TGF-β suppresses type 2 immunity to cancer

Abstract

The immune system uses two distinct defence strategies against infections: microbe-directed pathogen destruction characterized by type 1 immunity1, and host-directed pathogen containment exemplified by type 2 immunity in induction of tissue repair2. Similar to infectious diseases, cancer progresses with self-propagating cancer cells inflicting host-tissue damage. The immunological mechanisms of cancer cell destruction are well defined3,4,5, but whether immune-mediated cancer cell containment can be induced remains poorly understood. Here we show that depletion of transforming growth factor-β receptor 2 (TGFBR2) in CD4+ T cells, but not CD8+ T cells, halts cancer progression as a result of tissue healing and remodelling of the blood vasculature, causing cancer cell hypoxia and death in distant avascular regions. Notably, the host-directed protective response is dependent on the T helper 2 cytokine interleukin-4 (IL-4), but not the T helper 1 cytokine interferon-γ (IFN-γ). Thus, type 2 immunity can be mobilized as an effective tissue-level defence mechanism against cancer.

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Fig. 1: Blockage of TGF-β signalling in CD4+ T cells suppresses tumour development independently of CD8+ T cells.
Fig. 2: Blockage of TGF-β signalling in CD4+ T cells triggers cancer cell death associated with stromal T cell localization.
Fig. 3: Blockage of TGF-β signalling in CD4+ T cells induces tumour tissue healing, vessel reorganization and hypoxia-associated cancer cell death.
Fig. 4: Cancer immunity triggered by blockage of TGF-β signalling in CD4+ T cells is dependent on IL-4 but not on IFN-γ.
Fig. 5: IL-4 promotes a TH2 cell gene-expression program in TGFBR2-deficient CD4+ T cells.

Data availability

Data generated in this study are included within the paper (and its supplementary information files) or are available from the corresponding authors upon reasonable request. Raw gene-expression data that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession number GSE151406Source data are provided with this paper.

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Acknowledgements

We thank members of the M.O.L. laboratory for helpful discussions. This work was supported by a Howard Hughes Medical Institute Faculty Scholar Award (M.O.L.), an award from Mr. William H. and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research and the Center for Experimental Therapeutics at MSKCC (M.O.L.) and a Cancer Center Support Grant (P30 CA08748). B.G.N. and M.H.D. are recipients of F31 CA210332 and F30 AI29273-03 awards from National Institutes of Health. C.C. and S.L. are Cancer Research Institute Irvington Fellows supported by the Cancer Research Institute. E.G.S. is a recipient of a Fellowship from the Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center of MSKCC.

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Authors

Contributions

M.L. and M.O.L. were involved in all aspects of this study, including planning and performing experiments, analysis and interpretation of data and writing the manuscript. F.K. and T.A.C. processed and analysed all sequencing data and wrote the manuscript. I.T. provided the key mouse line. Y.C., J.J.H. and A.A.H. provided human renal cell carcinoma specimens. K.J.C., D.K., B.G.N., W.S., C.C., M.H.D., E.G.S., S.G. and S.L. assisted with mouse colony management and performed experiments.

Corresponding author

Correspondence to Ming O. Li.

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Competing interests

MSKCC has filed a patent application with the U.S. Patent and Trademark Office directed toward methods and compositions for targeting TGF-β signalling in CD4+ helper T cells for cancer immunotherapy.

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Peer review information Nature thanks Eduard Batlle, Carla Rothlin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Blockage of TGF-β signalling in CD8+ T cells affects their activation and differentiation.

a, Representative flow cytometry plots showing TGFBR2 expression on CD4+ T cells and CD8+ T cells from the tumour-draining lymph nodes of Tgfbr2fl/fl PyMT and Cd8a-cre;Tgfbr2fl/fl PyMT mice. The experiments were performed independently three times with similar results. b, Representative flow cytometry plots of CD62L and CD44 expression and statistical analyses of the gated populations among CD4+FOXP3 T cells (top panel), CD4+FOXP3+ T cells (middle panel) and CD8+ T cells (bottom panel) from the tumour-draining lymph nodes of Tgfbr2fl/fl PyMT (n = 4) and Cd8a-cre;Tgfbr2fl/fl PyMT (n = 4) mice. c, Representative flow cytometry plots and statistical analyses of programmed cell death protein 1 (PD-1) and granzyme B (GzmB) expression in tumour-infiltrating CD8+ T cells from Tgfbr2fl/fl PyMT (n = 6) and Cd8a-cre;Tgfbr2fl/fl PyMT (n = 10) mice. Data are represented as the mean ± s.e.m. (biologically independent mice in b, c). Two-tailed unpaired t-test (b, c). Source data

