Extended Data Fig. 7: GABA enhances the anti-inflammatory phenotype of IL-10 polarized macrophages partially via GABAA receptor signaling.
From: B cell-derived GABA elicits IL-10+ macrophages to limit anti-tumour immunity
a, Scheme of bone marrow cell differentiation in the presence of M-CSF without or with GABA for 6 days, followed by 6 h polarization with IL-10 (M-IL-10 and GABA-M-IL-10). qPCR or gene chip analysis was performed for GABA conditioned macrophages with IL-10 polarization (GABA-M-IL-10) and its control group (M-IL-10) (n = 3). b, c, Il10 mRNA level measured by q-PCR in monocytes (n = 3) (b) or BM (n = 4) (c)-derived M-IL-10 and GABA-M-IL-10. Results are represented relative to control group. d, Oxygen consumption rate (OCR) of M-IL-10 and GABA-M-IL-10 measured in real time by extracellular flux analyzer. Oligomycin (Oligo), FCCP and rotenone/antimycin A (Rot/AntiA) were added at the indicated time points (n = 2). e, f, M-IL-10 and GABA-M-IL-10 conditioned macrophages were co-cultured with CD8+ T cells stimulated for 72 h with anti-CD3 and anti-CD28 in the presence or absence of anti-IL-10 blocking antibodies (Abs). The percentage of granzyme B+ cells in CD8+ T cells analyzed by flow cytometry (e) and the concentration of IFNγ and TNF in supernatant measured by Cytometric Bead Array (CBA) (f) (n = 6 (M-IL-10 + CD8+), 5 (GABA-M-IL-10 + CD8+), 3 (M-IL-10 + CD8+ + IL-10 blocking Abs) or 4 (GABA-M-IL-10 + CD8+ + IL-10 blocking Abs)). g, Tumor volume (n = 6) or weight (n = 4) of WT mice co-injected with MC38 cells together with in vitro generated M-IL-10 or GABA-M-IL10. h, The DEGs representing cytokines and their receptors, MHC molecules and co-factors and OXPHOS between GABA and control (Ctr) group, obtained from gene chip analyses (n = 3) and shown for all groups as heatmap. Those genes, which are not differentially expressed between GABA + picrotoxin and Ctr group (two-tailed unpaired t-test; P > 0.05), are represented as red color (the transcript level is transformed to z-score). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t-test (b, c); one-way ANOVA (f, g (right)); two-way ANOVA (g (left)). Bars represent mean ±SEM. n indicates biological (g, h) or technical replicates (b-f). Data are pooled from two experiments (c) or representative of two experiments (d-f). Exact P values are in Source Data.
