High-dimensional phenotyping reveals novel macrophage-like and hybrid subsets within murine splenic conventional dendritic cells

Conventional dendritic cells (cDCs) are pivotal antigen-presenting cells (APCs) with critical roles in immune regulation, yet their subset classification remains ambiguous due to phenotypic overlap with macrophages and monocytes, particularly in the spleen. This study employed multi-parametric flow cytometry and clodronate liposome (CL) depletion to systematically re-evaluate splenic CD11chighMHCIIhigh cDCs in C57BL/6 mice. We identified three novel subsets: (1) a tissue-resident T-cell zone macrophage (TZM)-like population (F4/80inter-lowCX3CR1+MERTK+) constituting 0.59% of cDC2s with >10-fold CL-depletion resistance (p < 0.0001); (2) a resident F4/80high APC subset (CCR2 ⁻ Ly6C⁻) accounting for 2.7% of cDC2s with CL-sensitivity; (3) unconventional CD4⁺CD8α⁺ hybrids present in 2.57% of cDC2 and some cDC1s. These findings demonstrate unprecedented cDC plasticity driven by microenvironmental signals, revising conventional classification frameworks and proposing new targets for DC-based immunotherapies in autoimmunity and cancer. Our phenotypic mapping provides a foundational framework for future functional investigations into these novel subsets.