Schmitz, B. | bioRxiv Science

bioRxiv2025

T cell ectosomes promote antibody responses through cognate TCR-pMHC interactions

Protective antibody-mediated immunity requires effective T cell-mediated help. Recognition of peptide antigens presented by major histocompatibility complex class II molecules (pMHCII), via cognate T cell antigen receptors (TCR), activates CD4+ T helper cells to upregulate expression of CD40L and helper cytokines. CD40L-CD40 interactions at T-B cell synapses and secreted cytokines are well established mediators of T cell help. Whether engaged pMHCII also transmits signals that help B cells remains unresolved. Here, we show that TCR-enriched nanoscale vesicles shed by activated T cells (ectosomes) are transferred to antigen-primed B cells, where they engage and cluster cognate pMHCII, triggering signaling and specific IgG antibody production. Disruption of ectosome release attenuates B cell antibody production, while native and synthetic ectosomes boost antibody responses. We conclude that T cell ectosomes constitute a new modality of help for B cells, delivered through engagement of pMHCII by cognate ectosomal TCR.

immunology↗

bioRxiv2025

ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples

Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platforms performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of SARS-CoV-2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/647077v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a81075org.highwire.dtl.DTLVardef@d07f82org.highwire.dtl.DTLVardef@634beborg.highwire.dtl.DTLVardef@10d4b25_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