Fig. 2: Evaluation of immune cells in the GBM tumor before (day −4) and after (day +13) neoadjuvant ICI treatment.

a, Representative region of whole-slide hematoxylin and eosin (H&E) and IHC images of paired pretreatment and posttreatment GBM specimens for CD3⁺, CD8⁺ and CD4⁺ T cells. Representative images were taken from areas of increased tumor cellularity. b, Representative region of whole-slide high-plex immunofluorescence images and cellular neighborhood enrichment analysis showing the co-occurrence of specific immune cells (CD8⁺ T and CD4⁺ T cells, dendritic cells (DCs), macrophages and glia) with tumor cells in the paired pretreatment and posttreatment GBM specimens. One slide was prepared and representative images were taken from areas of increased tumor cellularity. c, The tumor-infiltrating CD45⁺ (immune) fraction was analyzed for T cell content (CD3⁺), T cell subsets (CD8⁺, CD4⁺FOXP3⁻ T_EM and CD4⁺FOXP3⁺ T_reg cells) and activation markers (PD-1⁺Ki-67⁺). PD-1 expression on T cells was detected with anti-PD-1-Brilliant Violet 421 (BV421) in the pretreatment tumor or anti-IgG4-PE (detects nivolumab bound to PD-1) in the posttreatment tumor. d, The fraction of activated and proliferating T cell subsets, along with their GzmB expression, were evaluated in the posttreatment GBM. There was insufficient material available for analysis of the T cell subsets in the pretreatment biopsy specimen. e, Heatmap showing the immune activation transcriptome gene set³⁶ scores (derived from the singscore method³⁷) in the pretreatment and posttreatment tumor specimens (RNA was analyzed in triplet for each tumor specimen using the NanoString PanCancer IO360 Panel). f, The posttreatment tumor was analyzed for drug/nivolumab occupancy of PD-1 sites (nivolumab detection, staining with anti-IgG4-PE) and residual unoccupied PD-1 sites (PD-1 detection, staining with anti-PD-1 (CD279)-BV421). Scale bars = 100 µm.

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