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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 6. — IL-3, IL-5, and GM-CSF cytokines mediate Th2 cell immunity against breast carcinogenesis. (A–C) 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; A), Tnf^KO (n = 6, 2/6 tumors were <0.5 cm³ at the endpoint; B), Ifng^KO (n = 10, 6/10 tumors were <0.5 cm³ at the endpoint; C) mutant CD4⁺ T cells compared to Tslpr^KO CD4⁺ T cells (n = 8, 3/8 tumors were <0.5 cm³; n = 5, 0/5 tumors were <0.5 cm³; and n = 8, 2/8 tumors were <0.5 cm³ at the endpoint, respectively; two-way ANOVA). (D and E) 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 Tslpr^KO (n = 7, 2/7 tumors were <0.5 cm³ at the endpoint) CD4⁺ T cells (two-way ANOVA; D) and WT (n = 9, 6/9 tumors were <0.5 cm³ at the endpoint) versus Tslpr^KO (n = 8, 1/8 tumors were <0.5 cm³ at the endpoint) CD4⁺ T cells (E) while both groups were treated with anti–IL-5 plus anti–GM-CSF blocking antibodies (ab; two-way ANOVA). (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 Tslpr^KO CD4⁺ T cells and rat IgG isotype control antibody (n = 14, 1/14 tumors were <0.5 cm³ at the endpoint; two-way ANOVA). (G) Schematic diagram of the experimental paradigm used to elucidate the role of IL-3/IL-5/GM-CSF receptors on breast cancer cells in mediating the antitumor effects of TSLP-stimulated CD4⁺ T cells against breast cancer. (H) Spider plot of PyMtOva^tg Csf2rb^KO Csf2rb2^KO primary tumor growth in Tslp^tg Tslpr^KO mice injected with WT (n = 7, 1/7 tumors were <0.5 cm³ at the endpoint) versus Tslpr^KO CD4⁺ T cells (n = 7, 1/7 tumors were <0.5 cm³ at the endpoint, two-way ANOVA). (I) PyMtOva^tg Csf2rb^KO Csf2rb2^KO tumor weights at the endpoint in Tslp^tg Tslpr^KO mice injected with WT (n = 7) versus Tslpr^KO (n = 7) CD4⁺ T cells (Mann-Whitney U test). (J) Percentage survival of Tslp^tg Tslpr^KO mice injected with WT (n = 7) versus Tslpr^KO (n = 7) CD4⁺ T cells followed by PyMtOva^tg Csf2rb^KO Csf2rb2^KO primary tumor implantation (log-rank test). (K) Distribution of histological grades of PyMtOva^tg Csf2rb^KO Csf2rb2^KO primary tumors developed in Tslp^tg Tslpr^KO mice injected with WT (n = 7) versus Tslpr^KO (n = 7) CD4⁺ T cells (Fisher’s exact test). (L) Representative images of H&E-stained PyMtOva^tg Csf2rb^KO Csf2rb2^KO breast tumors from Tslp^tg Tslpr^KO mouse injected with WT CD4⁺ T cells, Tslp^tg Tslpr^KO mouse injected with Tslpr^KO CD4⁺ T cells, and the donor tumor from PyMtOva^tg Csf2rb^KO Csf2rb2^KO mouse (scale bar: 100 μm). (M) Representative flow plots of transcription factor expression in PyMtOva^tg Csf2rb^KO Csf2rb2^KO breast tumor–infiltrating CD4⁺ T cells isolated from Tslp^tg Tslpr^KO mice injected with CD45.1⁺ WT versus TSLPR^KO CD4⁺ T cells. Numbers on the plots represent the percentage of cells within each gate. (N) Percentage of GATA3⁺ Th2 cells among CD45.1⁺ WT CD4⁺ T cells (test, n = 6) versus Tslpr^KO CD4⁺ T cells (control, n = 6) isolated from PyMtOva^tg Csf2rb^KO Csf2rb2^KO breast tumors in Tslp^tg Tslpr^KO mice (Mann–Whitney U test). Bar graph shows mean + SD. Each of the tumors in the studies is from a separate mouse. All experimental data verified in at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.