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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. 4 — a, Representative image of immunohistochemical staining for CD3, CD8, CD4 and FOXP3 on sequential sections of endpoint PDG-Ink4a tumors from cont, RT, IT, RT + Conc.IT and RT + Adj.IT treated mice (scale bar: 50 um; Cont is representative of n = 7, IT is representative of n = 8, RT is representative of n = 16, RT + Conc.IT is representative of n = 24 and RT + Adj.IT is representative of n = 21 independent repeats). b, Relative immune composition of primary (Prim) PDG-p53 glioblastoma as a percentage of CD45⁺ immune cells. 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 (Prim: CD8 n = 4, CD4 n = 8, Treg n = 8, Mono n = 2, MDM n = 2, MG n = 2, Neutro n = 2; d6 RT: CD8 n = 6, CD4 n = 10, Treg n = 10, Mono n = 9, MDM n = 9, MG n = 9, Neutro n = 9; d12 RT: CD8 n = 4, CD4 n = 7, Treg n = 6, Mono n = 3, MDM n = 3, MG n = 3, Neutro n = 3; d6 RT + Conc.IT: CD8 n = 5, CD4 n = 9, Treg n = 9, Mono n = 9, MDM n = 9, MG n = 9, Neutro n = 9; d12 RT + Conc.IT: CD8 n = 9, CD4 n = 8, Treg n = 9, Mono n = 5, MDM n = 5, MG n = 5, Neutro n = 5; d6 RT + Adj.IT: CD8 n = 4, CD4 n = 8, Treg n = 8, Mono n = 9, MDM n = 9, MG n = 9, Neutro n = 9; d12 RT + Adj.IT: CD8 n = 5, CD4 n = 4, Treg n = 5, Mono n = 5, MDM n = 5, MG n = 5, Neutro n = 5). c, Flow cytometry quantification of Ly6G⁺ neutrophils (gated from CD45⁺CD11b⁺Ly6c^int) from PDG-Ink4a treated tumors (Prim, RT, RT + Conc.IT or RT + Adj.IT) at the indicated time points post treatment initiation (Prim n = 6, d6 RT n = 5, d12 RT n = 10, d18 RT n = 10, d6 RT + Conc.IT n = 8, d12 RT + Conc.IT n = 8, d18 RT + Conc.IT n = 9, d6 RT + Adj.IT n = 12, d12 RT + Adj.IT n = 5, d18 RT + Adj.IT n = 5 mice). d-f, Flow cytometry quantification of PD-L1 mean fluorescence intensity (MFI) in myeloid cells in the TME of primary human (d), PDG-Ink4a (e) and PDG-p53 (f) glioblastoma. Tu = tumor cells (gated from CD45⁻CD11b⁻), Mono = monocytes (gated from CD45⁺CD11B⁺CD14⁺CD16⁺ (d) or CD45⁺CD11b⁺Ly6G⁻(e,f), MDM = monocyte-derived macrophages (gated from CD45⁺CD11B⁺CD14⁺CD16⁻CD49D⁺ (d) or CD45⁺CD11b⁺Ly6G⁻Ly6C⁻CD49d⁺ (e,f)), MG = microglia (gated from CD45⁺CD11B⁺CD14⁺CD16⁻CD49D⁻ (d) or CD45⁺CD11b⁺Ly6G⁻Ly6C⁻CD49d⁻ (e,f)), Neu = neutrophils (gated from CD45⁺CD11B⁺CD66B⁺ (d) or CD45⁺CD11b⁺Ly6G⁺Ly6C^int (e,f). d, n = 5 patients. e, n = 6 mice. f, n = 2 mice). g, Flow cytometry plots of CD4⁺ (gated CD45⁺CD11b⁻CD3⁺CD4⁺) and CD8⁺ T cell (gated CD45⁺CD11b⁻CD3⁺CD8⁺) FACS-isolation strategy of d12 RT, RT + Conc.IT and RT + Adj.IT PDG-Ink4a tumors. Sorted cells gated in red. Representative of n = 3 independent repeats. h-k, Normalized expression of indicated genes in CD8⁺ T cells (gated from CD45⁺CD11b⁻CD3⁺) FACS-purified from PDG-Ink4a tumors 12d post treatment initiation and subjected to RNA sequencing. l, Enriched pathways specific to RT + Adj.IT CD8⁺ T cells. (Supplementary Table S5). For h-l, RT n = 3, RT + Conc.IT n = 3 and RT + Adj.IT n = 3 mice. Statistics: one-way ANOVA with Benjamini, Krieger and Yekutieli correction for multiple testing (c,h-k) and Fisher’s exact test in combination with the Benjamini-Hochberg method for correction of multiple hypotheses testing (l). Data are represented as mean - S.E.M. (b), ± S.E.M. (c-f) or + S.E.M. (h-k).

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