7ad5ebae854f97844bae740229971003 pmc_11050013.pdf 884619780285592b074b49f49352e73d70e07ae0 pmc_11050013.pdf 361bbf68c4918b5120bf31dbe85404089289e92d5b900dba4120f131e2ad4a1a pmc_11050013.pdf Title: Precise Targeting of Autoantigen-Specific B Cells in Lupus Nephritis with Chimeric Autoantibody Receptor T Cells Subject: Despite conventional therapy, lupus nephritis (LN) remains a significant contributor to short- and long-term morbidity and mortality. B cell abnormalities and the production of autoantibodies against nuclear complexes like anti-dsDNA are recognised as key players in the pathogenesis of LN. To address the challenges of chronic immunosuppression associated with current therapies, we have engineered T cells to express chimeric autoantibody receptors (DNA-CAART) for the precise targeting of B cells expressing anti-dsDNA autoantibodies. T cells from LN patients were transduced using six different CAAR vectors based on their antigen specificity, including alpha-actinin, histone-1, heparan sulphate, or C1q. The cytotoxicity, cytokine production, and cell–cell contact of DNA-CAART were thoroughly investigated in co-culture experiments with B cells isolated from patients, both with and without anti-dsDNA positivity. The therapeutic effects were further evaluated using an in vitro immune kidney LN organoid. Among the six proposed DNA-CAART, DNA4 and DNA6 demonstrated superior selectively cytotoxic activity against anti-dsDNA+ B cells. Notably, DNA4-CAART exhibited improvements in organoid morphology, apoptosis, and the inflammatory process in the presence of IFN-stimulated anti-dsDNA+ B cells. Based on these findings, DNA4-CAART emerge as promising candidates for modulating autoimmunity and represent a novel approach for the treatment of LN. Keywords: lupus nephritis; CAART; anti-dsDNA; B cell Author: Cristina Solé, Maria Royo, Sebastian Sandoval, Teresa Moliné, Alejandra Gabaldón and Josefina Cortés-Hernández Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu Apr 11 11:10:24 2024 CEST ModDate: Thu Apr 11 11:15:41 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5207972 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ZZTQUO+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 ZFKCJT+EURM10 Type 1 Builtin yes yes yes 16 0 TMOOVW+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 JKPVFP+URWPalladioL-Roma Type 1 Custom yes yes yes 27 0 ZJDIYZ+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 CIQLLF+VnURWPalladioL Type 1 Custom yes yes 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TrueType WinAnsi yes yes no 324 0 IBOKFD+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 327 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-02-06 02:51:18 CET RepresentationInformation: pmc_11050013.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-01-23 02:37:01 CET Size: 5207972 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 596 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Patient Demographic Characteristics Destination: subsection.2.1 Item: Title: Design and Construction of Six DNA-CAART Destination: subsection.2.2 Item: Title: DNA4 and DNA6-CAART Show Higher Selective Depletion of Anti-dsDNA+ B Cells Destination: subsection.2.3 Item: Title: Confocal Live-Cell Imaging Provides Visualization of DNA4 and DNA6-CAART Interaction and Expansion with Anti-dsDNA+ B Cells Destination: subsection.2.4 Item: Title: Therapeutic Evaluation of DNA4 and DNA6-CAART Cells in 3D Immuno-Kidney Model Assay Destination: subsection.2.5 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Design and Construction of CAAR Vectors Destination: subsection.4.1 Item: Title: Patient Characteristics Destination: subsection.4.2 Item: Title: In Vitro Transduction and Expansion of CAART Cells Destination: subsection.4.3 Item: Title: In Vitro Cytotoxicity Assays Destination: subsection.4.4 Item: Title: Confocal Live-Cell Microscopy Interaction between B Cells and CAART Cells Destination: subsection.4.5 Item: Title: RNA Extraction and RT-qPCR Destination: subsection.4.6 Item: Title: Kidney Organoids B Cell Co-Culture to Evaluate Therapeutic CAART Cell Efficacy Destination: subsection.4.7 Item: Title: Immunofluorescence Analysis of Kidney Organoids after Exposure to CAART Cells Destination: subsection.4.8 Item: Title: Enzyme-Linked Immunosorbent Assay (ELISA) Destination: subsection.4.9 Item: Title: Statistical Analysis Destination: subsection.4.10 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Precise Targeting of Autoantigen-Specific B Cells in Lupus Nephritis with Chimeric Autoantibody Receptor T Cells Author: Cristina Solé, Maria Royo, Sebastian Sandoval, Teresa Moliné, Alejandra Gabaldón and Josefina Cortés-Hernández Subject: Despite conventional therapy, lupus nephritis (LN) remains a significant contributor to short- and long-term morbidity and mortality. B cell abnormalities and the production of autoantibodies against nuclear complexes like anti-dsDNA are recognised as key players in the pathogenesis of LN. To address the challenges of chronic immunosuppression associated with current therapies, we have engineered T cells to express chimeric autoantibody receptors (DNA-CAART) for the precise targeting of B cells expressing anti-dsDNA autoantibodies. T cells from LN patients were transduced using six different CAAR vectors based on their antigen specificity, including alpha-actinin, histone-1, heparan sulphate, or C1q. The cytotoxicity, cytokine production, and cell cell contact of DNA-CAART were thoroughly investigated in co-culture experiments with B cells isolated from patients, both with and without anti-dsDNA positivity. The therapeutic effects were further evaluated using an in vitro immune kidney LN organoid. Among the six proposed DNA-CAART, DNA4 and DNA6 demonstrated superior selectively cytotoxic activity against anti-dsDNA+ B cells. Notably, DNA4-CAART exhibited improvements in organoid morphology, apoptosis, and the inflammatory process in the presence of IFN-stimulated anti-dsDNA+ B cells. Based on these findings, DNA4-CAART emerge as promising candidates for modulating autoimmunity and represent a novel approach for the treatment of LN. 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