CD300f acts as an inducible inhibitory checkpoint that limits mast cell activation by suppressing MITF-dependent activation.

Mast cells (MCs) are key regulators of cutaneous immune responses and play a central role in allergic and non-IgE-mediated skin inflammation by activating the Mas-related G protein-coupled receptor X2 (MRGPRX2). The inhibitory receptor CD300f has been shown to modulate MC activation, but its role in regulating MRGPRX2-dependent signaling remains unclear. Here, we investigated CD300f expression and function in human skin MCs and its impact on MRGPRX2-mediated responses, with some comparison to IgE-mediated responses. CD300f was expressed on human skin MCs, and its surface expression increased following MRGPRX2 activation. Engagement of CD300f significantly attenuated MC activation induced by MRGPRX2-dependent stimuli, including substance P, vancomycin, and quinolone drugs. Transcriptomic analyses in CD300f-LAD2 revealed that CD300f signaling suppressed activation-associated gene programs after substance P stimulation, including NF-κB and MAPK pathways. Notably, CD300f engagement markedly downregulated microphthalmia-associated transcription factor (MITF), as confirmed at the protein level in LAD2 and skin MCs. MITF inhibition with TT012 and CD300f triggering enhance inhibitory responses, decreasing MITF and MITF-target expression more strongly. Conversely, lysyl-tRNA synthetase overexpression increases MITF expression and significantly reduces CD300f-mediated inhibition of both MRGPRX2- and FcεRI-dependent signaling. These findings reveal a CD300f-MITF regulatory axis that limits skin MC activation and inflammatory responses.