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Biology subjects

Ado, S.

Publications and source records attributed to Ado, S..

3 recordsLinked to original sources
bioRxiv2025

Clonal analysis of SepSecS-specific CD4 T cells reveals a new HLA-DPA1*02:01/HLA-DPB1*01:01-restricted immunodominant epitope in autoimmune hepatitis

Autoreactive CD4 T cells, recognizing liver-self-antigens such as SepSecS, are main drivers of the chronic inflammatory response during autoimmune hepatitis (AIH). Previous studies have uncovered immunodominant SepSecS epitopes often associated with HLA-DRB1*03 or HLA-DRB1*04 restriction, two alleles enriched in AIH population. However, HLA restriction of numerous SepSecS epitopes remains incomplete and it is still unclear if immunodominant epitopes could be presented by non-HLA-DR molecules. Here, we investigated epitope recognition of SepSecS-specific TCRs isolated from AIH patients, and their HLA restriction by generating TCR hybridoma cell lines. Seventeen TCRs recognized eight SepSecS epitopes with four distinct HLA restrictions, including a novel HLA-DPA1*02:01/DPB1*01:01-restricted SepSecS epitope. TCR clustering analysis using GLIPH2 algorithm suggested that this epitope is recognized by multiple distinct TCRs in HLA-DPA1*02:01[~]HLA-DPB1*01:01 patients. Our study provides new insights into liver-self-antigen T cell reactivity during AIH, which could offer potential therapeutic strategies by targeting autoreactive CD4 T cells.

immunology↗
bioRxiv2024

FB5P-seq-mAbs: monoclonal antibody production from FB5P-seq libraries for integrative single-cell analysis of B cells

Parallel analysis of phenotype, transcriptome and antigen receptor sequence in single B cells is a useful method for tracking B cell activation and maturation during immune responses. However, in most cases, the specificity and affinity of the B cell antigen receptor cannot be inferred from its sequence. Antibody cloning and expression from single B cells is then required for functional assays. Here we propose a method that integrates FACS-based 5-end single-cell RNA sequencing (FB5P-seq) and monoclonal antibody cloning for integrative analysis of single B cells. Starting from a cell suspension, single B cells are FACS-sorted into 96-well plates for reverse transcription, cDNA barcoding and amplification. A fraction of the single-cell cDNA is used for preparing 5-end RNA-seq libraries that are sequenced for retrieving transcriptome-wide gene expression and paired BCR sequences. The archived cDNA of selected cells of interest is used as input for cloning heavy and light chain variable regions into antibody expression plasmid vectors. The corresponding monoclonal antibodies are produced by transient transfection of a eukaryotic producing cell line and purified for functional assays. We provide detailed step-by-step instructions and describe results obtained on ovalbumin-specific murine germinal center B cells after immunization. Our method is robust, flexible, cost-effective, and applicable to different B cell types and species. We anticipate it will be useful for mapping antigen specificity and affinity of rare B cell subsets characterized by defined gene expression and/or antigen receptor sequence.

immunology↗
bioRxiv2024

Transcriptomic, clonal, and functional analyses reveal Liver tissue-imprinted immuno-profile of circulating autoreactive CD4 T cells in autoimmune liver diseases

Autoimmune liver diseases (AILD) are immune-mediated disorders in which CD4 T cells play a central role. However, the link between circulating self-antigen-specific CD4 T cells and the targeted tissue has not been extensively studied in AILD. We hypothesized that circulating autoreactive CD4 T cells were clonally and functionally related to dominant intra-hepatic pathogenic CD4 T cell clones. Single cell transcriptomic analysis of circulating self-antigen-specific CD4 T cells revealed a specific B-helper and immuno-exhausted transcriptional profile, which was conserved for different autoantigens, but distinct from several other types of foreign antigen specificities. In the blood, the dominant hepatic CD4 T cell clones had a similar transcriptomic signature and were enriched in the PD-1+ TIGIT+ HLA-DR+ CD4 T cell subset. In a mouse model, antigen-specific CD4 T cells acquired the immuno-exhausted transcriptional profile when they accumulated in the liver after local antigen reactivity. Locally, immune checkpoint molecules controlled the response of antigen-specific CD4 T cells responsible for liver damage. Our study reveals the origin and biology of liver-derived autoreactive CD4 T cells in the blood of AILD patients that are imprinted by the liver environment, and suggest a dysregulation of the immune checkpoint molecules pathways. Our study enables tracking and isolating circulating autoreactive CD4 T cells for future diagnostic and therapeutic purposes.

immunology↗