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Impaired humoral responses, as well as an increased propensity for autoimmunity, play an important role in the development of immune system dysfunction associated with aging. Accumulation of a subset of atypical B cells, termed age-associated B cells (ABCs), is one of the key age-related changes in B cell compartments. ABCs are characterized by their distinct phenotypes, gene expression profiles, special survival requirements, variations in B cell receptor repertoires, and unique functions. Here, we summarize recent progress in the knowledge base related to the features of ABCs, their potential role in immune senescence, and their relationship with autoimmune diseases.

High-throughput sequencing has the power to reveal the nature of adaptive immunity as represented by the full complexity of T-cell receptor (TCR) and antibody (IG) repertoires, but is at present severely compromised by the quantitative bias, bottlenecks, and accumulated errors that inevitably occur in the course of library preparation and sequencing. Here we report an optimized protocol for the unbiased preparation of TCR and IG cDNA libraries for high-throughput sequencing, starting from thousands or millions of live cells in an investigated sample. Critical points to control are revealed, along with tips that allow researchers to minimize quantitative bias, accumulated errors, and cross-sample contamination at each stage, and to enhance the subsequent bioinformatic analysis. The protocol is simple, reliable, and can be performed in 1-2 days.

PurposeAuto-antibodies (auto-abs) to type I interferons (IFNs) have been identified in patients with life-threatening coronavirus disease 2019 (COVID-19), suggesting that the presence of auto-abs may be a risk factor for disease severity. We therefore investigated the mechanism underlying COVID-19 exacerbation induced by auto-abs to type I IFNs.MethodsWe evaluated plasma from 123 patients with COVID-19 to measure auto-abs to type I IFNs. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from the patients with auto-abs and conducted epitope mapping of the auto-abs.ResultsThree of 19 severe and 4 of 42 critical COVID-19 patients had neutralizing auto-abs to type I IFNs. Patients with auto-abs to type I IFNs showed no characteristic clinical features. scRNA-seq from 38 patients with COVID-19 revealed that IFN signaling in conventional dendritic cells and canonical monocytes was attenuated, and SARS-CoV-2-specific BCR repertoires were decreased in patients with auto-abs. Furthermore, auto-abs to IFN-α2 from COVID-19 patients with auto-abs recognized characteristic epitopes of IFN-α2, which binds to the receptor.ConclusionAuto-abs to type I IFN found in COVID-19 patients inhibited IFN signaling in dendritic cells and monocytes by blocking the binding of type I IFN to its receptor. The failure to properly induce production of an antibody to SARS-CoV-2 may be a causative factor of COVID-19 severity.

B cells play a crucial role in the immune response and contribute to various autoimmune diseases. Recent studies have revealed abnormalities in the B cell receptor (BCR) repertoire of patients with autoimmune diseases, with distinct features observed among different diseases and B cell subsets. Classically, BCR repertoire was used as an identifier of distinct antigen-specific clonotypes, but the recent advancement of analyzing large-scale repertoire has enabled us to use it as a tool for characterizing cellular biology. In this review, we provide an overview of the BCR repertoire in autoimmune diseases incorporating insights from our latest research findings. In systemic lupus erythematosus (SLE), we observed a significant skew in the usage of VDJ genes, particularly in CD27 + IgD + unswitched memory B cells and plasmablasts. Notably, autoreactive clones within unswitched memory B cells were found to be increased and strongly associated with disease activity, underscoring the clinical significance of this subset. Similarly, various abnormalities in the BCR repertoire have been reported in other autoimmune diseases such as rheumatoid arthritis. Thus, BCR repertoire analysis holds potential for enhancing our understanding of the underlying mechanisms involved in autoimmune diseases. Moreover, it has the potential to predict treatment effects and identify therapeutic targets in autoimmune diseases.

IntroductionTCR and BCR repertoire diversity plays a critical role in tumor immunity. Thus, analysis of TCR and BCR repertoires might help predict the clinical efficacy of anti-PD-1 treatment.MethodsBlood samples from 30 patients with non-small cell lung cancer (NSCLC) treated with anti-PD-1 antibody were collected before and six weeks after treatment initiation. The clinical significance of TCR and BCR repertoire diversity in peripheral blood was evaluated in all the enrolled patients (n = 30) or in the subset with (n = 10) or without (n = 20) EGFR/ALK mutation.ResultsTCR and BCR diversity was significantly correlated at baseline (R = 0.65; P = 1.6 × 10–4) and on treatment (R = 0.72; P = 1.2 × 10–5). Compared to non-responders (SD or PD), responders (PR) showed significantly decreased TCR and BCR diversity after treatment in the EGFR/ALK wild-type subset (P = 0.0014 and P = 0.034, respectively), but not in all the enrolled patients. The post-treatment reduction in TCR and BCR repertoire diversity was also significantly associated with the occurrence of adverse events in the EGFR/ALK wild-type subset (P = 0.022 and P = 0.014, respectively). Patients with more reduced TCR diversity showed better progression-free survival (PFS) in the EGFR/ALK wild-type subset (P = 0.011) but not in the mutant subset.ConclusionsThese findings suggest the clinical significance of changes in peripheral TCR and BCR repertoire diversity after anti-PD-1 treatment in patients with NSCLC without EGFR/ALK mutation. Monitoring of the peripheral TCR and BCR repertoires may serve as a surrogate marker for the early detection of EGFR/ALK wild-type NSCLC patients who would benefit from anti-PD-1 treatment.

