Figure S3.

Th2 polarization and cytokine release play critical roles in TSLP-stimulated CD4⁺ T cell immunity against breast cancer. (A) Representative flow plots of transcriptional factor expression in tumor-infiltrating CD4⁺ T cells in Tslp-PyMt^tg Il4r^KO, PyMt^tg Il4r^KO, Tslp-PyMt^tg, and PyMt^tg mice. Numbers on the plots show the percentage of the cells within each gate. (B) Representative images of H&E-stained breast tumors in Tslp-PyMt^tg Il4r^KO, PyMt^tg Il4r^KO, Tslp-PyMt^tg, and PyMt^tg groups (scale bar: 100 μm). (C) Representative low (scale bars: 1 mm) and high (insets, scale bars: 100 μm) magnification images of H&E-stained lungs from Tslp-PyMt^tg Il4r^KO, PyMt^tg Il4r^KO, Tslp-PyMt^tg, and PyMt^tg mice. Breast cancer metastatic foci in each lung are highlighted by arrows. (D) The number of breast cancer metastatic foci in the lungs of Tslp-PyMt^tg Il4r^KO (test, n = 20) compared with PyMt^tg Il4r^KO mice (control, n = 23, Mann–Whitney U test). (E) Schematic diagram of the experimental paradigm used to elucidate the ability of IL4rα^KO compared with WT CD4⁺ T cells to suppress breast tumor growth in Rag1^KO mice in response to TSLP induction by topical calcipotriol treatment. Note that the calcipotriol treatment was started when implanted PyMt^tg tumors became palpable (∼5 mm in diameter) and repeated every 3 d until the conclusion of the study. Tumor/T cell donor and recipient mice are on the BALB/c background. (F) Spider plot of PyMt^tg primary tumor growth in Rag1^KO mice injected with Il4rα^KO (n = 5) versus WT (n = 4) CD4⁺ T cells and treated with calcipotriol (two-way ANOVA). The duration of calcipotriol treatment is indicated by purple bar on the graph. (G–I) Spider plots of PyMtOva^tg Tslpr^KO primary tumor growth in Tslp^tg Tslpr^KO mice injected with Il4^KO (n = 8, 2/8 tumors were <0.5 cm³ at the endpoint; G), Tnf^KO (n = 6, 2/6 tumors were <0.5 cm³ at the endpoint; H), Ifng^KO (n = 10, 6/10 tumors were <0.5 cm³ at the endpoint; I) mutant CD4⁺ T cells compared to WT CD4⁺ T cells (n = 6, 6/6 tumors were <0.5 cm³; n = 6, 5/6 tumors were <0.5 cm³; and n = 8, 6/8 tumors were <0.5 cm³ at the endpoint, respectively). Note that Il4^KO, Tnf^KO, and Ifng^KO tumor growth data are also shown in Fig. 6, A–C (two-way ANOVA). (J) Representative images of H&E-stained PyMtOva^tg Tslpr^KO breast tumors in Tslp^tg Tslpr^KO mice injected with Ifng^KO versus Tslpr^KO CD4⁺ T cells (scale bar: 100 μm). (K and L) Spider plots of primary PyMtOva^tg Tslpr^KO tumor growth in Tslp^tg Tslpr^KO mice injected with Il3^KO (n = 6, 4/6 tumors were <0.5 cm³ at the endpoint) versus WT (n = 6, 5/6 tumors were <0.5 cm³ at the endpoint) CD4⁺ T cells (two-way ANOVA; K) and WT CD4⁺ T cells in combination with anti–IL-5 plus anti–GM-CSF blocking antibodies (ab, n = 9, 6/9 tumors were <0.5 cm³ at the endpoint) versus WT CD4⁺ T cells and Rat IgG isotype control antibody (n = 10, 8/10 tumors were <0.5 cm³ at the endpoint; L; two-way ANOVA). Note that Il3^KO and WT CD4⁺ T cells plus anti–IL-5 plus anti–GM-CSF blocking antibodies tumor growth data are also shown in Fig. 6, D and E. (M) Representative images of HC11 mammosphere immunofluorescence staining after exposure to supernatants (sup.) derived from splenic Il3^KO versus WT CD4⁺ T cells stimulated once with anti-CD3, anti-CD28, and TSLP for 3 d ex vivo. Before addition to HC11 mammosphere culture, Il3^KO and WT CD4⁺ T cell supernatants were mixed with IL5/GM-CSF blocking and control antibodies, respectively. Mammospheres were stained for E-cadherin (green), Ki67 (red), and DAPI (blue, scale bar: 100 μm). (N) Quantification of HC11 mammosphere size in Il3^KO CD4⁺ T cell supernatant plus IL-5/GM-CSF antibodies (n = 14) versus WT CD4⁺ T cell supernatant plus control antibody (n = 14) group. Each dot represents one mammosphere (Mann–Whitney U test). (O) The number of Ki67⁺ cells per HC11 mammosphere exposed to Il3^KO CD4⁺ T cell supernatant plus IL-5/GM-CSF antibodies (n = 13) versus WT CD4⁺ T cell supernatant plus control antibody (n = 6). Each dot represents one mammosphere (Mann–Whitney U test). Bar graph shows mean + SD. All experimental data verified in at least two independent experiments. *, P < 0.05; ***, P < 0.0001.