Extended Data Fig. 6. Transcriptional and functional analyses of Treg subsets in RT + Conc.IT treated glioblastoma.

a, Representative flow cytometry plots depicting the FACS-isolation strategy for CD25⁻ T cells (gated CD45⁺CD11b⁻CD3⁺CD4⁺CD8⁻KLRG1⁻), CD25⁺ Tregs (gated CD45⁺CD11b⁻CD3⁺CD4⁺CD8⁻) and CD103⁺ Tregs (gated CD45⁺CD11b⁻CD3⁺CD4⁺CD8⁻KLRG1⁻) from tumors or spleens of PDG-Ink4a glioblastoma-bearing mice, 12d post RT and RT + Conc.IT treatment initiation, as well as CD8⁺ T cells from control spleens (gated CD45⁺CD11b⁻CD3⁺). Sorted cells gated in red. Representative of n = 5 independent repeats. b, Bar graphs showing the normalized expression of indicated genes in CD25⁺ Tregs (gated from CD45⁺CD11b⁺CD3⁺CD8⁻CD4⁺) and CD103⁺ Tregs (gated from CD45⁺CD11b⁺CD3⁺CD8⁻CD4⁺KLRG1⁻) FACS-purified from PDG-Ink4a glioblastoma 12d post treatment initiation (RT CD25⁺ n = 3, RT + Conc.IT CD25⁺ n = 3, RT + Conc.IT CD103⁺ n = 3 mice). c, Flowcytometry quantification of CD39⁺, Ki67⁺, IFNy⁺, GrzB⁺ and GrzA⁺ FACS-purified CD8⁺ T cells (CD45⁺CD11b⁻CD3⁺) from control spleen after 24 h of monoculture (mono) or co-culture (cocx) with CD25⁻ T cells (CD45⁺CD11b⁻CD3⁺CD4⁺CD8⁻KLRG1⁻), CD25⁺ Tregs (CD45⁺CD11b⁻CD3⁺CD4⁺CD8⁻) or CD103⁺ Tregs (CD45⁺CD11b⁻CD3⁺CD4⁺CD8⁻KLRG1⁻) isolated from spleens of tumor-bearing PDG-Ink4a mice 12d post RT + Conc.IT initiation. Cells were stimulated with anti-CD3/anti-CD28 antibodies, except for the monoculture control (mono unstim), and cultured in 1:1 and 1:2 ratios (Treg:CD8⁺ T cell; unstim n = 7, mono: n = 7, 1:1 CD25⁻ n = 5, 1:1 CD25⁺ n = 3, 1:1 CD103⁺ n = 2, 1:2 CD25⁻ n = 5, 1:2 CD25⁺ n = 5, 1:2 CD103⁺ n = 5 biologically independent samples). Statistics: one-way ANOVA with Benjamini, Krieger and Yekutieli correction for multiple testing (b,c). Data in this figure are represented as mean + S.E.M (b) and ± S.E.M (c). Gating strategies (c) depicted in Extended Data Fig. 6a.

Source data