Extended Data Fig. 2 Blockage of TGF-β signalling in CD4+ T cells affects their activation and differentiation, and represses tumour growth.

a, Representative flow cytometry plots of yellow fluorescent protein (YFP) expression in lymph node TCRβ+NK1.1CD4+, TCRβ+NK1.1CD8+, TCRβNK1.1+ and TCRγδ+ T cells isolated from Thpok-cre;Rosa26LSL-YFP (YFP) mice. The experiments were performed independently three times with similar results. b, TGFBR2 expression on CD4+ T cells and CD8+ T cells from the tumour-draining lymph nodes of Tgfbr2fl/fl PyMT and Thpok-cre;Tgfbr2fl/fl PyMT mice. The experiments were performed independently three times with similar results. c, Representative flow cytometry plots of CD62L and CD44 expression and statistical analyses of the gated populations among CD4+FOXP3 T cells (top panel), CD4+FOXP3+ T cells (middle panel) and CD8+ T cells (bottom panel) from the tumour-draining lymph nodes of Tgfbr2fl/fl PyMT (WT; n = 9) and Thpok-cre;Tgfbr2fl/fl PyMT (KO; n = 8) mice. d, Representative flow cytometry plots and statistical analyses of programmed cell death protein 1 (PD-1) and granzyme B (GzmB) expression in tumour-infiltrating CD8+ T cells from Tgfbr2fl/fl PyMT (n = 8) and Thpok-cre;Tgfbr2fl/fl PyMT (n = 8) mice. e, Representative flow cytometry plots of CD4 and CD8 expression in T cells from control mice or mice treated with anti-CD4 (αCD4). The experiments were performed independently twice with similar results. f, Tumour measurements of Tgfbr2fl/fl PyMT (n = 3) and Thpok-cre;Tgfbr2fl/fl PyMT (n = 3) mice treated with αCD4. Dashed lines denote tumour burden of individual mice and solid lines indicate mean of tumour burden in a group of mice. Data are represented as the mean ± s.e.m. (biologically independent mice in c, d). Two-tailed unpaired t-test (c, d, f). NS, not significant. Source data

Extended Data Fig. 3 CD8 deficiency does not affect cancer cell death triggered by blockage of TGF-β signalling in CD4+ T cells.

a, Representative immunofluorescence images of E-Cadherin (green), Ki67 (red) and cleaved Caspase 3 (CC3, cyan) in tumour tissues from 23-week-old Tgfbr2fl/fl PyMT and Cd8a-cre;Tgfbr2fl/fl PyMT mice. The percentage of Ki67+E-Cadherin+ cells over total E-Cadherin+ epithelial cells was calculated from 0.02 mm2 regions (n = 9). The percentage of CC3+ areas over total E-Cadherin+ areas was calculated from 0.02 mm2 regions (n = 9). b, Representative immunofluorescence images of E-Cadherin (green), Ki67 (red) and cleaved Caspase 3 (CC3, cyan) in tumour tissues from 23-week-old Cd8a/Tgfbr2fl/fl PyMT (Cd8a/) and Cd8a/Thpok-cre;Tgfbr2fl/fl PyMT (Cd8a/ knockout, Cd8a/ KO) mice. The percentage of Ki67+E-Cadherin+ cells over total E-Cadherin+ epithelial cells was calculated from 0.02 mm2 regions (n = 9). The percentage of CC3+ areas over total E-Cadherin+ areas was calculated from 0.02 mm2 regions (n = 9). Data are represented as the mean ± s.e.m. (biologically independent mice in a, b). Two-tailed unpaired t-test (a, b). Source data

Extended Data Fig. 4 Blockage of TGF-β signalling in CD4+ T cells causes leukocyte exclusion from the tumour parenchyma, and inhibits vasculature leakage in association with vasculature remodelling.

