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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 5. — Antigen specificity of Th2 cell immunity against breast carcinogenesis. (A) Schematic diagram of the experimental paradigm used to elucidate the effector mechanism of TSLP-stimulated CD4⁺ T cells against breast cancer. Note that only transferred CD4⁺ T cells in the test groups express TSLP receptor (Tslpr), but all other cells including the implanted tumor cells lack TSLP receptor (i.e., Tslpr^KO). (B) Spider plot of PyMt^tg Tslpr^KO primary breast tumor growth in Tslp^tg Tslpr^KO mice injected with WT (test, n = 10, 6/10 tumors were <0.5 cm³ at the endpoint) versus Tslpr^KO (control, n = 10, 4/10 tumors were <0.5 cm³ at the endpoint) CD4⁺ T cells (two-way ANOVA). (C) Spider plot of PyMtOva^tg Tslpr^KO primary tumor growth in Tslp^tg Tslpr^KO mice injected with WT (n = 10, 9/10 tumors were <0.5 cm³ at the endpoint) versus Tslpr^KO (n = 10, 2/10 tumors were <0.5 cm³ at the endpoint) CD4⁺ T cells (two-way ANOVA). (D) Survival rate of Tslp^tg Tslpr^KO mice injected with WT (n = 15) versus Tslpr^KO (n = 19) CD4⁺ T cells followed by PyMtOva^tg Tslpr^KO primary tumor implantation (log-rank test). (E) Distribution of histological grades of PyMtOva^tg Tslpr^KO primary tumors developed in Tslp^tg Tslpr^KO mice injected with WT (n = 15) versus Tslpr^KO (n = 20) CD4⁺ T cells (Fisher’s exact test). (F) Representative images of H&E-stained PyMtOva^tg Tslpr^KO breast tumors from (top to bottom): tumor donor, Tslp^tg Tslpr^KO mouse injected with WT CD4⁺ T cells, and mouse injected with Tslpr^KO CD4⁺ T cell. Note the degree of breast tumor differentiation in WT (test) group compared with the original tumor harvested from the donor mouse and Tslpr^KO (control) group. (G) Representative images of CD3/CD4, CD3/CD8, MHCII/CD4, and CD103/CD4 immunofluorescence staining on PyMtOva^tg Tslpr^KO primary tumors developed in Tslp^tg Tslpr^KO mice injected with WT versus Tslpr^KO CD4⁺ T cells. Note that CD103 colocalizes with CD4 and CD3 on few cells in the WT tumors (scale bars, 100 μm). (H and I) Representative flow plots showing percentage GATA3⁺ Th2 and Foxp3⁺ regulatory T cells (Tregs) in PyMtOva^tg Tslpr^KO breast tumors of Tslp^tg Tslpr^KO mice injected with CD45.1⁺ WT versus Tslpr^KO CD4⁺ T cells (H) and percentage GATA3⁺ and Ki67⁺ cells among tumor-infiltrating Foxp3⁻ CD4⁺ T effector cells in Tslp^tg Tslpr^KO mice injected with WT or Tslpr^KO CD4⁺ T cells (I). Injected CD45.1⁺ WT CD4⁺ T cells are highlighted in red; Tslpr^KO CD4⁺ T cells are in blue. Numbers on the plots represent the percentage of cells within each gate. (J and K) Percentage GATA3⁺ Th2 cells among CD45.1⁺ WT CD4⁺ T cells (test, n = 6) versus Tslpr^KO CD4⁺ T cells (control, n = 10; J) and Ki67⁺ cells among CD45.1⁺ WT Foxp3⁻ CD4⁺ T cells (test, n = 10) versus Tslpr^KO Foxp3⁻ CD4⁺ T cells (control, n = 10; K) isolated from PyMtOva^tg Tslpr^KO breast tumors developed in Tslp^tg Tslpr^KO mice (Mann-Whitney U test). Bar graphs show mean + SD. (L) H&E and immunofluorescence images show breast tumor and adjacent mammary gland of a Tslp^tg Tslpr^KO mouse injected with WT CD4⁺ T cells and implanted with PyMtOva^tg Tslpr^KO tumor. CD4 immunofluorescence staining highlights CD4⁺ T cells infiltrating the tumor, but not the adjacent normal mammary gland of the mouse (insets: scale bars, 100 μm). (M) Representative images of immunohistochemical staining for OVA in breast tumor and adjacent mammary gland of Tslp^tg Tslpr^KO mice implanted with high-grade PyMtOva^tg Tslpr^KO tumor. Note that CD4⁺ Th2 cell immunity suppressed an OVA-expressing tumor by transforming it to gland-like structures while sparing the OVA-negative adjacent mammary glands (scale bar: 100 μm). (N) Representative images of H&E-stained skin, lung, and mammary glands of Tslp^tg Tslpr^KO mice injected with WT versus Tslpr^KO CD4⁺ T cells and implanted with PyMtOva^tg Tslpr^KO tumor. No sign of inflammation is detected in the nontumor tissues at the endpoint (scale bar: 100 μm). (O) Spider plot showing PyMtOva^tg Tslpr^KO primary tumor growth in Tslp^tg Tslpr^KO mice injected with OTII (n = 9, 7/9 tumors were <0.5 cm³ at the endpoint) versus Tslpr^KO (n = 10, 2/10 tumors were <0.5 cm³ at the endpoint) CD4⁺ T cells (two-way ANOVA). (P) Representative images of H&E-stained PyMtOva^tg Tslpr^KO breast tumors from (top to bottom): tumor donor, tumor recipient mice injected with OTII, or Tslpr^KO CD4⁺ T cells (scale bar: 100 μm). (Q) Spider plot of PyMt^tg Tslpr^KO primary tumor growth in Tslp^tg Tslpr^KO mice injected with Ova-specific CD4⁺ T cells (OTII, n = 7, 3/7 tumors were <0.5 cm³ at the endpoint) versus Tslpr^KO CD4⁺ T cells (n = 8, 3/8 tumors were <0.5 cm³ at the endpoint, two-way ANOVA). (R) Representative images of H&E-stained PyMt^tg Tslpr^KO breast tumors from (top to bottom): tumor donor mouse, mouse injected with OTII CD4⁺ T cells, and mouse injected with Tslpr^KO CD4⁺ T cells (scale bar: 100 μm). 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.