Journal: Journal for Immunotherapy of Cancer

Article Title: Epigenetic modulation of neuroblastoma enhances T cell and NK cell immunogenicity by inducing a tumor-cell lineage switch

doi: 10.1136/jitc-2022-005002

Figure Lengend Snippet: Identification of MHC-I expression enhancing compounds in NBL. (A) Screening results of 3800 compounds are shown as standardized % of HLA-ABC expression compared with untreated control GIMEN NFκB reporter cells. Compounds marked in red are HDACis; compounds marked in blue reflect the IAPis. (B) MHC-I expression in GIMEN reporter cells treated with entinostat (5 µM) or IFN-γ (100 U/mL) as single agents and in combination (entinostat+IFN-γ). Control indicates untreated cells. (C) MHC-I expression in GIMEN reporter cells treated with AZD-5582 (250 nM) or IFN-γ (100 U/mL) as single agents and in combination (AZD-5582+IFN-γ). Control indicates untreated cells. HDACi, histone deacetylase inhibitor; IAPi, inhibitor of apoptosis inhibitor; IFN-γ, interferon gamma; MHC-I, major histocompatibility complex class I; NBL, neuroblastoma; NFκB, nuclear factor kappa B.

Article Snippet: MHC-I dependence was studied by incubation with 10 μg/mL of HLA-ABC antibody (W6/32, Biotechne) for an hour prior to coculture; IgG isotype (Invitrogen) was used as a control.

Techniques: Expressing, Histone Deacetylase Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Epigenetic modulation of neuroblastoma enhances T cell and NK cell immunogenicity by inducing a tumor-cell lineage switch

doi: 10.1136/jitc-2022-005002

Figure Lengend Snippet: Key regulators of NFκB signaling in NBL inhibit the effect of IAPi in the majority of cell lines and patient-derived organoids. (A) CD137 expression-based activation (left) and cytotoxic capacity (right) of PRAME SLLQHLIGL -directed T cells after coculture with GIMEN cells preincubated with 125 nM AZD-5582 for 48 hours (or respective controls, 100 U/mL IFN-γ or 1000 U/mL IFN-α). T cell activation and cytotoxicity were assessed after overnight coculture at an E:T of 1:1. Cytotoxicity was standardized to target only, treatment-matched controls (n=4). Data presented as mean±SEM. (B) Fold change in MHC-I expression on a panel of NBL cell lines and organoids after 48-hour incubation with 125 nM AZD-5582. Fold change reflects increases in HLA-ABC MFI relative to untreated control cells. Data presented as mean±SD. LAN5, n=3; GIMEN, n=5; rest: n=4, Statistical differences between log (fold changes) were calculated using a one-sample t-test. **P<0.01, ***P<0.001. (C) Histograms reflecting MHC-I expression on wild-type SK-N-SH (left) and SK-N-SH in which the NFκB pathway regulator N4BP1 is knocked out (right) after 48 hours of incubation with 125 nM AZD-5582. Control histograms are shown in black; histograms of AZD-5582 treated cells are shown in red. The top panel reflects cells without coincubation with MHC-I-enhancing cytokines; the lower panel shows expression of cells pretreated with 100 U/mL IFN-γ. Statistical differences were calculated using the Mann-Whitney U test. *P<0.05, ****P<0.0001. E:T, effector-to-target ratio; IAPi, inhibitor of apoptosis inhibitor; IFN-α, interferon alpha; IFN-γ, interferon gamma; MFI, median fluorescent intensity; MHC-I, major histocompatibility complex class I; NBL, neuroblastoma; NFκB, nuclear factor kappa B.

Article Snippet: MHC-I dependence was studied by incubation with 10 μg/mL of HLA-ABC antibody (W6/32, Biotechne) for an hour prior to coculture; IgG isotype (Invitrogen) was used as a control.

Techniques: Derivative Assay, Expressing, Activation Assay, Incubation, MANN-WHITNEY