a, Representative immunofluorescence images of E-Cadherin (green), CD45 (red) and cleaved Caspase 3 (CC3, cyan) in tumour tissues from 23-week-old Tgfbr2fl/fl PyMT (WT) and Thpok-cre;Tgfbr2fl/fl PyMT (KO) mice. Intratumoural (white arrows) and stromal (yellow arrows) CD45+ leukocytes were counted from 0.1 mm2 regions (n = 8 for Tgfbr2fl/fl PyMT and Thpok-cre;Tgfbr2fl/fl PyMT tumour tissues from biologically independent mice). b, Representative images of Sulfo-NHS-biotin (white), CD31 (red) and E-Cadherin (green) and quantification of Sulfo-NHS-biotin density and cancer cell-associated Sulfo-NHS-biotin+ events (magenta arrows) in tumour tissues from 23-week-old Tgfbr2fl/fl PyMT and Thpok-cre;Tgfbr2fl/fl PyMT mice. The percentage of Sulfo-NHS-biotin+ areas (n = 15) and cancer cell-associated deposition events (n = 9) were calculated from 0.8 mm2 regions. c, Quantification of the volume of avascular regions from three-dimensional confocal CD31 staining images of tumour tissues from 23-week-old Tgfbr2fl/fl PyMT (n = 9) and Thpok-cre;Tgfbr2fl/fl PyMT (n = 9) mice. Data are represented as the mean ± s.e.m. (biologically independent mice in ac). Two-tailed unpaired t-test (ac). Source data

Extended Data Fig. 5 Blockage of TGF-β signalling in CD4+ T cells promotes expansion of CD4+FOXP3 T cells in tumours.

Representative flow cytometry plots of TCRβ, NK1.1, CD4, CD8 and FOXP3 expression and statistical analyses of the gated populations in tumour-infiltrating leukocytes from 23-week-old Tgfbr2fl/fl PyMT (WT; n = 9) and Thpok-cre;Tgfbr2fl/fl PyMT (KO; n = 9) mice. Data are represented as the mean ± s.e.m. (biologically independent mice). Two-tailed unpaired t-test. Source data

Extended Data Fig. 6 IFN-γ deficiency does not impair cancer immunity triggered by blockage of TGF-β signalling in CD4+ T cells.

a, Representative flow cytometry plots of CD62L and CD44 expression and statistical analyses of the gated populations among CD4+FOXP3 T cells (top panel) and CD4+FOXP3+ T cells (bottom panel) from Ifng/Tgfbr2fl/fl PyMT (Ifng/; n = 3) and Ifng/Thpok-cre;Tgfbr2fl/fl PyMT (Ifng/ KO; n = 4) mice. b, Representative flow cytometry plots of TCRβ, NK1.1, CD4, CD8 and FOXP3 expression and statistical analyses of the gated populations in tumour-infiltrating leukocytes from 23-week-old Ifng/Tgfbr2fl/fl PyMT (Ifng/; n = 4) and Ifng/Thpok-cre;Tgfbr2fl/fl PyMT (Ifng/ KO; n = 5) mice. c, Representative immunofluorescence images of fibrinogen (Fg, white), CD31 (red), cleaved Caspase 3 (CC3, cyan) and E-Cadherin (green) in comparable individual tumours with sizes around 8x8 mm in length and width from 23-week-old Ifng/Tgfbr2fl/fl PyMT (Ifng−/−) and Ifng/Thpok-cre;Tgfbr2fl/fl PyMT (Ifng/ KO) mice. Extravascular (EV) Fg deposition events (magenta arrows) were calculated from 1 mm2 regions (n = 9 for Ifng/Tgfbr2fl/fl PyMT and Ifng/Thpok-cre;Tgfbr2fl/fl PyMT tumour tissues). Isolated CD31+ staining (yellow arrows) was counted from 1 mm2 regions (n = 9 for Ifng/Tgfbr2fl/fl PyMT and Ifng/Thpok-cre;Tgfbr2fl/fl PyMT tumour tissues). d, Representative immunofluorescence images of a hypoxia probe (HPP, white), CD31 (red), cleaved Caspase 3 (CC3, cyan) and E-Cadherin (green) in tumours from Ifng/Tgfbr2fl/fl PyMT (Ifng−/−) and Ifng/Thpok-cre;Tgfbr2fl/fl PyMT (Ifng/ KO) mice. The percentage of HPP+E-Cadherin+ regions over E-Cadherin+ epithelial regions was calculated from 1 mm2 regions (n = 9 for Ifng/Tgfbr2fl/fl PyMT and Ifng/Thpok-cre;Tgfbr2fl/fl PyMT tumour tissues). The shortest distance of HPP+ regions (magenta dashed lines) or CC3+ regions (yellow dashed lines) to CD31+ endothelial cells was measured in tumour tissues from Ifng/Thpok-cre;Tgfbr2fl/fl PyMT mice (n = 9). Data are represented as the mean ± s.e.m. (biologically independent mice in a–d). Two-tailed unpaired (a–d) or paired (d) t-test. Source data

Extended Data Fig. 7 IL-4 deficiency impairs cancer immunity triggered by blockage of TGF-β signalling in CD4+ T cells.

a, Representative flow cytometry plots of CD62L and CD44 expression and statistical analyses of the gated populations among CD4+FOXP3 T cells (top panel) and CD4+FOXP3+ T cells (bottom panel) from Il4/Tgfbr2fl/fl PyMT (Il4/; n = 4) and Il4/Thpok-cre;Tgfbr2fl/fl PyMT (Il4/ KO; n = 6) mice. b, Representative flow cytometry plots of TCRβ, NK1.1, CD4, CD8 and FOXP3 expression and statistical analyses of the gated populations in tumour-infiltrating leukocytes from 23-week-old Il4/Tgfbr2fl/fl PyMT (Il4/; n = 5) and Il4/Thpok-cre;Tgfbr2fl/fl PyMT (Il4/ KO; n = 7) mice. c, Representative immunofluorescence images of fibrinogen (Fg, white), CD31 (red), cleaved Caspase 3 (CC3, cyan) and E-Cadherin (green) in comparable individual tumours with sizes around 8x8 mm in length and width from 23-week-old Il4/Tgfbr2fl/fl PyMT (Il4/) and Il4/Thpok-cre;Tgfbr2fl/fl PyMT (Il4/ KO) mice. Extravascular (EV) Fg deposition events (magenta arrows) were calculated from 1 mm2 regions (n = 9 for Il4/Tgfbr2fl/fl PyMT and Il4/Thpok-cre;Tgfbr2fl/fl PyMT tumour tissues). Isolated CD31+ staining (yellow arrows) was counted from 1 mm2 regions (n = 9 for Il4/Tgfbr2fl/fl PyMT and Il4/Thpok-cre;Tgfbr2fl/fl PyMT tumour tissues). d, Representative immunofluorescence images of a hypoxia probe (HPP, white), CD31 (red), cleaved Caspase 3 (CC3, cyan) and E-Cadherin (green) in tumours from Il4/Tgfbr2fl/fl PyMT (Il4/) and Il4/Thpok-cre;Tgfbr2fl/fl PyMT (Il4/ KO) mice. The percentage of HPP+E-Cadherin+ regions over E-Cadherin+ epithelial regions was calculated from 1 mm2 regions (n = 9 for Il4/Tgfbr2fl/fl PyMT and Il4/Thpok-cre;Tgfbr2fl/fl PyMT tumour tissues). Data are represented as the mean ± s.e.m. (biologically independent mice in a–d). Two-tailed unpaired t-test (a–d). Source data

Extended Data Fig. 8 Antitumour immunity triggered by TGFBR2-deficient CD4+ T cells is dependent on IL-4.

a, 16- to 17-week-old PyMT mice bearing 5x5 mm tumours were transferred with CD4+CD25- T cells from Tgfbr2fl/fl (WT; n = 4), Thpok-cre;Tgfbr2fl/fl (KO; n = 4), Il4/Tgfbr2fl/fl (Il4/; n = 3) and Il4/Thpok-cre;Tgfbr2fl/fl (Il4/ KO; n = 3) mice on a weekly basis for 6 weeks. Tumour burden was measured and plotted. b, Representative immunofluorescence images of Ki67 (red) and cleaved Caspase 3 (CC3, cyan) in tumours from PyMT recipients transferred with Tgfbr2fl/fl (WT), Thpok-cre;Tgfbr2fl/fl (KO), Il4/Tgfbr2fl/fl (Il4/) or Il4/Thpok-cre;Tgfbr2fl/fl (Il4/ KO) CD4+CD25 T cells for 6 weeks. The percentage of Ki67+E-Cadherin+ cells over total E-Cadherin+ epithelial cells was calculated from 0.02 mm2 regions (n = 9). The percentage of CC3+ areas over total E-Cadherin+ areas was calculated from 0.02 mm2 regions (n = 9). Data are represented as the mean ± s.e.m. (biologically independent mice). Two-tailed unpaired t-test. c, Tumour measurements of Tgfbr2fl/fl (WT; n = 5), Thpok-cre;Tgfbr2fl/fl (KO; n = 4), anti-IL-4 (αIL-4)-treated WT (n = 5), αIL-4-treated KO (n = 4), anti-IFN-γ (αIFN-γ)-treated WT (n = 3) and αIFN-γ-treated KO mice (n = 3) inoculated with MC38 cancer cells. Dashed lines denote tumour burden of individual mice and solid lines indicate mean of tumour burden in a group of mice (a, c). Two-tailed unpaired t-test (a, c). ***: P < 0.001; **: P < 0.01; and NS: not significant. Source data

