Extended Data Fig. 10: FACS analysis of T-cell function in HSV-1-treated mice.

(a) Apoptosis in T cells co-cultured with IFNγ-activated astrocytes and anti-TRAIL single-chain antibody, which was incorporated into HSV-1^α-TRAIL (n = 5 anti-Isotype, n = 3 anti-TRAIL). (b) RT-qPCR detection of anti-TRAIL scFv sequence from RNA isolated from brains of GL261-bearing mice 24 h after treatment with HSV-1^α-TRAIL. (c) Immunofluorescence analysis of CD68⁺ myeloid cell infiltration into GL261 tumours following treatment with HSV-1^Empty or HSV-1^α-TRAIL. (d) Absolute T cell count from GL261-bearing mouse brains following treatment with HSV-1^Empty or HSV-1^α-TRAIL. (e,f) Quantification of activated/exhausted tumour-infiltrating HSV-1 MHC-I tetramer⁺ PD-1⁺ CD8⁺ (e) or GARC-1_77-85 tetramer⁺ PD-1⁺ CD8⁺ (f) following HSV-1 treatment (n = 8 each). (g,h) Quantification of activated/exhausted tumour-infiltrating PD-1⁺ CD8⁺ (g) or PD-1⁺ CD4⁺ (h) T cells following HSV-1 treatment (n = 8 each). (i,j) FACS analysis of T-cell exhaustion markers in tumour-infiltrating CD8⁺ (i) or CD4⁺ (j) T cells with relevance for anti-tumour immunity in the context of TRAIL blockade (n = 8 each). (k,l) FACS analysis (left) and exemplary plots (right) of cytokine expression in tumour-infiltrating CD8⁺ (k) or CD4⁺ (l) T cells (n = 8 each). Data shown as mean ± SEM. Unpaired two-tailed t-test used. (m) Graphical summary. In this study, we describe the induction of TRAIL⁺ astrocytes in GBM. TRAIL⁺ astrocytes are regulated by GBM-derived IL-11 via STAT3 signalling and limit anti-tumour immunity by inducing death receptor-mediated apoptosis in T cells. Treatment with an oncolytic HSV engineered to express TRAIL blocking single-chain antibodies in the TME improves the anti-tumour response. n indicates biologically independent samples. Schematic in (m) created in BioRender. Lee, J. (2025) (https://BioRender.com/pn72nh0).

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