interferon beta ifnβ  (R&D Systems)

Figure S1 ." title="T cell-depleted tumors and maturation trajectories of human DC vaccines (A) CIBERSORT deconvolution" property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: T cell-depleted tumors and maturation trajectories of human DC vaccines (A) CIBERSORT deconvolution across TCGA cancer types. Population abundances were row normalized (C1, n = 1,313; C2, n = 1,210, C3, n = 688; C4, n = 222, C5, n = 2; C6, n = 111). (B) Overall survival of cancer patients’ transcriptome profiled before ICBs treatment (anti-PD-1/CTLA4/PD-L1 ICBs, or combinations thereof) sub-grouped in T cell-depleted C4/C5 tumors (n = 667) and immunogenic C2/C3/C6 tumors (n = 474). Statistics: log rank test. (C) GISTIC 2.0 analysis with indicated 12 genes. Statistical significance: false discovery rate (FDR) < 0.05 (random permutations to background score distribution, BH adjusted). Bladder cancer, n = 136; breast cancer, n = 880; colorectal adenocarcinomas, n = 585; glioblastoma multiforme, n = 580; head and neck cancer, n = 310; kidney cancer, n = 497; acute myeloid leukemia, n = 200; lung adenocarcinoma, n = 357; lung squamous cell carcinoma, n = 344; ovarian cancer, n = 563; endometrial cancer, n = 496. (D–J) Single-cell trajectory reconstruction exploration and mapping (STREAM) DC vaccine trajectory of 93 DC vaccines from 18 prostate adenocarcinoma patients vaccinated with five to eight vaccines. (D) Overview of STREAM DC vaccine trajectory. (E and F) Pseudo-time inferred from DC vaccines’ transcriptome based on variable genes. Principal graph initiated with epg_alpha = 0.01, epg_mu = 0.2, epg_lambda = 0.03, and epg_n_nodes = 5. Dots depict individual DC vaccines and dot color represents (E) patient number or (F) DC vaccine batch/cycle (chi-squared test of independence of variables). (G and H) Signature scores overlaid on the graph as streamplots. Type I IFN/ISG-response signature (G) or mature regulatory DC signature (H) were used as color intensity. (I and J) Patient outcomes were overlaid on the graph as streamplots. PSA doubling time at week 48 (I) and intensity of IFNγ production of peripheral blood mononuclear cell after antigen restimulation (J) were used as color intensity. Here, “n” represents different patients (biological replicates). See also Figure S1 .

Techniques Used: Vaccines

Figures S2 and . " title="... analysis of DCs untreated or stimulated with LPS, IFNβ, or with untreated or dying TC1s (with/without IFNβ). ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Optimization of DCvax-IT for T cell-depleted tumors (A) Metagene expression for indicated signatures in different subcutaneous tumors (from GEO: GSE85509 ). (B) Flow cytometry analysis of CD45 + fraction from subcutaneous MC38/TC1 tumors on day 23 after injection (percentage of CD8 + of CD3 + cells, n = 6; two-tailed Student’s t test). (C) Tumor volume of TC1-tumor-bearing mice treated with anti-PD-1/CTLA4 on day 9/16 after injection (n = 6; area under curve; one-way ANOVA, Kruskal-Wallis test). (D) Survival of WT, Ripk3 −/− , and Mlkl −/− TC1 cells 24/48 h after treatment (three or four repeats). (E) Cell death of WT and Mlkl −/− TC1 cells 48 h after treatment. p values depict comparison WT vs. Mlkl −/− TC1 cells (n = 3; two-way ANOVA, Sidak’s multiple comparisons test). (F) Schematic overview of the vaccine formulation process. (G and H) Functional analysis of DCs untreated or stimulated with LPS, IFNβ, or with untreated or dying TC1s (with/without IFNβ). (G) Flow cytometry of DC maturation (MHCII + CD86 + frequency of CD11c + ). p values depict comparison vs. UT DCs (n = 3; one-way ANOVA, Dunnett’s multiple comparisons test). (H) IFN-signature expression (qPCR). p values depict comparison vs. UT DCs (n = 3; one sample t test). (I) Flow cytometry of frequency of PD-L1 + PD-L2 + CD200 + of CD11c + cells (moDCs alone/cocultured with untreated/dying WT/ Mlkl −/− TC1 cells). p values depict comparison vs. UT moDCs (n = 4, LPS/IFNβ n = 3; one-way ANOVA, Fischer least significant difference [LSD]). (J) Flow cytometry of frequency of CD11b + F4/80 + in moDCs (alone/cocultured with untreated/dying WT/ Mlkl −/− TC1 cells) or bone-marrow-derived macrophages (BMDMs). p values depict comparison vs. BMDMs (n = 3; one-way ANOVA, Dunnett’s multiple comparisons test). (K) Cytokine secretion via cytokine array. From all values, the background was subtracted. Normalization was done using moDCs + untreated cancer cells (n = 3). Here, “n” represents biological replicates and error bars represent SEM. See also Figures S2 and .

Techniques Used: Expressing, Flow Cytometry, Injection, Two Tailed Test, Comparison, Formulation, Functional Assay, Derivative Assay

Figure Legend Snippet:

Techniques Used: Control, Recombinant, Lysis, Protease Inhibitor, Western Blot, Staining, Stripping, Liposomes, CRISPR, MTS Assay, ATP Assay, Reverse Transcription, Cell Isolation, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Selection, Drug discovery, Vaccines, Single-cell Analysis, RNA Sequencing, Mutagenesis, Microarray, Purification, Software