Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 14:8:14649.
doi: 10.1038/ncomms14649.

Suppressive IL-17A+Foxp3+ and ex-Th17 IL-17AnegFoxp3+ Treg cells are a source of tumour-associated Treg cells

Stephanie Downs-Canner  1 , Sara Berkey  1 , Greg M Delgoffe  2   3   4 , Robert P Edwards  2   4   5 , Tyler Curiel  6 , Kunle Odunsi  7 , David L Bartlett  1 , Nataša Obermajer  1
Affiliations

Suppressive IL-17A+Foxp3+ and ex-Th17 IL-17AnegFoxp3+ Treg cells are a source of tumour-associated Treg cells

Stephanie Downs-Canner et al. Nat Commun. .

Abstract

Th17 and regulatory T (Treg) cells are integral in maintaining immune homeostasis and Th17-Treg imbalance is associated with inflammatory immunosuppression in cancer. Here we show that Th17 cells are a source of tumour-induced Foxp3+ cells. In addition to natural (n)Treg and induced (i)Treg cells that develop from naive precursors, suppressive IL-17A+Foxp3+ and ex-Th17 Foxp3+ cells are converted from IL-17A+Foxp3neg cells in tumour-bearing mice. Metabolic phenotyping of Foxp3-expressing IL-17A+, ex-Th17 and iTreg cells demonstrates the dissociation between the metabolic fitness and the suppressive function of Foxp3-expressing Treg cell subsets. Although all Foxp3-expressing subsets are immunosuppressive, glycolysis is a prominent metabolic pathway exerted only by IL-17A+Foxp3+ cells. Transcriptome analysis and flow cytometry of IL-17A+Foxp3+ cells indicate that Folr4, GARP, Itgb8, Pglyrp1, Il1rl1, Itgae, TIGIT and ICOS are Th17-to-Treg cell transdifferentiation-associated markers. Tumour-associated Th17-to-Treg cell conversion identified here provides insights for targeting the dynamism of Th17-Treg cells in cancer immunotherapy.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Th17 cells transdifferentiate into Foxp3+exTh17 cells in tumour-bearing mice.
ID8A ovarian cancer and MC38 colorectal cancer cells were injected intraperitoneally in IL-17aCreR26RReYFP fate reporter mice. TALs (ovarian cancer, n=4 mice per group), TILs (colorectal cancer, n=10 mice per group) and splenocytes were recovered at different time points of tumour progression and CD4+ T cells assessed for eYFP expression (a) as well as eYFP+ cells analysed for IL-17 production and Foxp3 expression (b) by flow cytometry. The fluorescence-activated cell sorting (FACS) gating strategy of live eYFP+ CD4+ T cells is presented in Supplementary Fig. 8a. We observed accumulation of eYFP+ cells (indicative of Th17 and/or exTh17 cells) over time (a) and the percentages of Foxp3+IL-17A− (that is, exTh17 Treg) cells increased in these mice with tumour progression. All data are mean±s.d. *P<0.05, **P<0.01 and ***P<0.001 by two-tailed Student's t-test. Similar results were obtained in an additional independent experiment.
Figure 2
Figure 2. Changes in tumour-associated Treg cells in Rorγt−/− mice.
Rorγt−/− and B6 mice were injected i.p. with ID8A cells. (a) The percentage of Foxp3+CD4+ cells of CD3+ cells in the tumour ascites and spleens were determined on day 35±2. Representative staining (left) and statistical analysis (right) of pooled data from two independent experiments with n=5 mice, respectively. The fluorescence-activated cell sorting (FACS) gating strategy of live T cells is presented in Supplementary Fig. 8b. (b,c) Percentages of Helios+ (b, left) and PD1+ (c, right) and representative staining of Foxp3+CD4+ cells in tumour ascites and spleens from n=5 mice. Black line indicates the staining of CD4+Foxp3+ cells from B6 mice