The CD8+ T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.

A novel network-based method identifies a cuproplasia-related pan-cancer gene signature to predict patient outcome.

Copper is a vital micronutrient involved in many biological processes and is an essential component of tumour cell growth and migration. Copper influences tumour growth through a process called cuproplasia, defined as abnormal copper-dependent cell-growth and proliferation. Copper-chelation therapy targeting this process has demonstrated efficacy in several clinical trials against cancer. While the molecular pathways associated with cuproplasia are partially known, genetic heterogeneity across different cancer types has limited the understanding of how cuproplasia impacts patient survival. Utilising RNA-sequencing data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) datasets, we generated gene regulatory networks to identify the critical cuproplasia-related genes across 23 different cancer types. From this, we identified a novel 8-gene cuproplasia-related gene signature associated with pan-cancer survival, and a 6-gene prognostic risk score model in low grade glioma. These findings highlight the use of gene regulatory networks to identify cuproplasia-related gene signatures that could be used to generate risk score models. This can potentially identify patients who could benefit from copper-chelation therapy and identifies novel targeted therapeutic strategies.

Chimeric Antigen Receptor T Cell Therapy Targeting Epithelial Cell Adhesion Molecule in Gastric Cancer: Mechanisms of Tumor Resistance.

Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is frequently overexpressed in various carcinomas. We have developed chimeric antigen receptor (CAR) T cells specifically targeting EpCAM for the treatment of gastric cancer. This study sought to unravel the precise mechanisms by which tumors evade immune surveillance and develop resistance to CAR T cell therapy. Through a combination of whole-body CAR T cell imaging and single-cell multiomic analyses, we uncovered intricate interactions between tumors and tumor-infiltrating lymphocytes (TILs). In a gastric cancer model, tumor-infiltrating CD8 T cells exhibited both cytotoxic and exhausted phenotypes, while CD4 T cells were mainly regulatory T cells. A T cell receptor (TCR) clonal analysis provided evidence of CAR T cell proliferation and clonal expansion within resistant tumors, which was substantiated by whole-body CAR T cell imaging. Furthermore, single-cell transcriptomics showed that tumor cells in mice with refractory or relapsing outcomes were enriched for genes involved in major histocompatibility complex (MHC) and antigen presentation pathways, interferon-γ and interferon-α responses, mitochondrial activities, and a set of genes (e.g., CD74, IDO1, IFI27) linked to tumor progression and unfavorable disease prognoses. This research highlights an approach that combines imaging and multiomic methodologies to concurrently characterize the evolution of tumors and the differentiation of CAR T cells.

CAR+ and CAR- T cells share a differentiation trajectory into an NK-like subset after CD19 CAR T cell infusion in patients with B cell malignancies.

Chimeric antigen receptor (CAR) T cell therapy is effective in treating B cell malignancies, but factors influencing the persistence of functional CAR+ T cells, such as product composition, patients' lymphodepletion, and immune reconstitution, are not well understood. To shed light on this issue, here we conduct a single-cell multi-omics analysis of transcriptional, clonal, and phenotypic profiles from pre- to 1-month post-infusion of CAR+ and CAR- T cells from patients from a CARTELL study (ACTRN12617001579381) who received a donor-derived 4-1BB CAR product targeting CD19. Following infusion, CAR+ T cells and CAR- T cells shows similar differentiation profiles with clonally expanded populations across heterogeneous phenotypes, demonstrating clonal lineages and phenotypic plasticity. We validate these findings in 31 patients with large B cell lymphoma treated with CD19 CAR T therapy. For these patients, we identify using longitudinal mass-cytometry data an association between NK-like subsets and clinical outcomes at 6 months with both CAR+ and CAR- T cells. These results suggest that non-CAR-derived signals can provide information about patients' immune recovery and be used as correlate of clinically relevant parameters.

Peripheral ameloblastoma: case report and our experience 20 years.

BACKGROUND AND AIM: The purpose of this study was to determine the clinical and histological features and treatment of peripheral ameloblastoma. Peripheral Ameloblastoma is a rare benign odontogenic tumor that concerns soft tissue and have a typical extraosseous localization. METHODS: Aim of this work is to show its clinical and histological characters, in order to define some useful information for differential diagnosis with other oral neoformations, comparing literature with our data, collected in ten years of clinical activity of Oral and Maxillofacial Surgery Unit of Policlinico Tor Vergata in Rome. RESULTS: Prognosis of PA is certainly favourable, with a restitutio ad integrum close to 100%. In the period between October 2011 and November 2021, we reported 8 diagnoses of P.A. Medium age of the group with diagnosis of PA was 71,4 y with a SD: 3,65. P.A.'s incidence in our sample of patients was 0,26%. CONCLUSIONS: PA is a benign odontogenic tumor that requires a careful diagnosis, a complete surgical eradication and adequate follow up, because malignant evolution is rare but possible.

