PD-L1 (programmed death-ligand 1) serves as a pivotal immune checkpoint in both the innate and adaptive immune systems. PD-L1 is expressed in macrophages in response to interferon-gamma (IFNgamma). We examined whether PD-L1 might regulate macrophage development. We established PD-L1-/- human pluripotent stem cells, differentiated them into macrophages, and observed a 60% reduction of CD11B+CD45+ macrophages in PD-L1-/-, orthogonally verified with PD-L1 inhibitor BMS-1166 reduced macrophages to

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... he same fold. Single-cell RNA sequencing further confirmed the 60% reduction of macrophages as well as the down-regulation of macrophage-defining transcription factors SPI1, KLF6, and MAFB. Further, PD-L1-/- macrophages reduced the level of inflammatory signals such as NFkappaB, TNF, and chemokines CXCL and CCL families. Whilst anti-inflammatory TGF-beta was upregulated. Finally, we identified that PD-L1-/- macrophages significantly down-regulated interferon-stimulated genes (ISGs) despite IFNgamma in differentiation media. Mechanistically, PD-L1-/- macrophages reduced IFNGR1 expression explaining that cells could not respond to IFNgamma. These data suggest that PD-L1 regulates inflammatory macrophage development by maintaining the IFNgamma signal.