Figure 3. Modified CDNs potently activate STING and signal through all human STING allelic variants.

(A) Purified human STING and murine STING binding to CDNs were analyzed by thermal shift assay. Temperature curves are the average from a representative experiment of three independent experiments performed in duplicate. Tm shift values are mean values + s.e.m. (B) Domain structure of hSTING is shown with the positions of the amino acid variations (bottom). The allelic frequencies of the hSTING isoforms shown on the left hand column were obtained from the 1000 Genome Project database as previously described (Yi et al., 2013). (C) HEK293T cells were stably transfected with the indicated STING alleles. Whole cell lysates from HEK293T cells stably expressing the indicated full length STING-HA proteins were analyzed by Western blot with anti-HA antibodies. (D) HEK293T cells expressing the indicated STING alleles were transfected with an IFN-β-luciferase reporter construct. After 24 hours, cells were stimulated for 6 hours with the indicated CDN compound (10 μM), and assessed for IFN-β-reporter activity. (E–F) Human PBMCs from donors with the indicated STING alleles were stimulated with 10 μM of the indicated CDN, or 100 μg/ml DMXAA (E), or human PBMCs from a donor homozygous for the reference variant (STING^REF/REF) was stimulated with 10 μM and 50 μM of the indicated CDN or 100 μg/ml DMXAA (F). After a 6 hour stimulation, fold induction of IFN-β was measured by q-RT-PCR and relative normalized expression was determined by comparison with untreated controls. Results are representative of at least two independent experiments.