Nanoscale artificial antigen presenting cells to expand human antigen-specific cytotoxic CD4 T cells for immunotherapy 2258982

Abstract

CD4 T cells play key roles in immune modulation and cancer immunity. Increasing evidence shows they also exhibit cytotoxicity and can be harnessed for cancer immunotherapy. However, methods to reliably expand antigen-specific CD4 T cells remain limited. Here, we developed a nanoscale platform to expand antigen-specific CD4 T cells with cytotoxicity against cognate target cells. We generated nanoscale artificial antigen-presenting cells (aAPCs) composed of iron-dextran nanoparticles conjugated with peptide-MHC II dimers and anti-CD28 antibodies to provide antigen-specific and co-stimulatory signals. HLA-DP4 dimers were loaded with the DP4-restricted tetanus toxoid p30 (947—967) or HSV (283—302). CD4 T cells isolated from healthy donor PBMCs were co-cultured with aAPCs and cytokines for 14 days. Antigen-specific populations were quantified by tetramer staining and analyzed by flow cytometry, bulk RNA-seq, and TCR-seq to assess phenotype, transcriptome, and clonality. p30-specific cells expanded 1000-fold and HSV-specific cells expanded 100-fold by day 14. The most robust donor reached over 75% antigen-specific cells within the CD4 compartment. The expanded cells included stem cell memory, central memory, and effector memory subsets and produced TNF-α, IFN-γ, IL-2, granzyme B, and perforin. Functionally, the expanded CD4 T cells exhibited antigen-specific cytotoxicity, killing nearly 90% of cognate targets at a CD4:target ratio of 1. Blocking assays demonstrated that cytotoxicity required HLA-II recognition and granzyme B activity. Transcriptomic profiling revealed a Th1-skewed, NK-like cytotoxic phenotype, while TCR sequencing confirmed antigen-driven clonal selection. Together, our data show that nanoscale aAPCs reliably expand functional antigen-specific CD4 T cells from human PBMCs, providing both mechanistic insight into cytotoxic CD4 biology and a translational foundation for antigen-specific cell immunotherapy. PhRMA foundation, NIH, Taiwan Government Funding for Study Abroad Technological Innovations in Immunology (TECH)