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. 2023 Apr 20;4(5):665–681. doi: 10.1038/s43018-023-00547-6

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 2 — a, Representative flow cytometry histograms displaying ovalbumin (OVA) fluorescence intensity in DF1-OVA cells used to generate PDG-Ink4a-OVA glioblastoma. b, Longitudinal individual tumor volumes measured by weekly MRI in PDG-Ink4a (black) and PDG-Ink4a-OVA (red) tumor-bearing mice. Each dot is a tumor volume quantification. Lines indicate matched tumor progression per animal (PDG-Ink4a n = 6, PDG-Ink4a-OVA n = 17 mice). c, Representative image of immunohistochemical OVA staining in endpoint, treatment-naïve PDG-Ink4a (upper panel) and PDG-Ink4a-OVA glioblastoma (lower panel; scale bar: 50μm; representative of n = 6 PDG-Ink4a and n = 12 PDG-Ink4a-OVA independent repeats). d,e Flow cytometry quantification of OVA⁺ T cells (gated from CD45⁺CD11b^-CD3⁺CD8⁺) in tumor (d) and superficial cervical lymph nodes (LN), spleen (SP) and blood (e) in end⁻stage, treatment-naive PDG-Ink4a-OVA tumor-bearing mice (FMO = fluorescence minus one; d: Tumor FMO n = 6, Tumor = 12 mice. e: SP FMO n = 8, LN FMO n = 3, Blood FMO n = 6, SP n = 9, LN n = 8, Blood n = 12 mice). f, Relative immune composition in the glioblastoma TME of primary, treatment-naive tumors. Treg = regulatory T cells, CD8 = CD8⁺ T cells, CD4 = CD4⁺ T cells, Mono = Ly6C⁺ monocytes, MDM = CD49d⁺ monocyte-derived macrophages, MG = CD49d^- microglia, Neutro = Ly6G⁺ neutrophils, cDC1 = CD24⁺CD11b⁻dendritic cells, cDC2 = CD24⁻CD11b⁺ dendritic cells (PDG-Ink4a: CD8 n = 2, CD4 n = 7, Treg n = 7, Mono n = 6, MDM n = 6, MG n = 6, Neutro n = 6, cDC1 n = 5, cDC2 n = 5; PDG-p53: CD8 n = 4, CD4 n = 8, Treg n = 8, Mono n = 2, MDM n = 2, MG n = 2, Neutro n = 2, cDC1, cDC2 = N/A; PDG-Ink4a-OVA: CD8 n = 9, CD4 n = 9, Treg n = 9, Mono n = 9, MDM n = 9, MG n = 9, Neutro n = 9, cDC1 n = 9, cDC2 n = 9; GL261: CD8 n = 5, CD4 n = 5, Treg n = 6, Mono n = 5, MDM n = 5, MG n = 5, Neutro n = 5, cDC1 n = 5, cDC2 n = 5). g-k, Flow cytometry quantification of CD8⁺ T cells in end-stage, treatment-naive PDG-Ink4a, PDG-p53, PDG-Ink4a-OVA and GL261 tumors. g, total CD8⁺ T cells (gated from CD45⁺CD11b⁻CD3⁺ cells). h, Ki67⁺ CD8⁺ T cells from (g). i, CD69⁺ CD8⁺ T cells from (g). j, CD44⁺ CD8⁺ T cells from (g). k, PD-1⁺ CD8⁺ T cells from (g). For g: PDG-Ink4a n = 5, PDG-p53 n = 7, PDG-Ink4a-OVA n = 9 mice, GL261 n = 5 mice. For h-k: PDG-Ink4a n = 5, PDG-p53 n = 4, PDG-Ink4a-OVA n = 9 mice, GL261 n = 5 mice. l, Schematic overview of the experimental design. GL261 and PDG-Ink4a-OVA tumors were initiated as described in Methods. Tumor size was quantified by MRI. Based on tumor volume, mice were distributed into treatment groups by block randomization (rIgG2a isotype control (Cont), anti-PD-1 (IT), 5x2Gy radiotherapy (RT), or adjuvant combination treatment (RT + Adj.IT)), followed⁻up weekly by MRI and sacrificed at 80d or at humane endpoint. Schematic created using BioRender.com. m, Distribution of GL261 tumor volume at the time of inclusion into treatment (Cont n = 8, IT n = 10, RT n = 13, RT + Adj.IT n = 13 mice). n, Longitudinal individual tumor volumes measured by weekly MRI in Cont, RT, IT, and RT + Adj.IT treated GL261 tumor-bearing mice (Cont n = 4, IT n = 7, RT n = 7, RT + Adj.IT n = 6 mice). o, Distribution of PDG-Ink4a-OVA tumor volume at the time of inclusion into treatment (Cont n = 10, IT n = 9, RT n = 6, RT + Adj.IT n = 7 mice). p, Longitudinal individual tumor volumes measured by weekly MRI in Cont, RT, IT and RT + Adj.IT treated PDG-Ink4a-OVA tumor-bearing mice (Cont n = 5, IT n = 4, RT n = 6, RT + Adj.IT n = 7 mice). For (n,p), each line indicates matched tumor progression per mouse. The vertical dashed line indicates start of treatment (Tx start). Statistics: Two-sided unpaired t-test (d), one-way ANOVA with Benjamini, Krieger and Yekutieli correction for multiple testing (e,g-k). Data are represented as mean ± S.E.M. (d,e,g-k,m,o) or - S.E.M. (f).

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