Fig 2: Upregulation of IP-10 and IL-8 in HUVECs overexpressing lnc-SLC15A-1. (A,B) Total lysates from HUVECs transfected with 1.5 µg of lnc-SLC15A1-1 expression plasmid or empty vector (pcDNA3.1(+)) were analyzed by Western blotting. Loading control: ß-actin. (C,D) Quantitative results of Western blotting. Analysis and quantification of Western blotting results of IP-10 and IL-8. Data are the means ± SDs (n = 3; * p < 0.05, ** p < 0.01). (E,F) Enzyme-linked immunosorbent assay (ELISA) of IP-10 and IL-8 in conditioned medium. Culture medium of HUVECs with/without overexpression of lnc-SLC15A1-1 was collected, followed by ELISA application. All data shown are the means ± SDs (* p < 0.05).

Fig 3: Monocytes are the main source of IFN-a in response to cGAMP. Impact of aging on monocyte counts (a) and IFN-a response (b) after cGAMP stimulation for males and females. Correlation coefficients “R” and probabilities of correlation were calculated using Pearson test. (a) Correlation between the absolute number of monocytes/mm3 (log10) and age (years). (b) Correlation between the cGAMP-stimulated release of IFN-a (log10) and the absolute number of monocytes/mm3 (log10). (c) PBMC from 5 donors were depleted of CD14+ cells (> 98% depletion) using magnetic particles or mock-depleted and stimulated with cGAMP (20 µg/ml) or R848 (1 µg/ml) for 24 h. IFN-a production was measured by ELISA. Paired Student's t-test was used for statistical analysis. (d, e) Total PBMCs (5 × 105 cells/well) or monocyte-enriched cells (% CD14+ cells > 77% at 3 × 105 cells/well) were stimulated with cGAMP or left untreated (NS). The production of IFN-a (d) or IP-10/CXCL-10 (e) was then assessed in 24 h-supernatants. Median values in pg/ml for [IFN-a] or [IP-10] concentrations normalized to 3 × 105 cells/well are shown in parentheses. (f, g) Intracellular analysis of IFN-a synthesis in 18 h-stimulated monocytes, gated as CD11cpos HLA-DRpos cells, from male or female donors. Brefeldin A was added for the last 3 h of culture. Frequencies of IFN-apos monocytes are shown from individual female or male donors stimulated or not with cGAMP. Statistical differences between groups were assessed using the Kruskal-Wallis's test corrected for multiple comparisons.

Fig 4: Fully human L19-IFN? KRG functional characterization. (A) SPR of L19-IFN? WT on human IFN?R1. (B) SPR of L19-IFN? KRG on human IFN?R1. (C) IP-10 release on THB-1 cells exposed to titration of L19-IFN? WT in coated EDB (+) and non-coated EDB (-) wells. (D) IP-10 release on THB-1 cells exposed to titration of L19-IFN? KRG in coated EDB (+) and non-coated EDB (-) wells. (E) IP-10 release on THB-1 cells exposed to recombinant IFN?. (F) Pharmacokinetic analysis conducted in cynomolgus monkeys injected at the dose of 0.1 mg/kg of L19-IFN? KRG and L19-IFN? WT. (G) Pharmacokinetic analysis conducted in cynomolgus monkeys injected at the dose of 0.5 mg/kg of L19-IFN? KRG. (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).