High-dimensional spectral flow cytometry uncovers progressive T-cell exhaustion and myeloid cell reprogramming in a CLL mouse model.

Chronic lymphocytic leukemia (CLL) exhibits marked clinical heterogeneity that cannot be fully explained by genetic alterations, underscoring the critical role of the tumor microenvironment in disease progression and therapeutic resistance. To delineate the temporal evolution of immune dysregulation in CLL, we used the Eµ-TCL1 adoptive transfer (TCL1-AT) mouse model combined with high-dimensional spectral flow cytometry to longitudinally profile lymphoid and myeloid compartments. To assess the reproducibility across different disease kinetics, we analyzed seven independent TCL1-AT cohorts generated from distinct primary tumors. CLL progression was associated with coordinated restructuring of adaptive and innate immune cell populations. Within the T-cell compartment, early activation and effector expansion transitioned to a progressive accumulation of terminally exhausted CD8+ T cells. Concurrently, immunoregulatory populations expanded, including regulatory type 1 (Tr1)-like CD4+ T cells, activated regulatory T cells (Tregs) and, importantly, a previously underappreciated Eomes-expressing Treg subset. Natural killer (NK) cells exhibited transient expansion followed by differentiation and acquisition of exhaustion markers. Myeloid cell profiling revealed an expansion of immunosuppressive PD-L1high patrolling monocytes, mature regulatory dendritic cells (mregDCs), and phenotypic reprogramming of dendritic cells (DCs) consistent with impaired antigen presentation and promotion of T-cell exhaustion and regulatory T-cell subsets. Despite substantial variation in leukemia kinetics, expansion of patrolling monocytes, exhausted CD8+ T cells, and Tregs was consistently observed across all seven independent TCL1-AT cohorts, demonstrating robust immune remodeling. Collectively, our findings demonstrate a stepwise transition toward a suppressive microenvironment involving innate and adaptive immune compartments in the Eµ-TCL1 mouse model, providing a framework for understanding immune failure in CLL and informing strategies to restore anti-leukemic immunity.