Fig 2: Src-anchored IDO1 delivered by mRNA protects host B cells in aGVHD(A) C57BL/6xDBA/2 F1 recipients received C57BL/6 donor cells and were injected i.v. with 0.5 mg/kg of LNP A-formulated mRNA on days 0 and 6.(B–E) Donor CD8 T cell engraftment (B and D) and host B cell (C and E) percentages were determined by flow cytometry on (B and C) day 7 in peripheral blood and (D and E) day 14 in the spleen.(F and G) On day 14, splenic CD4 and CD8 T cells within the donor and host compartments were analyzed for expression of (F) active caspase-3 (apoptosis) or (G) Ki67 (proliferation).(H) The percentages of CD4 T cells expressing FoxP3 within the spleen (top) and blood (bottom) were determined to quantify Treg populations. Data are individuals and median. Data are of n = 3–8 mice/group and representative of 3 similar experiments. Significance was determined by one-way ANOVA with a secondary Tukey’s multiple comparisons test. ∗∗p < 0.005, ∗∗∗p < 0.0005, and ∗∗∗∗p < 0.0001.

Fig 3: Major histocompatibility complex (MHC) class I-directed and II-directed indoleamine 2,3-dioxygenase 1 (IDO1) peptides cooperate together but not with IDO1 enzyme inhibition. (A) Growth curves of CT26 tumors in wild-type (WT) mice immunized with the MHC class I-directed and II-directed peptides EP2 and EP6 either separately or together beginning 7 days after tumor engraftment. Responses to adjuvant alone, individual peptides (gray lines) and combined peptides (black lines) are plotted as means±SEM (n=8 tumors/cohort). (B) Evaluation of EP2 and EP6 both separately and in combination in Ido1-/- mice as described in A (n=8 tumors/cohort). (C) Growth curves of CT26 tumors in WT mice treated with EP2 and epacadostat either separately or together beginning 7 days after tumor engraftment. Responses to adjuvant alone, EP2 and epacadostat individually (gray lines), and combined treatment (black lines) are plotted as means±SEM (n=10 tumors/cohort). (D) Evaluation of EP6 and epacadostat both separately and in combination as described in B (n≥9 tumors/cohort). P values for longitudinal tumor growth comparisons between combined and individual peptide vaccine-treated groups are included on each graph.

Fig 4: Metabolites levels from the kynurenine, nicotinamide, purine and pyrimidine metabolic pathways are affected by IDO1 expression by the tumor. On Day 28 of tumor progression, whole tumor was harvested from IE9mp1-mIDO1 (n=4) and IE9mp1-Empty Vector control (n=4) tumor-bearing mice. On Day 47 of tumor progression, whole tumor was harvested from IE9mp1-mIDO1 (n=4) and IE9mp1-Empty Vector control (n=5) tumor-bearing mice. Mean log 2 fold changes in metabolites from (A) tryptophan amino acid degradation, (B) NAD, (C) purine and (D) pyrimidine metabolism pathways measured in IE9mp1-mIDO1 and IE9mp1-EV tumor from WT C57BL/6 mice on Days 28 and 47. These data presented are the relative abundance measurements of the metabolite level and sample t test statistical analysis reveals the 95% confidence interval of the mean log-fold change in metabolites. The p-values of all the metabolites are listed in Supplemental Table 1 .

Fig 5: Indoleamine 2,3-dioxygenase 1 (IDO1) expression is localized to infiltrating immune cells within CT26 tumors. (A) Confocal images of CT26 tumor sections. (top row, left to right) Immunofluoresence staining of sections from wild-type (WT) and Ido1-/- mice for IDO1 (Cy3, red), and from WT mice for programmed death-ligand 1 (PDL1) (Cy3, red) and the combination of IDO1 (fluorescein isothiocyanate (FITC), green) and PDL1 (Cy3, red). Nuclei were stained on all sections (DAPI, blue). (bottom row, left to right) Staining of CT26 tumor sections from WT mice for combinations of IDO1 (Cy3, red) with CD45, CD11b, Gr1 and CD11c (FITC, green). Nuclei were stained on all sections (DAPI, blue). (B) Confocal images of a field of FACS-isolated CD45+, CD11b+, CD11c+ cells from a CT26 tumor in a WT host. (left to right) Immunofluoresence staining for nuclei (DAPI, blue), CD11c (FITC, green), IDO1 (Cy3, red), CD19 (Cy5, purple) and the composite image.