Sustained antigenic engagement programs multidimensional CTL states with distinct cytotoxic capacities

Cytotoxic T lymphocytes (CTLs) eliminate infected or malignant cells through repeated target-cell encounters, yet how they sustain cytotoxic activity during extended antigenic stimulation remains poorly understood. In particular, it remains unclear whether preserved population-level killing reflects maintenance of a uniform effector phenotype or diversification into functionally distinct CTL populations. Here, we combined live-cell imaging, high-dimensional cytometry, single-cell RNA sequencing, phenotypic trajectory analysis, population sorting, and rechallenge killing assays to define how human clonal CTLs remodel during sustained antigenic engagement. Prior antigen encounter altered the kinetics and pattern of target-cell membrane permeabilization but did not compromise overall subsequent killing upon rechallenge. Single-cell transcriptomic profiling after prolonged engagement revealed broad remodeling of antigen-experienced CTLs and heterogeneous cytotoxic transcriptional programs. Time-resolved scRNA-seq further showed that these functional dimensions remodeled with distinct kinetics, with rapid induction of activation- and cytotoxicity-associated programs, later remodeling of vesicular-trafficking programs, and progressive reduction of adhesion/ contact interaction -associated programs. High-dimensional cytometry showed that this temporal remodeling was accompanied by progressive redistribution of CTLs across a multidimensional protein-expression landscape defined by distinct combinations of cytotoxic, vesicular/degranulation, activation, cytokine, adhesion, and regulatory features. Phenotypic trajectory analysis organized this protein landscape along a temporal remodeling axis, with early lytic-granule-rich profiles giving way to multiple late-engaged phenotypes with distinct combinations of functional protein features. Surface-marker-defined late CTL populations displayed distinct killing capacities upon rechallenge, demonstrating that preserved population-level cytotoxicity can coexist with substantial functional heterogeneity. Finally, CAR T cells exposed to CD19+ targets retained cytotoxic activity upon rechallenge and acquired multidimensional phenotypic features that partially overlapped with the remodeling framework identified in clonal CTLs. Reanalysis of longitudinal patient-derived CAR T-cell transcriptomes further showed that related functional programs were dynamically organized after infusion, with the features distinguishing responder and non-responder tisa-cel samples changing between the infusion product and day 7. Together, these findings indicate that sustained CTL killing is accompanied by progressive multidimensional remodeling and diversification rather than maintenance of a fixed effector phenotype. Our findings support a model in which population-level cytotoxic activity can be maintained while the CTLs composing that population progressively diversify into functionally distinct states during sustained antigenic engagement.