associates with an IGHJ gene before associating with an IGHD gene partner.

Ability to develop broadly neutralizing HIV-1 antibodies is not restricted by the germline Ig gene repertoire.

The human Ig repertoire is vast, producing billions of unique Abs from a limited number of germline Ig genes. The IgH V region (IGHV) is central to Ag binding and consists of 48 functional genes. In this study, we analyzed whether HIV-1-infected individuals who develop broadly neutralizing Abs show a distinctive germline IGHV profile. Using both 454 and Illumina technologies, we sequenced the IGHV repertoire of 28 HIV-infected South African women from the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 002 and 004 cohorts, 13 of whom developed broadly neutralizing Abs. Of the 259 IGHV alleles identified in this study, approximately half were not found in the International Immunogenetics Database (IMGT). This included 85 entirely novel alleles and 38 alleles that matched rearranged sequences in non-IMGT databases. Analysis of the rearranged H chain V region genes of mAbs isolated from seven of these women, as well as previously isolated broadly neutralizing Abs from other donors, provided evidence that at least eight novel or non-IMGT alleles contributed to functional Abs. Importantly, we found that, despite a wide range in the number of IGHV alleles in each individual, including alleles used by known broadly neutralizing Abs, there were no significant differences in germline IGHV repertoires between individuals who do and do not develop broadly neutralizing Abs. This study reports novel IGHV repertoires and highlights the importance of a fully comprehensive Ig database for germline gene usage prediction. Furthermore, these data suggest a lack of genetic bias in broadly neutralizing Ab development in HIV-1 infection, with positive implications for HIV vaccine design.

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy.

BACKGROUND: Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood. OBJECTIVE: We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT. METHODS: We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT. RESULTS: Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. CONCLUSION: In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.

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.

Human immunoglobulin classes and subclasses show variability in VDJ gene mutation levels.

Somatic point mutations provide glimpses into B-cell histories, and mutation numbers generally correlate with antibody affinity. We recently proposed a model of human isotype function, based in part on mutation analysis, in which the dominant pathway of isotype switching involves B cells moving sequentially through the four immunoglobulin (Ig) G subclasses. This should result in predictable differences in affinity between isotypes, and this helps explain how different isotypes work together. The model built on analysis of rearranged immunoglobulin heavy chain sequences amplified from Papua New Guinean villagers, which showed highly significant differences in the mean number of V-REGION mutations in sequences, associated with the different IgG subclasses. To determine whether this relationship between mutation levels and isotypes is a more general phenomenon, the present study was conducted in healthy, urban residents of Sydney, Australia. VDJ sequences were generated from eight individuals, using 454 pyrosequencing, from cells expressing all isotypes except IgD and IgE. This resulted in 35 118 unique, productive VDJ sequences for the study. The data confirm that VDJ genes associated with progressively more 3' Ig heavy chain gamma (IGHG) constant region genes show increasing levels of point mutation. Mean V-REGION mutations in IgA1 and IgA2 sequences were similar. Patterns of mutations also differed between isotypes. Despite their association with T-independent responses, IgG2 sequences showed significantly more mutational evidence of antigen selection than other IgG isotypes. Antigen selection was also significantly higher in IgA2 than in IgA1 sequences, raising the possibility of a preferential switch pathway from IGHG2 to IGHA2.

Improvement of immune dysregulation in individuals with long COVID at 24-months following SARS-CoV-2 infection.

This study investigates the humoral and cellular immune responses and health-related quality of life measures in individuals with mild to moderate long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24 months. LC participants show elevated nucleocapsid IgG levels at 3 months, and higher neutralizing capacity up to 8 months post-infection. Increased spike-specific and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells were observed at 3 and 8 months, but these differences do not persist at 24 months. Some LC participants had detectable IFN-γ and IFN-ß, that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at the 24 month timepoint shows similar immune cell proportions and reconstitution of naïve T and B cell subsets in LC and MC. No significant differences in exhaustion scores or antigen-specific T cell clones are observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24 months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count are associated with improvements in health-related quality of life.