Fig 1: Effects of radiation on cancer-associated fibroblast (CAF)-derived immunomodulators. (A, B) Protein levels of secreted TGF-β and PGE2 in culture supernatants measured by ELISA. (C) Intracellular IDO protein expression in CAF cell lysates analyzed by ELISA. In (C), positive control is non-irradiated CAFs stimulated with IFN-γ and negative control non-irradiated CAFs cultured without stimulation. Data represents mean (± SD) values from three (A), five (B) or four (C) different CAF donors. P-values between NFs and CAFs in (A, B) were determined using one-way ANOVA with Tukey correction for multiple comparisons, whereas P-values between groups of monocultures and co-cultures in (C) were determined using two-way ANOVA with Dunnett correction for multiple comparisons. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 2: SRC.IDO1 expression in liver is required for efficacy in autoimmune models(A) Summary table of expression, metabolite changes, and efficacy of mRNA constructs containing miRNA ts.(B–E) EAE-induced mice were injected i.v. with 0.5 mg/kg of LNP A-formulated mRNA on days −1 and 6. The mRNAs included either a miR122 or miR142 ts to extinguish expression in either hepatocytes or myeloid cells, respectively. (B and C) Mean clinical score (B) and percentage of disease-free mice (C) were calculated. (D and E) Mean disease intensity (D) and mean peak score (E) were calculated between days 10 and 21. Data are mean and SEM of 12 mice/group and representative of 2 similar experiments. (B) Significance was determined compared to dead SRC.IDO by one-way ANOVA with a secondary Dunnett’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and #p < 0.0001. (C) Significance was determined by log-rank (Mantel-Cox) test to dead SRC.IDO. ∗∗p < 0.01 and ∗∗∗∗p < 0.0001.(F–I) C57BL/6xDBA/2 F1 recipients received C57BL/6 donor cells to induce aGVHD and were injected i.v. with 0.5 mg/kg of LNP A-formulated mRNA on days 0 and 6. (F and G) On day 14, splenic (F) donor CD8 T cell engraftment and (G) host B cell percentages were determined by flow cytometry. (H and I) The percentages of CD4 T cells expressing FoxP3 within the spleen were determined in the (H) donor and (I) host compartments.(D–I) Data are individuals and median. Data are n = 3–8 mice/group and representative of 2 similar experiments. Significance was determined by one-way ANOVA with a secondary Tukey’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.0001.See also Figure S4.
Fig 3: mRNA-encoded anchored IDO1 is protective in EAE(A) Mice were induced with EAE with MOG peptide and pertussis toxin (PT) and then injected i.v. with 0.5 mg/kg of LNP A-formulated mRNA on days −1 and 6.(B and C) Mean clinical score (B) and percentage of disease-free mice (C) were tracked over time.(D and E) Mean disease intensity (D) and mean peak score (E) were calculated between days 10 and 21.(B) Data are mean and SEM of 12 mice/group and representative of 4 similar experiments. Significance was determined compared to dead SRC.IDO by two-way ANOVA with a secondary Dunnett’s multiple comparisons test. ∗∗p < 0.01 and #p < 0.0001. (C) Significance was determined by log-rank (Mantel-Cox) test to dead SRC.IDO. ∗∗∗∗p < 0.0001. (D and E) Data are individual mice and median of 12 mice/group and representative of 4 similar experiments. Significance was determined by one-way ANOVA with secondary Tukey’s multiple comparisons test. ∗∗∗∗p < 0.0001.
Fig 4: Engineered SRC.IDO treatment alters metabolite levels up to 96 h post-injection(A–E) Naive C57BL/6 mice were injected i.v. with 0.5 mg/kg of LNP A-formulated mRNA and then monitored 6–192 h post-injection. Human IDO1 expression was determined in the (A, B, and D) liver and (A, C, and E) spleen. (A) Representative images of liver and spleen from a V5-tagged SRC.IDO-treated mouse at the indicated time stained with anti-V5 for IDO1 expression compared with PBS-treated controls. Scale bars indicate 100 μm. (B and C) IDO1 expression was determined as percentage of cells positive for V5 tag from (A) in (B) liver and (C) spleen. (D and E) Human IDO1 expression was determined in (D) liver and (E) spleen by ELISA. Dotted line indicates the levels found in PBS-treated or untreated samples.(F–H) KYN (F) and TRP (G) levels and KYN:TRP ratios (H) were measured in serum by ELISA. Data are mean and SD of n = 5 mice/group and representative of 5 similar experiments. Significance was determined by two-way ANOVA between SRC.IDO and Dead SRC.IDO, followed by Sidak’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.005,∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.See also Figures S1–S3.
Fig 5: Engineered IDO1 ameliorates rat CIA(A) Sprague-Dawley rats were injected intradermally with type II chicken collagen/IFA on day 0 to induce collagen-induced arthritis (CIA). Rats were treated with 0.5 mg/kg LNP A-formulated mRNA i.v.(B–E) For prophylactic dosing, administration of mRNA occurred weekly with dosing on days 0, 7, and 14.(F–I) To dose therapeutically, rats were injected weekly starting on day 0, 3, 7, 10, or 14 as indicated in the figure legend.(B and F) Mean clinical score and (C and G) percentage of disease-free animals were calculated. (D and H) Mean disease intensity and (E and I) mean peak score were calculated from day 10 to 21. (B and F) Data are mean and SEM of 5–8 rats/group and representative of 3 similar experiments. Significance was determined compared to dead SRC.IDO by two-way ANOVA with secondary Dunnett’s multiple comparisons tests. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, and #p < 0.0001. (C and G) Significance was determined compared to dead SRC.IDO by log-rank (Mantel-Cox) test. ∗∗p < 0.01. (D, E, H, and I) Data are individual rats and median. Significance was determined compared to dead SRC.IDO by one-way ANOVA and secondary Dunnett’s multiple comparisons tests. ∗∗∗∗p < 0.0001.
Supplier Page from Abcam for Human IDO ELISA Kit