High activation levels maintained in receptor-binding domain-specific memory B cells in people with severe coronavirus disease 2019.

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen-specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)-specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at ~2 months and ~4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor-kappa B in the severe group, involving CD80, FOS, CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80hi TNFAIP3hi and CD11chi CD95hi at the transcriptome level. Both groups revealed an increase in somatic hypermutation over time, indicating progressive evolution of humoral memory. This study revealed distinct molecular signatures of long-term RBD-specific MBCs in convalescence, indicating that the longevity of these cells may differ depending on acute COVID-19 severity.

Identification of human progenitors of exhausted CD8+ T cells associated with elevated IFN-γ response in early phase of viral infection.

T cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of individuals with primary infection using single-cell multi-omics to identify the functions and phenotypes of HCV-specific CD8+ T cells. Early elevated IFN-γ response against the transmitted virus is associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome, we find heterogeneous subsets of progenitors of exhaustion, based on the level of PD-1 expression and loss of AP-1 transcription factors. Intra-clonal analysis shows distinct trajectories with multiple fates and evolutionary plasticity of precursor cells. These findings challenge the current paradigm on the contribution of CD8+ T cells to HCV disease outcome and provide data for future studies on T cell differentiation in human infections.

Haematopoietic Stem Cell Transplantation Results in Extensive Remodelling of the Clonal T Cell Repertoire in Multiple Sclerosis.

Autologous haematopoietic stem cell transplantation (AHSCT) is a vital therapeutic option for patients with highly active multiple sclerosis (MS). Rates of remission suggest AHSCT is the most effective form of immunotherapy in controlling the disease. Despite an evolving understanding of the biology of immune reconstitution following AHSCT, the mechanism by which AHSCT enables sustained disease remission beyond the period of lymphopenia remains to be elucidated. Auto-reactive T cells are considered central to MS pathogenesis. Here, we analyse T cell reconstitution for 36 months following AHSCT in a cohort of highly active MS patients. Through longitudinal analysis of sorted naïve and memory T cell clones, we establish that AHSCT induces profound changes in the dominant T cell landscape of both CD4+ and CD8+ memory T cell clones. Lymphopenia induced homeostatic proliferation is followed by clonal attrition; with only 19% of dominant CD4 (p <0.025) and 13% of dominant CD8 (p <0.005) clones from the pre-transplant repertoire detected at 36 months. Recovery of a thymically-derived CD4 naïve T cell repertoire occurs at 12 months and is ongoing at 36 months, however diversity of the naïve populations is not increased from baseline suggesting the principal mechanism of durable remission from MS after AHSCT relates to depletion of putative auto-reactive clones. In a cohort of MS patients expressing the MS risk allele HLA DRB1*15:01, public clones are probed as potential biomarkers of disease. AHSCT appears to induce sustained periods of disease remission with dynamic changes in the clonal T cell repertoire out to 36 months post-transplant.

Investigation of product-derived lymphoma following infusion of piggyBac-modified CD19 chimeric antigen receptor T cells.

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter-driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell-derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene-modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance.

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

Human CD8 T-stem cell memory subsets phenotypic and functional characterization are defined by expression of CD122 or CXCR3.

Long-lived T-memory stem cells (TSCM ) are key to both naturally occurring and vaccine-conferred protection against infection. These cells are characterized by the CD45RA+ CCR7+ CD95+ phenotype. Significant heterogeneity within the TSCM population is recognized, but distinguishing surface markers and functional characterization of potential subsets are lacking. Human CD8 TSCM subsets were identified in healthy subjects who had been previously exposed to CMV or Influenza (Flu) virus in flow cytometry by expression of CD122 or CXCR3, and then characterized in proliferation, multipotency, self-renewal, and intracellular cytokine production (TNF-α, IL-2, IFN-γ), together with transcriptomic profiles. The TSCM CD122hi -expressing subset (versus CD122lo ) demonstrated greater proliferation, greater multipotency, and enhanced polyfunctionality with higher frequencies of triple positive (TNF-α, IL-2, IFN-γ) cytokine-producing cells upon exposure to recall antigen. The TSCM CXCR3lo subpopulation also had increased proliferation and polyfunctional cytokine production. Transcriptomic analysis further showed that the TSCM CD122hi population had increased expression of activation and homing molecules, such as Ccr6, Cxcr6, Il12rb, and Il18rap, and downregulated cell proliferation inhibitors, S100A8 and S100A9. These data reveal that the TSCM CD122hi phenotype is associated with increased proliferation, enhanced multipotency and polyfunctionality with an activated memory-cell like transcriptional profile, and hence, may be favored for induction by immunization and for adoptive immunotherapy.

