Background Persistent production of type I interferons (IFN-Is) is one of the hallmarks of lupus skin disease that is exacerbated by ultraviolet (UV) light. We previously showed IFN-I induction by UV occurs in a cGAS-STING-dependent manner, but the IFN-I signature returns to baseline levels in healthy skin, unlike in lupus. Here, we propose that the immune checkpoint VISTA is a regulator of skin IFN-I production in keratinocytes of therapeutic relevance to lupus.

Methods Skin biopsies from B6, B6.Vsir-/- (VISTA-deficient), B6.Vsir-/-Sting-/-, KRT14creVsirfl/fl (Vsir-/- in keratinocytes), and cre-Vsirfl/fl female mice (3 mo) were collected prior to, 3 and 24h after UVB (500mJ/cm2). Gene expression was quantified by RNA-seq (Rosalind). Skin infiltrating cells were quantified by flow cytometry. Human keratinocytes were isolated from healthy skin and cultured in vitro. Cells were treated with agonistic anti-VISTA (803) or isotype IgG2a (20ug/ml) prior to UVB (50mJ/cm2), in the presence or absence of IFNa (100U). Expression of IFN-Is and IFN-I stimulated genes (ISGs) were quantified by qPCR (4hr after UV) and IFN-I score derived.

Results At baseline, B6.Vsir-/- skin had increased IFN-k mRNA, IFN-b protein, as well as total STING protein levels. RNA-seq revealed increased expression of Sting and upstream cytosolic DNA sensors in VISTA-deficient mouse skin before and after UV. B6.Vsir-/- mice exhibited a 5- fold higher skin IFN-I score after UV compared to B6 mice. Higher baseline and UV-induced IFN- I response in VISTA-deficient skin was suppressed in the absence of STING (Vsir-/-Sting-/- mice). Moreover, fewer skin-infiltrating neutrophils and inflammatory monocytes were recruited to Vsir-/-Sting-/- vs. STING-sufficient Vsir-/- skin post UV. RNAseq also revealed decreased expression of DNA repair genes like Ogg1, Xpd and Pole in B6.Vsir-/- skin, which are essential for repair of UV-induced DNA damage. VISTA deficient mouse keratinocytes produced 2-fold higher IFN-k at baseline ex vivo and accumulated higher levels of oxidized DNA (8-OHdG) compared with B6 cells. Mice with a conditional VISTA deletion only in keratinocytes exhibited a 10-fold higher baseline IFN-I signature and increased IFN-b protein levels compared to controls. Expression of VISTA on human keratinocytes decreased 4 hr after UV, but increased 5-fold 24 hr after UV. Pre- treatment of human keratinocytes with an agonistic anti-VISTA antibody (803) suppressed UV- induced IFN-k, Stat1 phosphorylation and IFN-I score, even in keratinocytes with a pre-existing IFN-I signature.

Conclusion These studies identify VISTA as a suppressor of IFN-I production in keratinocytes and demonstrate STING-dependent IFN-I production in the absence of VISTA. As oxidized DNA is resistant to degradation in the cytosol where it can serve as a potent trigger of cGAS-STING, VISTA promotion of DNA repair may indirectly suppress STING signals. Antibody- mediated activation of VISTA in keratinocytes demonstrates its potential as a target to suppress IFN-I production in the context of photosensitivity and lupus.