and grey filled line CD4+Foxp3+ cells from Rorγt−/− mice. (d) Survival time was monitored in untreated and α-PD1 antibody (Ab)-treated Rorγt−/− (n=5) and B6 mice (n=10). Mice were treated with either PBS (200 μl i.p.) or RMP1–14 (α-PD1 Ab, 5 mg kg−1, BioXcell, 200 μl i.p.) on days 3, 6, 9 and 12. Spleens and ascites from untreated B6 and Rorγ−/− mice (n=5 per group) were collected at day 35±2 and cells purified. Survival curves were compared using log-rank (Mantel–Cox) test. All data (a–c) are mean±s.d. *P<0.05, **P<0.01 and ***P<0.001 by two-tailed Student's t-test.
Figure 3
Figure 3. Transdifferentiation of adoptively transfered Foxp3neg/+IL-17A+ T cells.
(a) Timeline of the experiment: CD4+ T cells from Foxp3GFP reporter mice were cultured under Th17 (IL-6, IL-23, TGF-β, 3 days)/Treg (TGF-β, +1 day)-driving conditions. Foxp3negIL-17A+ (i), Foxp3+IL-17A+ (ii), Foxp3negIL-17Aneg (iii) and Foxp3+IL-17Aneg (iv) CD4+ T cells were sorted and injected i.p. into ID8A-tumour bearing mice on days 5, 12 and 19. (b) Following staining with IL-17A-detection kit, live/CD4+/Th17–Treg subsets (i–iv) were flow sorted using the presented gating strategy (note that the 3 h activation with PMA and ionomycin before cell sorting results in a reduced mean fluorescence intensity of Foxp3-GFP; see Supplementary Fig. 9). The fluorescence-activated cell sorting (FACS) gating strategy for live CD4+ cells is presented in Supplementary Fig. 8c. (c) Th17–Treg subsets (i–iv) were injected i.p. into ID8A-luc tumour-bearing Cd45.1 mice on days 5, 12 and 19 and tumour growth monitored by bioluminescence (day 25, n=4 mice per group) compared with control tumour-bearing mice. (d) CD4+CD45.1neg cells from ID8A-luc tumour-bearing Cd45.1 mice, receving adoptively transfered Th17–Treg subsets (groups i–iv, n=4 mice per group), were analysed on day 40±1 for IL17A production and Foxp3 expression. Statistical analysis of the percentages of Foxp3negIL-17A+, Foxp3+IL-17A+ and Foxp3+IL-17Aneg of CD4+CD45.1neg cells in each group of mice (top) and representative staining (bottom). The FACS gating strategy of live cells is presented in Supplementary Fig. 8d. All data (c,d) are mean±s.d. *P<0.05 and **P<0.01 by two-tailed Student's t-test.
Figure 4
Figure 4. Tumour-associated TGF-β and PGE2 promote ex-Th17 Foxp3+IL-17Aneg cells.
Splenocytes from IL-17aCreR26RReYFP fate reporter mice were stimulated with CD3/CD28 microbeads under control, Th17 (IL-6, IL-23, TGF-β) and Treg (TGF-β)-driving conditions. (a–c) Representative flow cytometry analysis (day 3, a) and time-dependent induction (b,c) of eYFP+ cells (b) and Th17–Treg subsets (c) from IL-17aCreR26ReYFP fate reporter mice (CD4+ gated). Cells were cultured in the presence of Th17 (IL-6, IL-23 and TGF-β)- and Treg (TGF-β)-driving conditions and analysed on days 3, 4, 5 and 7 by flow cytometry for the expression of eYFP. The fluorescence-activated cell sorting (FACS) gating strategy of live cells is presented in Supplementary Fig. 8e (b) and the expression of Foxp3 and IL-17A production by eYFP+ cells is shown. Similar results were obtained in an additional independent experiment. (d) CD4+ T cells from IL-17aCreR26RReYFP reporter mice were cultured under Treg (TGF-β)-driving conditions or conditioned medium of ID8A cells (aggregate data of 3–7 independent experiments; mean±s.d., left). COX2 inhibitor celecoxib was added during preparation of conditioned medium and celecoxib and TGF-β blocking antibody were added to cell cultures during CD3/CD28 stimulation (right). (e) Representative flow cytometry analysis (day 7) of CD4+ T cells from Th17eYFP-Foxp3mRFP fate reporter mice cultured in the presence of Th17/Treg (IL-6, IL-23 and TGF-β, 3 days and TGF-β, +4 days)- and Treg (TGF-β, 7 days)-driving conditions. The FACS gating strategy of live cells is presented in Supplementary Fig. 8e. All data are mean±s.d. *P<0.05, **P<0.01 and ***P<0.001.