Prophylactic antigen-specific T-cells targeting seven viral and fungal pathogens after allogeneic haemopoietic stem cell transplant.

OBJECTIVES: Adoptive immunotherapy using donor-derived antigen-specific T-cells can prevent and treat infection after allogeneic haemopoietic stem cell transplant (HSCT). METHODS: We treated 11 patients with a prophylactic infusion of 2 × 107 cells per square metre donor-derived T-cells targeting seven infections (six viral and one fungal) following HSCT. Targeted pathogens were cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, varicella zoster virus, influenza, BK virus (BKV) and Aspergillus fumigatus. RESULTS: T-cell products were successfully generated in all patients with 10 products responsive to 6 or 7 infections. T-cell infusions were associated with increases in antigen-experienced activated CD8+ T-cells by day 30. CMV, EBV and BKV reactivation occurred in the majority of patients and was well controlled except where glucocorticoids were administered soon after T-cell infusion. Three patients in that circumstance developed CMV tissue infection. No patient required treatment for invasive fungal infection. The most common CMV and EBV TCR clonotypes in the infusion product became the most common clonotypes seen at day 30 post-T-cell infusion. Donors and their recipients were recruited to the study prior to transplant. Grade III/IV graft-versus-host disease developed in four patients. At a median follow-up of 390 days post-transplant, six patients had died, 5 of relapse, and 1 of multi-organ failure. Infection did not contribute to death in any patient. CONCLUSION: Rapid reconstitution of immunity to a broad range of viral and fungal infections can be achieved using a multi-pathogen-specific T-cell product. The development of GVHD after T-cell infusion suggests that infection-specific T-cell therapy after allogeneic stem cell transplant should be combined with other strategies to reduce graft-versus-host disease.

Quantitative analysis of the splice variants expressed by the major hepatitis B virus genotypes.

Hepatitis B virus (HBV) is a major human pathogen that causes liver diseases. The main HBV RNAs are unspliced transcripts that encode the key viral proteins. Recent studies have shown that some of the HBV spliced transcript isoforms are predictive of liver cancer, yet the roles of these spliced transcripts remain elusive. Furthermore, there are nine major HBV genotypes common in different regions of the world, these genotypes may express different spliced transcript isoforms. To systematically study the HBV splice variants, we transfected human hepatoma cells, Huh7, with four HBV genotypes (A2, B2, C2 and D3), followed by deep RNA-sequencing. We found that 13-28 % of HBV RNAs were splice variants, which were reproducibly detected across independent biological replicates. These comprised 6 novel and 10 previously identified splice variants. In particular, a novel, singly spliced transcript was detected in genotypes A2 and D3 at high levels. The biological relevance of these splice variants was supported by their identification in HBV-positive liver biopsy and serum samples, and in HBV-infected primary human hepatocytes. Interestingly the levels of HBV splice variants varied across the genotypes, but the spliced pregenomic RNA SP1 and SP9 were the two most abundant splice variants. Counterintuitively, these singly spliced SP1 and SP9 variants had a suboptimal 5' splice site, supporting the idea that splicing of HBV RNAs is tightly controlled by the viral post-transcriptional regulatory RNA element.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.

Ex vivo enrichment of PRAME antigen-specific T cells for adoptive immunotherapy using CD137 activation marker selection.

