Fate induction through asymmetric T cell division is modulated by chimeric antigen receptor co-stimulatory domains

Summary: Abstract Body: Chimeric antigen receptor T (CART) cell therapy has revolutionized the treatment of certain blood cancers and shows emerging promise in autoimmune diseases. These receptors frequently include 4-1BB or CD28 costimulatory domains. However, how these signals govern early T cell fate remains incompletely understood. Using single-cell multi-omic analyses of CD8 CART expressing 4-1BB (BBζ) or CD28 (28ζ) costimulatory domains, we show these domains distinctly inform asymmetric T cell division (ATCD) into proximal (PD) and distal daughters (DD) with divergent effector and memory phenotypes, respectively. BBζ induces more pronounced transcriptional and epigenetic remodeling following ATCD, with 1,100 differentially expressed genes (fold change >1.2, FDR < 0.05) versus 270 for 28ζ, and 2,276 differentially accessible chromatin regions versus 163 in 28ζ daughters (fold change > 1.5, FDR < 0.05). In a humanized leukemia model, BBζ daughters persisted at higher levels and provided sustained tumor control, while 28ζ daughters failed to prevent leukemic outgrowth (n=4-9/group, p<0.01). Mechanistically, BBζ daughters displayed heightened oxidative metabolism, coupled with elevated anti-apoptotic gene expression. Single-cell gene regulatory network analysis revealed both BBζ and 28ζ utilize similar transcription factors (e.g. PRDM1, TBX21 in PD; BCL11B, STAT1 in DD), but BBζ drives greater asymmetry, with DD acquiring an epigenetic phenotype like stem cell memory T cells marked by reduced activity of chromatin architecture factors (CTCF, RAD21, SMC3). In contrast, 28ζ daughters exhibit less molecular and functional polarization and diminished persistence. By integrating transcriptomics and chromatin accessibility, these findings highlight how costimulatory domains fine-tune fate-determining factors during ATCD to shape early CART outcomes and inform the design of improved cellular immunotherapies for cancer and autoimmune diseases. Corbett Berry¹, Caitlin Frazee¹, Casey Lee¹, Sisi Chen¹, Patrick Herman¹, Roderick O'Connor², Christoph Ellebrecht^{1, 2} 1. Dermatology, University of Pennsylvania, Philadelphia, PA, United States. 2. Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, United States. Adaptive and Auto-Immunity