Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions.

Background: Apolipoprotein C-III (APOC3) plays a crucial role in triglyceride metabolism and is closely associated with cardiovascular disease risk. Elevated APOC3 levels contribute to higher plasma triglycerides and increased risk of atherosclerosis, making APOC3 expression an attractive and logical therapeutic target. Methods: While studying various APOC3 transcript isoforms expressed in hepatoma cell lines (HepG2, Huh7) and healthy liver tissue using publicly available long-read RNA sequencing, we found three novel APOC3 isoforms. These isoforms were validated through RT-PCR and Sanger sequencing. Results: All three novel isoforms are splicing variants of the MANE transcript, APOC3-201. Isoforms 1 and 2 exhibit splicing patterns similar to APOC3-201 from exons 2–4; however, isoform 1 shares its exon 1 splicing pattern with APOC3-203, while isoform 2 features an extended exon 1 that includes exon 1a, the adjacent intronic region, and exon 1b. The third isoform closely resembles APOC3-201, but lacks exon 2, which contains the translation start codon. Remarkably, similar APOC3 splicing patterns and transcript variants were observed in Caco-2 cells, a model of the small intestine, indicating that these isoforms are not liver-specific. Conclusions: This study identifies three novel APOC3 isoforms and highlights their expression in both hepatic and intestinal cell models. Further studies are needed to elucidate the functional roles of these novel isoforms and their contribution to the regulation of APOC3 gene expression.

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases, including inclusion body myositis (IBM), Sjögren’s syndrome, and systemic lupus erythematosus. While they have diagnostic relevance for IBM, little is known about the autoreactive B cells that produce these antibodies. To address this, we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in Escherichia coli. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase, preserving the protein’s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation, this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A, the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases.