Fig. 5: Increased p27, IFNγ, and IFNγR1 in intratumoral Siah2^−/− Tregs.

a, b Lymphocytes from draining lymph nodes of Siah2 WT and Siah2^−/− mice were cultured, stimulated with CD3/CD28 antibodies plus IL2, and 3 days later percentages of CD4⁺Foxp3⁻, CD4⁺Foxp3⁺, and CD8⁺ cells among CD45⁺ cells were determined by flow cytometry (n = 4). c Lysates from lymphocytes stimulated as in a were immunoblotted for p27 and cleaved caspase 3 plus GAPDH as a loading control (left; n = 4). Graph at right shows represents relative intensity of p27 protein between genotypes. d qPCR analysis of indicated transcripts from lymphocytes cultured as in a (N = 4, both genotypes). e T cells isolated from CD45.2⁺ Siah2^−/− spleen and CD45.1 WT spleen were cocultured (1:1), stimulated with CD3/CD28 antibodies plus IL2, and 3 days later percentages of CD45.1⁺ WT and CD45.2⁺ Siah2^−/− cells in CD4⁺Foxp3⁻, CD4⁺ Foxp3⁺, and CD8⁺ populations were determined by flow cytometry (n = 4). f, g The experiment was carried on as in e, and the percentage of CD45.1⁺ WT and CD45.2⁺ Siah2^−/− in Ki67⁺ CD4⁺Foxp3⁺ g, Ki67⁺CD4⁺ Foxp3⁻, and Ki67⁺CD8⁺ populations f was determined by flow cytometry (n = 4). h p27(red)/Foxp3(green) immunostaining (left panels) of tissues from tumors grown in Siah2 WT and Siah2^−/− mice collected 11 days after melanoma cell injection, plus quantification (right; n = 3). Scale bar, 100 μm. i Frequencies of tumor-infiltrating IFN-γ⁺ or IFNγR1⁺ among CD8⁺, CD4⁺, and CD4⁺/CD25⁺ T cells 11 days after tumor inoculation and following overnight stimulation in vitro with PMA and Ionomycin (n = 5). j Expression of indicated mRNAs, as identified by single-cell RNAseq within the Treg cluster (cluster 13) in both genotypes. k Violin plot comparing expression levels of IL12/IFNγ-regulated genes, based on data obtained from single-cell RNAseq k; mean ± s.e.m. Data were analyzed by unpaired t-test in a–i, and by Wilcoxon rank-sum test in j–k. ***P < 0.0005, **P < 0.005, and *P < 0.05 compared with WT.