Fig 2: Expression of T-cell activation/exhaustion markers on the CD8+ TIL. a Expression of Nrp-1, PD-1, LAG-3, CTLA-4 and Tim-3 on CD8+ T cells from B16F10 TIL isolated at day 15. Right: percentages of Nrp-1 among CD8+ T cells expressing or not PD-1 (n = 26), Tim-3 (n = 19), LAG-3 (n = 19) or CTLA-4 (n = 16). b Expression of Nrp-1 on CD8+ T cells correlated with that of PD-1 (n = 38). c Expression of Nrp-1 on CD8+ TIL is restricted to a PD-1hi T-cell population. Left, Expression of Nrp-1 and PD-1 on CD8+ TIL; identification of three T-cell subsets: Nrp-1+PD-1hi, Nrp-1−PD-1+and Nrp-1−PD-1−. Right: expression of exhaustion/activation markers, functional proteins and transcription factors in Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− CD8+ T-cell subsets. Heat maps including percentages of cells positive for expression of PD-1, Nrp-1, Tim-3, CTLA-4, LAG-3, granzyme B (GrzB) and Ki-67, and gMFI for NFATc1, IRF-4, Helios, Blimp-1 and T-bet (n = 5). d gMFI of PD-1 on Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− TIL subsets (n = 16). Results are representative of three to five independent experiments. Means ± SEM two-tailed Student’s unpaired t test a or one-way ANOVA test with Bonferroni correction d. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file c

Fig 3: NK1R-signaling Promotes Activation of NFAT1, NFAT2, and NF?b-p65, and T Cell Survival Dependent on IL-2 Secretion(A) Calcineurin activity in WT and NK1RKO T cells incubated with SarSP or CD3 Ab, measured 15 min after treatment. Means ± 1 SD of duplicated results of 1 representative experiment of 2.(B) Western blot analysis of NFAT1, NFAT2, NF?ß-p65, cFos, and cJun, in nuclear extracts of WT and NK1RKO T cells, untreated or after incubation with CD3 and CD28 Ab. Controls were stimulated with ionomycin (Iono). One representative experiment of 3.(C) Concentrations of IL-2 by ELISA in supernatants of WT and NK1RKO T cells untreated or after 24-h stimulation with CD3 and CD28 Ab. Means ± 1 SD, 1 representative of 3 experiments.(D) Surface IL-2Ra expression by FACS in WT and NK1RKO T cells untreated or after 24-h stimulation with CD3 and CD28 Ab. One representative of 3 experiments.(E) Comparison by FACS of T cell proliferation (CFSE dilution), cell death (FVD incorporation), and activation (CD44High) between WT and NK1RKO T cells after 4-day stimulation with CD3 and CD28 Ab. Numbers are cell percentages per quadrant. One representative out of 6 experiments.(F) Comparison by FACS of cycles of cell division (histograms) and percentages of cell proliferation (bar diagram) between WT and NK1RKO T cells. Numbers in histograms are cell percentages. Each dot in the bar diagrams represents a mouse. Means ± 1 SD.(G) Percentages of cell death (by FVD incorporation) of WT and NK1RKO T cells stimulated for 4 days with CD3 and CD28 Ab, alone or plus IL-2. Means ± 1 SD, 6 mice per condition.Results were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test (A, C, F, and G). ***p < 0.001, ****p < 0.0001, NS: not significant.

Fig 4: Single nuclei RNA-sequencing (snRNA-seq) uncovers transcriptional changes of GBM infiltrating T and dendritic cells in the post-vaccination tumor immune microenvironment.A) Representative gating strategy for T cell enrichment by NFATc1 nuclear staining. An example pre- and post-GNOS-PV01 sample is shown as well as an example positive control thymus from an unrelated, healthy donor. B) Dotplot showing expression of key marker genes for each annotated cell type via the GBmap reference. Data is scaled. C) Dotplot showing expression of key marker genes for each annotated T cell subtype. Data is scaled. D) GZMK+ activated and exhausted cells were computationally subset and differential expression analysis using the FindMarkers function in Seurat v5 was performed comparing patient 2 post- to pre-GNOS-PV01. Four significant pathways of interest with negative enrichment scores are displayed and colored by their normalized enrichment score (NES). The top ten differentially expressed genes (DEG) shared among the pathways are shown and colored by log2 fold change. Genes contributing to each pathway are connected by chords. E) Dendritic cells were computationally subset and differential expression analysis using the FindMarkers function in Seurat v5 was performed comparing patient 2 post- to pre-GNOS-PV01. The top ten significant pathways after gene set enrichment analysis (Gene Ontology Human Biological Processes reference) are displayed in a bubble plot. The size of the circles represents the pathway gene set size and the color of the dots shows the adjusted p-value. Differentially expressed genes were identified using a non-parametric Wilcoxon rank-sum test with Benjamini–Hochberg false discovery rate (FDR) correction. Gene set enrichment analysis was performed using a permutation-based enrichment score statistic with FDR adjustment. Abbreviations: GBM, glioblastoma; AC-like, astrocyte-like; MES- like, mesenchymal-like; OPC-like, oligodendrocyte progenitor cell-like; OPC, oligodendrocyte progenitor cell; DC, dendritic cell; TAM- BDM, tumor associated macrophage that originate from bone marrow; TAM-MG, tumor associated macrophage that originate from microglia; NK, natural killer cells; Treg, regulatory T cells. Source data

Fig 5: snRNA-seq defines transcriptional profiles of the GBM microenvironment before and after GNOS-PV01.a, Schematic of nuclei isolation from pre-GNOS-PV01 and post-GNOS-PV01 frozen tumor samples for patients 2 and 9, followed by staining for NFATc1 to enrich for T cells. Nuclei were then used as input for 5′ single-nuclei gene expression library preparation (10× Genomics) and sequencing (NovaSeq6000). Created in BioRender; Garfinkle, E. https://biorender.com/celopys (2026). b, Uniform manifold approximation and projection (UMAP) visualization of 40,959 total nuclei (eight total Harmony integrated samples; pre-GNOS-PV01 and post-GNOS-PV01 samples from patients 2 and 9 each prepared in biological replicate). Cell types were annotated using the GBmap reference. c, Bar graph showing percentage composition of each cell type grouped by category (tumor, normal brain or immune) and patient (2, 9) pre GNOS-PV01 (solid bar) and post GNOS-PV01 (striped bar). d, T cells from each sample were computationally subset and re-integrated using Harmony for subtyping based on key gene expression markers. UMAP visualization of 1,439 total T cells split by patient and timepoint is displayed. e, Expression of CD69 and PDCD1 for patients 2 and 9 post-GNOS-PV01 samples in GZMK+ activated and exhausted CD8+ T cells is displayed in boxplots. f, All GBM cells were computationally subset, and expression of B2M is shown in a violin plot across patients 2 and 9 pre GNOS-PV01 and post GNOS-PV01. For boxplots in e and f, boxes represent the interquartile range (IQR), center line designates the median, whiskers extend to 1.5× IQR and each dot represents a single cell. Statistical significance was calculated across timepoints using unpaired two-tailed Wilcoxon rank test. AC-like, astrocyte-like; DC, dendritic cell; MES-like, mesenchymal-like; NPC-like, neural progenitor cell-like; OPC-like, oligodendrocyte progenitor cell-like; OPC, oligodendrocyte progenitor cell; TAM-BDM, tumor-associated macrophage that originates from bone marrow; TAM-MG, tumor-associated macrophage that originates from microglia; Treg, regulatory T cells.