Fig. 3: LiSmore enables photo-inducible maturation and antigen presentation in BMDCs. | Nature Communications

Fig. 3: LiSmore enables photo-inducible maturation and antigen presentation in BMDCs.

From: Optogenetic engineering of STING signaling allows remote immunomodulation to enhance cancer immunotherapy

Fig. 3

a Experimental setup. Bone marrow cells from C57BL/6J mice were cultured in GM-CSF to induce BMDCs. On days 7 and 8, BMDCs were infected with Control, LiSmore, or left untreated (blank). After 36 h, transduced BMDCs were either shielded (dark) or exposed to pulsed blue light (light; 470 nm, 1 mW/cm2; 20 s ON, 5 min OFF) for 18 h. Blank BMDCs were stimulated with cGAMP and PBS. b, c Flow cytometry profiles (b) and mean fluorescent intensity (MFI) quantification (c) of H2Kb or MHCI, IA/IE, MHCII, CD86, CD80, CD40) and CCR7 in the indicated BMDCs. Plots were gated on viable CD11c+GFP+ events. n = 4 independent biological replicates (mean ± S.D.); one-way ANOVA. d Experimental setup for ex vivo OT-1 CD8+ T cell activation. OVAp pulsed control or LiSmore BMDCs were co-cultured with OT-I CD8+ T cells. Cells were exposed to pulsed blue light for 18 h (470 nm, 20 s ON, 5 min OFF, 1 mW/cm2). Untransduced BMDCs/OVAp were also co-cultured with OT-1 CD8+ T cells and treated with 2’3’-cGAMP (2 μg/ml) or PBS. e Proliferation (CellTrace Violet, CTV) of OT-1 CD8+ T cells on day 4. The division index is shown in the right bar graph (e). n = 4 independent biological replicates (mean ± S.D.); one-way ANOVA. f Left, representative flow cytometry profiles indicative of IFN-γ production from OT-1 CD8+ T cells 18 h. Right, quantification of IFN-γ production in OT-1 CD8+ T cells. n = 6 independent biological replicates (mean ± S.D.); one-way ANOVA test. g Experimental design for assessing cross-presentation and CTL-mediated cytotoxicity. OT-1 CD8+ T cells, LiSmore-BMDCs, and B16-OVAp cells were mixed at a 2:1:1 ratio with or without light illumination (470 nm, 1 mW/cm2; 20 s ON, 5 min OFF) for 24 h. h Quantification of OVAp presentation on SIINFEKL–MHC-I in LiSmore-BMDCs or control BMDCs with and without photostimulation. n = 4 independent biological replicates (mean ± S.D.); one-way ANOVA. i LDH release assay to evaluate OVA-specific CD8+ T cell killing. n = 6 independent biological replicates from three experiments (mean ± SD); one-way ANOVA.

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