VISTA downregulation in fibroblasts may upregulate IFN-1 expression, but more research is needed.

I conducted my first research experiment in the summer of 8th grade to develop a simple qualitative test using surface tension to detect detergent in glyphosate-based herbicides. I continued research the next summer from home using protein modeling to explore the binding of the SARS-CoV-2 spike protein with various animal ACE-2 receptors. I conducted this experiment last summer looking at the expression of VISTA in UVB-exposed fibroblasts at the Dartmouth-Hitchcock Medical Center. This is my third year participating in the AJAS annual conference, and I'm excited to learn more about everyone's research!

Supplemental Figure 1. Fibroblasts Surface Expression of VISTA Following UVB Exposure Regardless of Set of Experiments

This graph shows the level of expression of VISTA on the surface of fibroblasts after no UV exposure, 4 hours after UV exposure (1x50mJ/cm2), 24 hours after UV exposure, and 48 hours after UV exposure. The y axis indicates the median fluorescence intensity (MFI) of VISTA. The bars indicate the mean VISTA expression levels.

Supplemental Figure 2. Fibroblasts Intracellular Expression of VISTA Following UVB Exposure Regardless of Set of Experiments

This graphs shows the level of expression of VISTA intracellularly of fibroblasts after no UV exposure, 4 hours after UV exposure (1x50mJ/cm2), 24 hours after UV exposure, and 48 hours after UV exposure. The y axis indicates the median fluorescence intensity (MFI) of VISTA. The bars indicate the mean VISTA expression levels.

Detecting VISTA Expression in Fibroblasts

Systemic lupus erythematosus (SLE) is an autoimmune disease, which affects approximately 5 million people worldwide. Most SLE patients experience photosensitivity to ultraviolet (UV) rays from sunlight or artificial light, which can worsen disease or activate lupus flares. There are no long-term treatments that address this highly prevalent feature of SLE. Type I Interferon (IFN-1) is abnormally elevated in >75% of SLE patients and is produced in response to apoptotic and necrotic cells, which can be caused by exposure to UV radiation. Fibroblasts, found in the dermis, have been shown to be susceptible to UVB-induced apoptosis and previous literature suggests that fibroblasts express interferon-related pathways. Here, we hypothesize that V-domain Ig Suppressor of T cell Activation (VISTA), a negative immune checkpoint regulator, controls IFN-1 response to UV light in fibroblasts after UVB (310 nm) exposure. Fibroblasts were extracted from skin biopsies of healthy donors and plated in a 12-well plate for experimentation. Fibroblasts were exposed to UVB light using Tyler Research UV-2 irradiation and dosimetry system (1x50 mJ/cm2) to trigger cytokine production or left unexposed (control). VISTA protein expression levels inside and on the surface of fibroblasts at different time points (4, 24, and 48 hours after UV) were measured using flow cytometry. We found that skin fibroblasts express VISTA and that its surface levels decreased over 48 hours after UVB exposure, while intracellular VISTA expression decreased initially (4 hours after UVB) and subsequently increased 48 hours following UVB exposure. Given that UV light triggers an initial increase and subsequent decrease in the skin IFN-1 response to UV light and the requirement for VISTA in IFN-1 production in different cell types, our data suggest that downregulation of VISTA in dermal fibroblasts may upregulate IFN-1 expression. Future studies will examine this mechanism. The main limitation of our studies is high variability in the mean VISTA expression levels in/on fibroblasts across donors. Future studies will control for cell culture passage and be extended to SLE fibroblasts.

Sora Shirai, Hanover High School, Hanover, NH

Mentor: Dr. Sladjana Skopelja-Gardner

A 2 minute video briefly summarizing my experiment!