(448) Gli3 Regulates PD-L1 Expression in Macrophages and Alters Maturation and Cytokine Profiles in Plasmacytoid Dendritic Cells

Introduction/Rationale: Our lab previously demonstrated that TLR–TRIF–IRF3 signaling induces the transcription factor GLI3, revealing a non-canonical regulatory axis beyond Hedgehog (HH) signaling. However, how GLI3 influences myeloid cell function downstream of innate activation remains unclear.

Methods: Bone-marrow-derived macrophages (BMDM) and dendritic cells (DC) were generated from wild-type (Gli3fl/fl LysMcre-/-) and myeloid-specific Gli3 knockout (Gli3fl/fl LysMcre+/-, referred to as M-Gli3-/-) mice. Macrophages were in vitro polarized to M2 and analyzed for Cd274 (PD-L1) expression by qPCR and flow cytometry. DC subsets (moDCs, cDC1, cDC2, pDC) were in vitro derived and assessed for lineage differentiation, CD86 expression, and cytokine production.

Results: M2 macrophages from WT mice showed robust PD-L1 expression, whereas MGli3⁻/⁻ macrophages exhibited markedly reduced PD-L1 transcript and surface expression by flow, independent of HH/SMO signaling, as supplementation with sonic hedgehog (Shh) ligand did not restore PD-L1 levels. GLI3 deletion did not impair DC subset differentiation, indicating normal myeloid development. However, matured pDCs from MGli3⁻/⁻ cultures displayed reduced TNF-α and CD86 expression compared with WT DCs, suggesting a selective role for GLI3 in pDC activation rather than lineage specification.

Conclusion: Building upon our prior demonstration that TLR-TRIF–IRF3 signaling induces GLI3, these findings reveal that GLI3 functions downstream to regulate macrophage PD-L1 and pDC activation markers, positioning GLI3 as a context-dependent modulator of myeloid immunoregulatory functions independent of canonical Hedgehog signaling.