Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. Superantigen specificity for different Vβ chains results in Vβ skewing, whereby T cells with specific Vβ chains and diverse antigen specificity are overrepresented in the T cell receptor (TCR) repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCRβ variable gene 11-2 (TRBV11-2), with up to 24% of clonal T cell space occupied by TRBV11-2 T cells, which correlated with MIS-C severity and serum cytokine levels. Analysis of TRBJ gene usage and complementarity-determining region 3 (CDR3) length distribution of MIS-C expanded TRBV11-2 clones revealed extensive junctional diversity. Patients with TRBV11-2 expansion shared HLA class I alleles A02, B35, and C04, indicating what we believe is a novel mechanism for CDR3-independent T cell expansion. In silico modeling indicated that polyacidic residues in the Vβ chain encoded by TRBV11-2 (Vβ21.3) strongly interact with the superantigen-like motif of SARS-CoV-2 spike glycoprotein, suggesting that unprocessed SARS-CoV-2 spike may directly mediate TRBV11-2 expansion. Overall, our data indicate that a CDR3-independent interaction between SARS-CoV-2 spike and TCR leads to T cell expansion and possibly activation, which may account for the clinical presentation of MIS-C.

Multisystem inflammatory syndrome in children (MIS-C) manifests as a severe and uncontrolled inflammatory response with multiorgan involvement, occurring weeks after SARS-CoV-2 infection. Here, we utilized proteomics, RNA sequencing, autoantibody arrays, and B cell receptor (BCR) repertoire analysis to characterize MIS-C immunopathogenesis and identify factors contributing to severe manifestations and intensive care unit admission. Inflammation markers, humoral immune responses, neutrophil activation, and complement and coagulation pathways were highly enriched in MIS-C patient serum, with a more hyperinflammatory profile in severe than in mild MIS-C cases. We identified a strong autoimmune signature in MIS-C, with autoantibodies targeted to both ubiquitously expressed and tissue-specific antigens, suggesting autoantigen release and excessive antigenic drive may result from systemic tissue damage. We further identified a cluster of patients with enhanced neutrophil responses as well as high anti-Spike IgG and autoantibody titers. BCR sequencing of these patients identified a strong imprint of antigenic drive with substantial BCR sequence connectivity and usage of autoimmunity-associated immunoglobulin heavy chain variable region (IGHV) genes. This cluster was linked to a TRBV11-2 expanded T cell receptor (TCR) repertoire, consistent with previous studies indicating a superantigen-driven pathogenic process. Overall, we identify a combination of pathogenic pathways that culminate in MIS-C and may inform treatment.

A considerable fraction of B cells recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with germline-encoded elements of their B cell receptor, resulting in the production of neutralizing and nonneutralizing antibodies. We found that antibody sequences from different discovery cohorts shared biochemical properties and could be retrieved across validation cohorts, confirming the stereotyped character of this naive response in coronavirus disease 2019 (COVID-19). While neutralizing antibody sequences were found independently of disease severity, in line with serological data, individual nonneutralizing antibody sequences were associated with fatal clinical courses, suggesting detrimental effects of these antibodies. We mined 200 immune repertoires from healthy individuals and 500 repertoires from patients with blood or solid cancers — all acquired prior to the pandemic — for SARS-CoV-2 antibody sequences. While the largely unmutated B cell rearrangements occurred in a substantial fraction of immune repertoires from young and healthy individuals, these sequences were less likely to be found in individuals over 60 years of age and in those with cancer. This reflects B cell repertoire restriction in aging and cancer, and may to a certain extent explain the different clinical courses of COVID-19 observed in these risk groups. Future studies will have to address if this stereotyped B cell response to SARS-CoV-2 emerging from unmutated antibody rearrangements will create long-lived memory.

Tumor cells shed DNA into the plasma. “Liquid biopsy” analysis of mutations or other genomic alterations in circulating cell-free DNA (cfDNA) may provide us with a tool to detect minimal residual cancer, comprehensively profile the genomic tumor landscape in search of druggable targets, and monitor cancers non-invasively over time for treatment failure or emerging treatment-resistant tumor subclones. While liquid biopsies have not yet entered routine clinical management in patients with gastric and gastroesophageal junction cancers, this group of diseases may benefit from such advanced diagnostic tools due to their pronounced genetic spatiotemporal heterogeneity and limitations in imaging sensitivity. Moreover, as the armamentarium of targeted treatment approaches and immunotherapies expands, cfDNA analyses may reveal their utility not only as a biomarker of response but also for precision monitoring. In this review, we discuss the different applications of cfDNA analyses in patients with gastric and gastroesophageal junction cancer and the technical challenges that such liquid biopsies have yet to overcome.

Background: Chronic HCV infection leads to a complex interplay with adaptive immune cells that may result in B cell dyscrasias like cryoglobulinemia or lymphoma. While direct-acting antiviral therapy has decreased the incidence of severe liver damage, its effect on extrahepatic HCV manifestations such as B cell dyscrasias is still unclear. Methods: We sequenced B cell receptor (BCR) repertoires in patients with chronic HCV mono-infection and patients with HCV with a sustained virological response (SVR) after direct-acting antiviral therapy. This data set was mined for highly neutralizing HCV antibodies and compared to a diffuse large B cell lymphoma data set. The TKO model was used to test the signaling strength of selected B-BCRs in vitro. Single-cell RNA sequencing of chronic HCV and HCV SVR samples was performed to analyze the transcriptome of B cells with HCV-neutralizing antigen receptors. Results: We identified a B cell fingerprint with high richness and somatic hypermutation in patients with chronic HCV and SVR. Convergence to specific immunoglobulin genes produced high-connectivity complementarity-determining region 3 networks. In addition, we observed that IGHV1-69 CDR1 and FR3 mutations characterizing highly neutralizing HCV antibodies corresponded to recurrent point mutations found in clonotypic BCRs of high-grade lymphomas. These BCRs did not show autonomous signaling but a lower activation threshold in an in vitro cell model for the assessment of BCR signaling strength. Single-cell RNA sequencing revealed that B cells carrying these point mutations showed a persisting oncogenic transcriptome signature with dysregulation in signaling nodes such as CARD11, MALT1, RelB, MAPK, and NFAT. Conclusions: We provide evidence that lymphoma-like cells derive from the anti-HCV immune response. In many patients, these cells persist for years after SVR and can be interpreted as a mechanistic basis for HCV-related B cell dyscrasias and increased lymphoma risk even beyond viral elimination. Export