IMMU-76. From borders to brain: determinants of monocyte and BAM access to the microglial niche

BACKGROUND Recent findings show that immune cells from the skull bone marrow and peripheral blood can migrate through the meninges and engraft as long-lived, monocyte-derived microglia—challenging traditional views of central nervous system (CNS) immune privilege. While the impact of such engraftment remains unclear, it opens therapeutic opportunities for microglial replacement, CAR-macrophages, or, if harmful, blockade of monocyte entry. Understanding the dynamics of the CNS macrophage niche is critical to guide these approaches. METHODS We assessed CNS macrophage repopulation across distinct depletion models (CSF1R inhibition, Cx3cr1CreERT2/wtCsf1rfl/fl, Sall1CreERT2/wtCsf1rfl/fl) using immunofluorescence and high-dimensional single-cell analyses (scRNA-seq, spectral flow cytometry) of dissected CNS compartments, and lineage tracing (ZMAN-FACS, skull bone marrow transplantation). RESULTS Using a published scRNA-seq atlas, we derived and validated a high-dimensional FACS panel that distinguishes all CNS macrophage subsets in CNS macrophage reporter lines (Sall1GFP, Cx3cr1GFP,Pf4-Cre;Ai14). Pharmacological CSF1R inhibition depleted all subsets, including choroid plexus MHCII-high BAMs, but spared MHCII-high BAMs in the dura and leptomeninges, likely due to ongoing monocyte input from nearby skull bone marrow. Sall1CreERT2/wt; Ai14-lineage tracing showed that microglia repopulation post-BLZ945 originates from residual Sall1⁺ cells. In contrast, permanent CSF1R deletion (Cx3cr1CreERT2/wtCsf1rfl/fl) delayed repopulation and led to engraftment of CD49d⁺Clec12a⁺non-microglial macrophages. Microglia-selective CSF1R deletion (Sall1CreERT2/wtCsf1rfl/fl)) allowed rapid niche reconstitution by CSF1R-sufficient peripheral macrophages. scRNA-seq and flow cytometry identified repopulating cells with BAM-like signatures within the brain. Blood monocyte lineage tracing confirmed contribution, while skull marrow grafts did not contribute under rapid-repopulation conditions. CONCLUSION Monocyte engraftment into the brain requires failed microglial self-renewal and the ontogeny is defined by the presence of CSF1R-sufficient cells at niche opening. In rapid, microglia-specific depletion, only blood and border-associated macrophages—not skull marrow monocytes—repopulate the niche, revealing tight spatial and temporal constraints on CNS access. These insights redefine the rules of monocyte engraftment in the brain.