OBJECTIVE: Adoptive immunotherapy with ex vivo expanded tumor-specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP-compliant manufacturing method for PRAME-specific T cells from healthy donors for adoptive immunotherapy. METHODS: Mononuclear cells were pulsed with PRAME 15-mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137neg fraction in medium supplemented with interleukin (IL)-2, IL-7 and IL-15. Cultured T cells were restimulated with antigen-pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re-exposure were assessed with flow cytometry, enzyme-linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T-cell receptor (TCR) beta clonality studies were performed on selected cultures. RESULTS: PRAME-stimulated cultures (n = 10) had mean expansion of 2500-fold at day 18. Mean CD3+ percentage was 96% with CD4:CD8 ratio of 4:1. Re-exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)-γ (mean: 12.2%) and TNF-α (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype (CXCR3+/CCR4neg/CCR6neg/Tbet+, mean: 73%) and cytokine production including IL-2, IL-4, IL-8, IL-13 and GM-CSF (2%, 6%, 8%, 4% and 11%, respectively). CONCLUSION: PRAME-specific T cells for adoptive immunotherapy were enriched from healthy donor mononuclear cells. The products were oligoclonal, exhibited Th1 phenotype and produced multiple cytokines.

Rapidly expanded partially HLA DRB1-matched fungus-specific T cells mediate in vitro and in vivo antifungal activity.

Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4+ T cells with potent antifungal characteristics, including production of tumor necrosis factor α, interferon γ, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice-compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen-matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.

Mass cytometry reveals immune signatures associated with cytomegalovirus (CMV) control in recipients of allogeneic haemopoietic stem cell transplant and CMV-specific T cells.

OBJECTIVES: Cytomegalovirus (CMV) is known to have a significant impact on immune recovery post-allogeneic haemopoietic stem cell transplant (HSCT). Adoptive therapy with donor-derived or third-party virus-specific T cells (VST) can restore CMV immunity leading to clinical benefit in prevention and treatment of post-HSCT infection. We developed a mass cytometry approach to study natural immune recovery post-HSCT and assess the mechanisms underlying the clinical benefits observed in recipients of VST. METHODS: A mass cytometry panel of 38 antibodies was utilised for global immune assessment (72 canonical innate and adaptive immune subsets) in HSCT recipients undergoing natural post-HSCT recovery (n = 13) and HSCT recipients who received third-party donor-derived CMV-VST as salvage for unresponsive CMV reactivation (n = 8). RESULTS: Mass cytometry identified distinct immune signatures associated with CMV characterised by a predominance of innate cells (monocytes and NK) seen early and an adaptive signature with activated CD8+ T cells seen later. All CMV-VST recipients had failed standard antiviral pharmacotherapy as a criterion for trial involvement; 5/8 had failed to develop the adaptive immune signature by study enrolment despite significant CMV antigen exposure. Of these, VST administration resulted in development of the adaptive signature in association with CMV control in three patients. Failure to respond to CMV-VST in one patient was associated with persistent absence of the adaptive immune signature. CONCLUSION: The clinical benefit of CMV-VST may be mediated by the recovery of an adaptive immune signature characterised by activated CD8+ T cells.

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.

Exploring and analysing single cell multi-omics data with VDJView.

BACKGROUND: Single cell RNA sequencing provides unprecedented opportunity to simultaneously explore the transcriptomic and immune receptor diversity of T and B cells. However, there are limited tools available that simultaneously analyse large multi-omics datasets integrated with metadata such as patient and clinical information. RESULTS: We developed VDJView, which permits the simultaneous or independent analysis and visualisation of gene expression, immune receptors, and clinical metadata of both T and B cells. This tool is implemented as an easy-to-use R shiny web-application, which integrates numerous gene expression and TCR analysis tools, and accepts data from plate-based sorted or high-throughput single cell platforms. We utilised VDJView to analyse several 10X scRNA-seq datasets, including a recent dataset of 150,000 CD8+ T cells with available gene expression, TCR sequences, quantification of 15 surface proteins, and 44 antigen specificities (across viruses, cancer, and self-antigens). We performed quality control, filtering of tetramer non-specific cells, clustering, random sampling and hypothesis testing to discover antigen specific gene signatures which were associated with immune cell differentiation states and clonal expansion across the pathogen specific T cells. We also analysed 563 single cells (plate-based sorted) obtained from 11 subjects, revealing clonally expanded T and B cells across primary cancer tissues and metastatic lymph-node. These immune cells clustered with distinct gene signatures according to the breast cancer molecular subtype. VDJView has been tested in lab meetings and peer-to-peer discussions, showing effective data generation and discussion without the need to consult bioinformaticians. CONCLUSIONS: VDJView enables researchers without profound bioinformatics skills to analyse immune scRNA-seq data, integrating and visualising this with clonality and metadata profiles, thus accelerating the process of hypothesis testing, data interpretation and discovery of cellular heterogeneity. VDJView is freely available at https://bitbucket.org/kirbyvisp/vdjview.