Extended Data Fig. 9 A TH2 cell gene-expression signature stratifies patients with cancer for survival probability.

a, A TH2 gene-expression signature was used to perform survival analysis in TCGA Pan Cancer cohort data (n = 10,002) and survival curves were plotted for the signature high group (top 50%; n = 4,996) and low group (bottom 50%; n = 5,006). The corresponding censored patient numbers are included in major time points. Two-tailed log-rank test. b, The TH2 gene signature enrichment score was estimated and plotted for each RNA-sequencing sample of TCGA Pan Cancer patients (n = 10,050). The middle line in the box indicates median and the bound indicates 25% quartile (Q1) and 75% quartile (Q3). The whisker reaches to the max/min point within the 1.5x interquartile range from either Q3 or Q1, respectively. c, The TH2 gene signature was used to perform survival analysis in patients with low-grade glioma (LGG; n = 514) and glioblastoma multiforme (GBM; n = 160), kidney chromophobe (KICH; n = 65) and kidney renal clear cell carcinoma (KIRC; n = 533). The survival curves were plotted for the signature high group (top 50%) and low group (bottom 50%). The corresponding censored patient numbers are included in major time points. Two-tailed log-rank test. d, Representative immunofluorescence images of E-Cadherin (green) and CD31 (red) in KICH (n = 4 patients) and KIRC (n = 8 patients) tumour tissues. Isolated CD31+ staining was counted from 0.2-mm2 regions (n = 9). The stromal regions are marked by dotted lines in KICH samples. Data are shown as mean ± s.e.m. Two-tailed unpaired t-test. Source data

Extended Data Fig. 10 Blockage of TGF-β signalling in CD4+ T cells reprograms tumour vasculature and halts cancer progression.

In untransformed mammary glands, developmentally wired blood vasculature maintains a layer of healthy epithelium. In mammary tumours from control PyMT mice, sprouting angiogenesis is induced with an immature and leaky vasculature in support of cancer cell survival and tumour growth, which co-occurs with leukocyte infiltration to the tumour parenchyma. Blockage of TGF-β signalling in CD4+ T cells remodels the tumour vasculature with the endothelium ensheathed by pericytes and fibroblasts, concomitant with leukocyte exclusion from the tumour parenchyma. In addition, the vasculature pattern is reconfigured, which creates large avascular regions leading to cancer cell hypoxia and cancer cell death. Such a host-directed tissue-level cancer defence response is dependent on the type 2 cytokine IL-4.

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Supplementary Table

Supplementary Table 1 | Differentially expressed transcripts among tumor-infiltrating CD4+CD25- T cells from Tgfbr2fl/fl PyMT (WT; n=2 biological replicates), Thpok-cre;Tgfbr2fl/fl PyMT (KO; n=2 biological replicates with 2 technical repeats), Il4-/-Tgfbr2fl/fl PyMT (Il4-/-; n=2 biological replicates) and Il4-/-Thpok-cre;Tgfbr2fl/fl PyMT (Il4-/-KO; n=2 biological replicates) mice. According to the implementation of DESeq2 package, the test used for testing if a differential expression is significant is Wald test and a 2-tailed p value (pvalue) is reported. A multiple test corrected p value (padj) by Benjamini & Hochberg method (1995) is also reported. Please reference DESeq2 manual for more details.

Supplementary Table

Supplementary Table 2 | A TH2 gene-expression signature curated from the upregulated transcripts of tumor-infiltrating CD4+CD25- T cells in Thpok-cre;Tgfbr2fl/fl PyMT vs. Tgfbr2fl/fl PyMT and Thpok-cre;Tgfbr2fl/fl PyMT vs. Il4-/-Thpok-cre;Tgfbr2fl/fl PyMT comparisons.

Video 1

| Reconstructed three-dimensional video of CD31+ endothelial cells (red) from confocal tumor tissue images (500 x 500 x 180 μm in length x width x height) of 23-week-old Tgfbr2fl/fl PyMT mice. The experiments were performed independently three times with similar results.

Video 2

| Reconstructed three-dimensional video of CD31+ endothelial cells (red) from confocal tumor tissue images (500 x 500 x 180 μm in length x width x height) of 23-week-old Thpok-cre;Tgfbr2fl/fl PyMT mice. The experiments were performed independently three times with similar results.

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Liu, M., Kuo, F., Capistrano, K.J. et al. TGF-β suppresses type 2 immunity to cancer. Nature 587, 115–120 (2020). https://doi.org/10.1038/s41586-020-2836-1

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