Xynapse-T — an antigen-specific scaffold for functional stimulation and expansion of T-cells 2259137

Abstract Introduction Precise and reproducible activation of antigen-specific T cells to evaluate functionality and efficient proliferation of specific cells is critical across diverse research areas, including CAR/TCR cell therapy monitoring, infectious disease response, vaccination, and cancer immunology. We previously demonstrated that MHC class I—based Xynapse-T reagents, which co-display MHC-peptide (MHCp) complexes and CD28 co-engagers, selectively stimulate antigen-specific CD8+ T cells and provide a cell-free platform for in vitro functional assessment. In this study, we extend the Xynapse-T technology to MHC class II artificial antigen-presenting cell (aAPC) scaffolds to enable targeted stimulation and expansion of antigen-specific CD4+ T cells. Methods Artificial antigen-presenting cell (aAPC) scaffolds were engineered by conjugating MHC class II peptide (MHCIIp ) complexes and CD28 co-engagers to a synthetic scaffold. These Xynapse-T reagents simultaneously engage the T cell receptor (TCR) and CD28 on CD4+ T cells, facilitating selective activation within a polyspecific T cell population. T cell activation was assessed through surface marker expression and proliferation assays. Results Xynapse-T reagents displaying MHCIIp and CD28 co-engagers effectively stimulated antigen-specific CD4+ T cells, resulting in: 1. Upregulation of activation markers (CD69, CD134) 2. Enhanced cytokine production (e.g. IFN-γ) 3. Robust T cell expansion No activation was observed with control Xynapse-T reagents displaying irrelevant MHCp. Conclusion Xynapse-T technology enables precise ex vivo stimulation, activation, and expansion of antigen-specific CD4+ T cells. This platform offers a versatile and highly specific tool for: • Potency assessment of engineered T cells • Generation of antigen-specific T cell lines • Enrichment of rare T cell populations Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)