Journal: Science (New York, N.Y.)

Article Title: Rewiring STAT signaling from the cell surface with Trikine immunotherapeutics

doi: 10.1126/science.adx9954

Figure Lengend Snippet: ( A-C ), B6129SF1/J (F1) mice (n = 8 animals) bearing established s.c. KP1 tumors were administered cytokine (3 μg functional cytokine), PBS, or left NT i.p. on day 5 and every other day until day 19. For combination therapy, anti-PD-1 antibody (200 μg per dose) was i.p. administered on days 7, 11, 15, and 19; the experimental timeline (A), average tumor growth curves (B), and survival curves (C). Statistical significance in (C) was calculated using a Mantel-Cox test, with **** representing the statistical significance (p < 0.0001) of the IL-10/2-Trikine + αPD-1 survival curve over those of NT, IL-10/2-Trikine, and αPD-1. Results representative of 2 independent experiments. ( D-H ), Experimental setup is described in . Mice were sacrificed on day 19, and CD45 + TILs were sorted for scRNA-seq analysis. Gene Set Enrichment Analysis (GSEA) for differentially expressed genes (DEGs) between T cells from IL-10/2-Trikine and mono-IL-10 treatment groups using Hallmark gene sets (left); GSEA for DEGs between T cells from IL-10/2-Trikine + anti-PD-1 and IL-10/2-Trikine treatment groups using Hallmark gene sets (right) (D). Merged UMAP of unsupervised cell clusters (E). Cluster composition for each treatment group (F). Distribution of each treatment group across the UMAP space (G). Relative abundance of T, Mki67+ T, NK, and Mki67+ NK cells among CD45+ cells by treatment group (H). scRNAseq data are the result of one independent experiment. ( I-J ), F1 mice bearing KP1 tumors were administered i.p. mono-IL-10, IL-10/2-Trikine (3 μg functional cytokine per dose), or PBS every other day from day 7 to day 17. Mice also received i.p. injections of anti-PD-1 antibody (200 μg per dose) on days 9, 13, and 17. On day 18, mice were sacrificed, and tumors were collected for flow cytometry analysis (n = 7 animals). Frequencies of PD-1 + ICOS + cells among CD4 + TILs (I). Frequencies of IFN-γ+TNF-α+ cells among PD-1/ICOS quadrant-gated subsets (J). Results representative of 2 independent experiments. ( K ), B6129SF1/J mice bearing KP1 tumors were treated intraperitoneally with IL-10/2-Trikine (3 μg functional cytokine per dose) every other day from day 7 to day 17. Anti–PD-1 antibody (200 μg per dose) was administered i.p. on days 9, 13, and 17. Depleting antibodies (400 μg per dose) were administered i.p. on days 6, 9, and 13 (n = 6 animals). Shown are the average tumor growth curves. ( L ), Counts of viable KP1 tumor cells. Results representative of 2 independent experiments. All data represent mean ± s.e.m. and are analyzed by one-way (I, J, L) or two-way (B, K) ANOVA with Tukey’s post-test. Schematic in A created using BioRender.com .

Article Snippet: For experiments performed at Stanford University, five- to six-week-old Thy1.2 + C57BL/6 (C57BL/6J) mice and B6129SF1/J (F1, Strain #101043) mice were purchased from Jackson Laboratory.

Techniques: Functional Assay, Flow Cytometry