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. 2022 Jun 3;219(7):e20201963. doi: 10.1084/jem.20201963

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© 2022 Boieri et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S4. — IL-3, IL-5, and GM-CSF receptors expression and function in breast normal epithelial and cancer cells. (A) Histogram showing expression levels of mouse IL-3Rα (CD123), common β chain receptor (CD131), IL-5Rα (CD125), and GM-CSFRα (CD116) on the surface of EpCAM⁺ CD45⁻ PyMt^tg tumor cells. Gray histograms with dashed outline show fluorescence minus one (FMO) control. (B) Box plots of human IL3RA (IL-3 receptor α chain), CSF2RA (GM-CSF receptor α chain), CSF2RB (common β chain receptor), and CRLF2 (TSLPR) gene expression in normal mammary glands versus breast cancer samples across TCGA/GTEx datasets (one-way ANOVA, Gene Expression Profiling Interactive Analysis database). (C) IL-3Rα immunohistochemical stain of human mammary gland and breast tumor. Images were obtained from the Human Protein Atlas website (https://v18.proteinatlas.org). (D) Representative images of common β chain receptor (CD131) IHC staining on human breast tumor and adjacent normal breast glands. Control staining with no primary antibody is also shown. (E) Representative images of common β chain receptor (CD131) and cytokeratin (CK) immunofluorescence staining on human breast tumor and adjacent normal breast glands. (F) Spider plot of PyMtOva^tg Tslpr^KO primary tumor growth in Tslp^tg Tslpr^KO mice injected with Il3^KO CD4⁺ T cells in combination with anti–IL-5/GM-CSF antibodies (n = 13, 1/13 tumors were <0.5 cm³ at the endpoint) versus WT CD4⁺ T cells and Rat IgG isotype control antibody (n = 10, 9/10 tumors were <0.5 cm³ at the endpoint; two-way ANOVA). Note that Il3^KO CD4⁺ T cells plus anti–IL-5/GM-CSF antibodies tumor growth data are also shown in Fig. 6 F. (G) Representative H&E and CD4-immunostained images of PyMtOva^tg Tslpr^KO primary tumors developed in Tslp^tg Tslpr^KO mice injected with Il3^KO CD4⁺ T cells plus anti–IL-5 and anti–GM-CSF blocking antibodies (test) versus Tslpr^KO CD4⁺ T cells plus isotype control antibody (negative control) and WT CD4⁺ T cells plus isotype control antibody (positive control). (H) Distribution of histological grades of PyMtOva^tg Tslpr^KO primary tumors developed in Tslp^tg Tslpr^KO mice injected with Il3^KO CD4⁺ T cells plus anti–IL-5 and anti–GM-CSF blocking antibodies (test, n = 13) versus Tslpr^KO CD4⁺ T cells plus isotype control antibodies (negative control, n = 14) and WT CD4⁺ T cells plus isotype control antibodies (positive control, n = 4, Fisher’s exact test). (I) Quantification of CD4⁺ T cells in PyMtOva^tg Tslpr^KO primary tumors developed in Tslp^tg Tslpr^KO mice injected with Il3^KO CD4⁺ T cells plus anti–IL-5 and anti–GM-CSF blocking antibodies (test, n = 13) versus Tslpr^KO CD4⁺ T cells plus isotype control antibody (negative control, n = 12) and WT CD4⁺ T cells plus isotype control antibody (positive control, n = 4). CD3/CD4 double-positive cells were counted in 10 HPF images per tumor sample. HPF images for each sample were chosen randomly across the tumor section. Each dot represents one HPF image (unpaired t test). (J) PyMtOva^tg Tslpr^KO tumor weight developed in Tslp^tg Tslpr^KO mice injected with Il3^KO CD4⁺ T cells plus anti–IL-5 and anti–GM-CSF blocking antibodies (n = 13) versus Tslpr^KO CD4⁺ T cells plus isotype control antibody (n = 12). Note that WT CD4⁺ T cells plus isotype control antibody (positive control) were not included in this analysis, as only one of four tumors had an appreciable mass (Mann–Whitney U test). (K) Flow plot showing percentage GATA3⁺ Th2 and Foxp3⁺ regulatory T cells (Tregs) among tumor-infiltrating CD4⁺ T cells in Il3^KO CD4⁺ T cells plus anti–IL-5 and anti–GM-CSF blocking antibodies versus Tslpr^KO CD4⁺ T cells plus isotype control antibody group. (L) Percentage GATA3⁺ Th2 cells among tumor-infiltrating CD4⁺ T cells in Il3^KO CD4⁺ T cells plus anti–IL-5 and anti–GM-CSF blocking antibodies (n = 13) versus Tslpr^KO CD4⁺ T cells plus isotype control antibody (n = 13) group (Mann–Whitney U test). (M) Tumor weights compared between the tumors with high (n = 7) versus low (n = 6) percentage GATA3⁺ Th2 cells in Il3^KO CD4⁺ T cells plus anti–IL-5 and anti–GM-CSF blocking antibodies (test) group. Red arrows point to the tumor with the highest percentage of tumor-infiltrating Th2 cells (Mann–Whitney U test). (N) Percentage Foxp3⁺ Tregs among tumor-infiltrating CD4⁺ T cells in Il3^KO CD4⁺ T cells plus anti–IL-5 and anti–GM-CSF blocking antibodies (n = 13) versus Tslpr^KO CD4⁺ T cells plus isotype control antibody (n = 13) group (Mann–Whitney U test). Scale bars 100 μm. Bar graph shows mean + SD. All experimental data verified in at least two independent experiments. **, P < 0.01; ***, P < 0.0001.