Fig 1: Screen for genes that modulate dendritic cell phenotype and functionCAS9 expressing-inducible immortalized dendritic cells (iniDC CAS9) were transfected with specific CRISPR RNAs (crRNA) to perform gene knockouts. Dexamethasone and doxycycline (DEX/DOX) were removed from the culture system during transfection and recovery of cells to differentiate iniDC CAS9 cells into primary de-iniDC CAS9 which were then used for assessing the phagocytosis of dying cancer cells (Phago), quantification of IL-1β, IL-6, and TNFα production upon stimulation with LPS, as well as the assessment of antigen cross-presentation capacity upon ovalbumin (OVA) treatment by interleukin-2 (IL-2) quantification in B3Z hybridoma co-culture supernatants. For each parameter, the readouts of crRNA-transfected wells were normalized to non-target control crRNA. Averaged ratios are summarized as a heatmap (A), in which Fpr1 is highlighted as a candidate gene whose KO leads to a loss of function phenotype in DC. Fpr1 KO de-iniDC CAS9 cells (Fpr1−/−) were analyzed for cytokine production (B–D) upon stimulation with LPS or MCA205 cells lysate, cross-presentation of OVA antigen or SINFKEL peptide (SL8) (E), and phagocytosis of MCA205 cells that were either untreated (UT) or crizotinib (CRIZO)-treated (F) Cytokine concentration and phagocytic events (CD11C+ CellTracker+ cells) are reported as bar charts (means ± SD of triplicates). Statistical differences were calculated by means of the ANOVA test (Dunnett's multiple comparisons test), with ∗∗p < 0.01, ∗∗∗p < 0.001 as compared to UT, ###p<0.001 or $$$p<0.001 as compared between Fpr1−/− and wild type (wt) de-iniDC CAS9 cells with the same stimuli. Differentiated iniDC and iniDC CAS9 cells (de-iniDC) were pulsed with tumor cell lysate and intratumorally (i.t.) injected into subcutaneous MCA205 tumors grafted on immunocompetent mice to test their (immuno)therapeutic potential. Tumor growth curves are reported in (G) as mean ± SEM (n = 6). Statistical significance was calculated by means of the ANOVA Type 2 (Wald test) and significant results are indicated as ∗P<0.05 compared to the PBS group.
Fig 2: The amino acid environment tunes NLRP3 inflammasome activation and inflammatory cytokine production.(A) Schematic of experimental approach to limit environmental amino acids in BMDM cell culture. (B) to (E) BMDMs were cultured for 24 hours in custom-formulated RPMI 1640 media containing all amino acids at standard concentrations (Control) or lacking a single amino acid (Table S1), as indicated. After 24 hours, cells were primed with LPS (100 ng/mL) for 4 hours and stimulated with nigericin (5 μM) for 2 hours. Supernatants were analyzed for (B) IL-1β, (C) IL-6, and (D) TNF release by ELISA, and (E) LDH release was quantified as a measure of cytotoxicity. (B to E) Data represent the mean values of biological triplicates pooled from 3 independent experiments +/− SD. (B to D) Cytokine values from amino acid dropout conditions were normalized to control values (set at 100%) and expressed as percent change. (E) LDH release was expressed as percent cytotoxicity relative to control samples, where untreated BMDMs and detergent-permeabilized BMDMs cultured in control medium were defined as 0% and 100% cell death, respectively. AA=amino acid. OHP=hydroxyproline.
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