Extended Data Fig. 6: Analysis of T-cell activation and myeloid cells in after Tnfsf10 knockdown in astrocytes.
From: Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity
(a,b) Activation/exhaustion profiling by flow cytometry of tumour-infiltrating PD-1+ CD8+ (a) or PD-1+ CD4+ (b) T cells following genetic perturbation of TRAIL in astrocytes (n = 7 NTsgRNA, n = 9 sgTnfsf10). (c) Flow cytometry analysis of T-cell exhaustion markers in tumour-infiltrating CD8+ (left) or CD4+ (right) T cells following genetic perturbation of TRAIL in astrocytes (n = 7 NTsgRNA, n = 9 sgTnfsf10). (d) Tox expression measured by qPCR in GL261-infiltrating CD4 or CD8 T cells following genetic inactivation of TRAIL in astrocytes. (e,f) Flow cytometry analysis and representative plots of cytokines after restimulation with PMA/Ionomycin in tumour-infiltrating CD8+ (e) or CD4+ (f) T cells following genetic perturbation of TRAIL in astrocytes (n = 7 NTsgRNA, n = 9 sgTnfsf10). (g) Nuclear translocation of NFAT2 analysed by imaging flow cytometry in splenic CD4+ T cells activated in vitro with anti-CD3/CD28 alone or in combination with recombinant TRAIL as indicated. Violin plot of similarity score between DAPI and NFAT2 analysed using the Mann-Whitney U-test with Sidak’s correction (left) and representative images (right) are shown. (h) DR5 expression median fluorescence intensity (MFI) and histogram distribution in T cell and myeloid compartments from GL261-bearing mice following genetic perturbation with control NTsgRNA (n = 7 mice). (i) Percentage of apoptotic microglia and monocyte-derived macrophages (MDM) in GL261-bearing mice 15 days following genetic inactivation of TRAIL in astrocytes. (j,k) Differential expression analysis by bulk RNA-seq in microglia (j) and flow cytometry analysis of microglia and MDM isolated from GL261-bearing Rag2-/- mice undergoing sgTnfsf10 or NTsgRNA genetic perturbation of astrocytes (k). (l) DR5 median fluorescence intensity and histogram in GL261-infiltrating dendritic cell subtypes. (m) Purity analysis of mixed glia culture, using GFAP, CD45, CD11b, O4, Ter119 as lineage markers. Data shown as mean ± SEM. Unpaired two-tailed t-test. n indicates biologically independent samples.
