ZNRF1 deficiency disrupts Fas ligand trafficking and immune balance

Fas ligand (FasL)-mediated apoptosis constrains immune responses by eliminating activated lymphocytes, yet how FasL is delivered to the plasma membrane in myeloid cells remains unclear. We identify the RING E3 ligase ZNRF1 as a macrophage-intrinsic checkpoint that licenses terminal trafficking and surface exposure of FasL. Myeloid-specific Znrf1 deletion caused age-dependent spontaneous splenomegaly and, upon allosensitization, elevated CD4⁺/CD8⁺ T-cell ratios, enlarged germinal centers with heightened IL-21/Tfh activity, and augmented alloantibody production. In macrophages, ZNRF1 deficiency disrupted the link between total and surface FasL. Although cellular FasL levels increased upon stimulation, there was no corresponding elevation in surface FasL, indicating a defect in terminal trafficking or docking. Confocal imaging showed preserved peripheral polarization of LAMP1⁺ lysosome-related organelles while FasL cargo did not co-accumulate at the cortex, indicating a late docking/fusion defect. Biochemically, ZNRF1 deficiency weakened the Munc18-2 (Stxbp2)-Syntaxin-3 (Stx3) interaction; Stxbp2 knockdown reduced surface FasL, and reconstitution with wild-type ZNRF1-but not the catalytically inactive C184A mutant-restored surface FasL despite similar complex assembly, establishing a requirement for ZNRF1 E3 activity. Functionally, Znrf1-deficient macrophages displayed impaired FasL-dependent killing of activated CD4⁺ T cells and Fas-sensitive targets, not rescued by stronger LPS priming. These findings define a ZNRF1-Munc18-2-Stx3 axis that couples lysosome-related organelle polarization to fusion, ensuring timely FasL availability at the macrophage surface and suggesting a tractable node to modulate immune hyperactivation.