Celiac disease (CeD) is a common autoimmune disorder caused by an abnormal immune response to dietary gluten proteins. The disease has high heritability. HLA is the major susceptibility factor, and the HLA effect is mediated via presentation of deamidated gluten peptides by disease-associated HLA-DQ variants to CD4+ T cells. In addition to gluten-specific CD4+ T cells the patients have antibodies to transglutaminase 2 (autoantigen) and deamidated gluten peptides. These disease-specific antibodies recognize defined epitopes and they display common usage of specific heavy and light chains across patients. Interactions between T cells and B cells are likely central in the pathogenesis, but how the repertoires of naïve T and B cells relate to the pathogenic effector cells is unexplored. To this end, we applied machine learning classification models to naïve B cell receptor (BCR) repertoires from CeD patients and healthy controls. Strikingly, we obtained a promising classification performance with an F1 score of 85%. Clusters of heavy and light chain sequences were inferred and used as features for the model, and signatures associated with the disease were then characterized. These signatures included amino acid (AA) 3-mers with distinct bio-physiochemical characteristics and enriched V and J genes. We found that CeD-associated clusters can be identified and that common motifs can be characterized from naïve BCR repertoires. The results may indicate a genetic influence by BCR encoding genes in CeD. Analysis of naïve BCRs as presented here may become an important part of assessing the risk of individuals to develop CeD. Our model demonstrates the potential of using BCR repertoires and in particular, naïve BCR repertoires, as disease susceptibility markers.

Dynamic changes of the paired heavy and light chain B cell receptor (BCR) repertoire provide an essential insight into understanding the humoral immune response post-SARS-CoV-2 infection and vaccination. However, differences between the endogenous paired BCR repertoire kinetics in SARS-CoV-2 infection and previously recovered/naïve subjects treated with the inactivated vaccine remain largely unknown. We performed single-cell V(D)J sequencing of B cells from six healthy donors with three shots of inactivated SARS-CoV-2 vaccine (BBIBP-CorV), five people who received the BBIBP-CorV vaccine after having recovered from COVID-19, five unvaccinated COVID-19 recovered patients and then integrated with public data of B cells from four SARS-CoV-2-infected subjects. We discovered that BCR variable (V) genes were more prominently used in the SARS-CoV-2 exposed groups (both in the group with active infection and in the group that had recovered) than in the vaccinated groups. The VH gene that expanded the most after SARS-CoV-2 infection was IGHV3-33, while IGHV3-23 in the vaccinated groups. SARS-CoV-2-infected group enhanced more BCR clonal expansion and somatic hypermutation than the vaccinated healthy group. A small proportion of public clonotypes were shared between the SARS-CoV-2 infected, vaccinated healthy, and recovered groups. Moreover, several public antibodies had been identified against SARS-CoV-2 spike protein. We comprehensively characterize the paired heavy and light chain BCR repertoire from SARS-CoV-2 infection to vaccination, providing further guidance for the development of the next-generation precision vaccine.

Cancer recurrence and chemoresistance are the leading causes of death in high-grade serous ovarian cancer (HGSOC) patients. However, the unique role of the immune environment in tumor progression for relapsed chemo-resistant patients remains elusive. In single-cell resolution, we characterized a comprehensive multi-dimensional cellular and immunological atlas from tumor, ascites, and peripheral blood of a chemo-resistant patient at different stages of treatment. Our results highlight a role in recurrence and chemoresistance of the immunosuppressive microenvironment in ascites, including MDSC-like myeloid and hypo-metabolic γδT cells, and of peripheral CD8 + effector T cells with chemotherapy-induced senescent/exhaustive. Importantly, paired TCR/BCR sequencing demonstrated relative conservation of TCR clonal expansion in hyper-expanded CD8 + T cells and extensive BCR clonal expansion without usage bias of V(D)J genes after chemotherapy. Thus, our study suggests strategies for ameliorating chemotherapy-induced immune impairment to improve the clinical outcome of HGSOC.

The vast diversity of mammalian adaptive antigen receptors allows for robust and efficient immune responses against a wide number of pathogens. The antigen receptor repertoire is built during the recombination of B and T cell receptor (BCR, TCR) loci and hypermutation of BCR loci. V(D)J recombination rearranges these antigen receptor loci, which are organized as an array of separate V, (D), and J gene segments. Transcription activation at the recombining locus leads to changes in the local three-dimensional architecture, which subsequently contributes to which gene segments are utilized for recombination. The endogenous retrovirus (ERV) mouse mammary tumor provirus 8 ( Mtv8 ) resides on mouse chromosome 6 interposed within the large array of light chain kappa V gene segments. As ERVs contribute to changes in genomic architecture by driving high levels of transcription of neighboring genes, it was suggested that Mtv8 could influence the BCR repertoire. We generated Mtv8 -deficient mice to determine if the ERV influences V(D)J recombination to test this possibility. We find that Mtv8 does not influence the BCR repertoire.

Notably, the majority of intracellular proteins are processed and presented on tumour cell surfaces as peptide-MHC class I complexes, representing a vast and largely untapped resource for immunotherapeutic intervention. The study bySnyder et al.reports a large-scale, high-resolution analysis of SARS-CoV-2-specific T-cell responses across over 1,400 individuals, identifying more than 70,000 TCRs linked to viral antigens through MIRA (1,2) and repertoire sequencing (3). Through comprehensive BCR repertoire analysis, they tracked the emergence and evolution of complementarity-determining region 3 sequences pre- and post-immune checkpoint inhibitor (ICI) treatment, with an emphasis on de novo clonotypes. Wang and colleaguesevaluated the efficacy and safety of camrelizumab combined with platinum-based chemotherapy (TP or FP) as a first-line treatment for advanced oesophageal squamous cell carcinoma (ESCC) and explored treatment response mechanisms using immune repertoire sequencing (IRS).