Zachary Peters1, Lindsay Mendyka1, Sicong Shan1, Angelique Cortez1, William Rigby2, Christopher Burns1, Randolph Noelle3 and Sladjana Skopelja-Gardner1, 1Dartmouth Hitchcock Medical Center, Lebanon, NH, 2Dartmouth Hitchcock Medical Center, Lebanon, PA, 3Geisel School of Medicine at Dartmouth, Lebanon, NH
Background/Purpose: Most lupus patients chronically exhibit higher IFN-I scores in the skin, peripheral blood, and kidneys that is exacerbated by ultraviolet (UV) light. In normal skin, IFN-I response to UV returns to baseline over time, provoking the questions: what controls IFN-I response and why do cells in lupus skin fail to downregulate IFN-I production? Antibody-mediated activation of the immune checkpoint VISTA on monocytes suppresses their IFN-I signature and absence of VISTA enhances TLR7-stimulated IFNa production by pDCs. These findings prompted us to test the hypothesis that VISTA expression by keratinocytes regulates basal and UV light-triggered IFN-I production in the skin.
Methods: Skin biopsies from B6, B6.Vsir-/- (VISTA-deficient), 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 and pathway analysis performed (Rosalind). Skin infiltrating cells were quantified by flow cytometry. IFNb and IFNk levels were quantified by immunoblotting of skin lysates. Human keratinocytes were isolated from healthy skin and culturedin 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 pre-treatment. Expression of IFN-Is, Vsir, and IFN-I stimulated genes (ISGs) were quantified by qPCR (4hr after UV) and IFN-I score derived.
Results: VISTA-deficient mice have a ~50-fold higher skin IFN-I score at baseline that is associated with greater IFNb protein levels. This increased IFN-I response in the absence of VISTA is not limited to the skin, as increased ISG expression was seen in the kidney. A single exposure of skin to UVB light resulted in a significantly higher IFN-I score in Vsir-/- vs. B6 skin 3 and 24hr after exposure, which was accompanied by significantly increased IFNb levels in the skin and blood. The higher IFN-I response in the absence of VISTA was not due to excess dead cells. While Vsir-/- mice demonstrated increased immune cell infiltration and chemokine production 24hr after UV, the elevated IFN-I score in the absence of VISTA was independent of immune cell influx at 3hr. RNA-seq analysis demonstrated suppressed DNA repair pathways in the absence of VISTA and upregulation of DNA sensing pathways ZBPI-STING and AIM2. The IFN-I score was also elevated in the skin of mice with conditional VISTA deletion (KRT14creVISTAfl/fl) both at baseline and 3hr after UV. Exposure of human primary keratinocytes to UVB light upregulated VISTA gene (4hr) and protein (24hr) expression. Pre-treatment of keratinocytes with an agonistic anti-VISTA IgG suppressed UV-induced IFNk and ISG expression. The suppressive effect of VISTA agonism on IFN-I response was maintained in keratinocytes with a pre-existing IFN-I signature.
Conclusion: These studies identify VISTA as a novel regulator of the IFN-I response and a potential therapeutic target to suppress IFN-I production in lupus skin and photosensitive reactions. The novel keratinocyte-intrinsic role of VISTA is particularly relevant to lupus pathogenesis as these cells demonstrate a high IFN-I signature, express high IFNk levels, and are direct targets of UVB rays.
Disclosures: Z. Peters, None; L. Mendyka, None; S. Shan, None; A. Cortez, None; W. Rigby, Bristol Myers Squibb, AbbVie; C. Burns, None; R. Noelle, None; S. Skopelja-Gardner, None.