Figure 5
Figure 5. Suppressive Foxp3+IL-17A+ cells are metabolically active.
(a,b) YFP+Foxp3negIL-17A+, YFP+Foxp3+IL-17A+, YFP+Foxp3negIL-17Aneg, YFP+Foxp3+IL-17Aneg and YFPnegFoxp3negIL-17Aneg, YFP+Foxp3negIL-17Aneg CD3+ T cells were sorted using the strategy presented in Fig. 4e and analysed with XFe Extracellular Flux Analyzer. (a) Cumulative data of two independent experiments evaluating basal respiration (OCR) versus glycolysis (basal extracellular acidification rate (ECAR)) of individual Th17–Treg subsets (mean values of each real-time run, n=2 for YFP+IL-17A+Foxp3neg, YFP+IL-17A+Foxp3+, YFP+IL-17AnegFoxp3neg, YFP+IL-17AnegFoxp3+ and YFPnegIL-17AnegFoxp3+, n=7 YFPnegIL-17AnegFoxp3neg). (b) CD4+ T cells, isolated from Cd45.1 mice and stained with CFSE, were analysed after 72 h of stimulation with αCD3 Ab, in the presence of irradiated CD4neg fraction and Th17–Treg subsets, sorted as presented in Fig. 4e. One-way analysis of variance (ANOVA) of immunosuppressive effects of the subsets at 1:4 ratio subset:CD4+. Similar results were obtained in an additional independent experiment. (c,d) The dose-dependent effects of 2DG (c) and etomoxir (d) on the transdifferentiation of Th17 cells (the percentage of IL-17AnegFoxp3+ cells of CD4+eYFP+) is shown. Similar results were obtained in an additional independent experiment. All data are mean±s.d. *P<0.05, **P<0.01 and ***P<0.001 by one-way ANOVA.
Figure 6
Figure 6. Transcriptome analysis of IL-17A+Foxp3+/neg cells.
(a–c) Foxp3negIL-17A+ (i), Foxp3+IL-17A+ (ii), Foxp3negIL-17Aneg (iii) and Foxp3+IL-17Aneg (iv) CD3+ T cells were sorted using the strategy presented in Fig. 3a. (a) Heat map from hierarchical clustering of differentially expressed genes (n=3 per subset). (b) Volcano plot demonstrates increase in defined transmembrane markers (Tigit, Tmem154, Folr4, Iikzf2, Pglyrp1, Cd86, Lrrc32, Il1rl1, Itgb8) in Foxp3+IL-17A+ cells compared with Foxp3negIL-17A+ T cells. (c) Cumulative data of flow cytometric staining (ICOS, CD73, Lag3, neuropilin1) of Th17-Treg subsets (i–iv) from three independent experiments. Additional data (TIGIT, ST2, Folr4, GARP, PGLYRP1) and representative flow cytometric staining are presented in Supplementary Fig. 7. All data are mean±s.d. *P<0.05, **P<0.01 and ***P<0.001 by one-way analysis of variance (ANOVA).
Figure 7
Figure 7. Tumour induction of Treg-associated characteristics in human Th17 TALs.
(a–c) Foxp3-expressing IL-17A+/− cells from ovarian cancer patients are also characterized by expression of Th17–Treg cell plasticity-related targets. (a,b) Representative intracellular staining of IL-17A and Foxp3 in TALs (CD3+CD4+-gated) from two ovarian cancer ascites—note the inclination towards either Foxp3-expressing (left) or IL-17-producing (right) phenotype (a) and a comparison of the percentages of individual subsets (b) in TALs from ovarian cancer ascites (n=15) compared with the lymphocytes from peripheral blood of the matched donors (n=5). The fluorescence-activated cell sorting (FACS) gating strategy of live CD4+ T cells is presented in Supplementary Fig. 8f. (c) Flow cytometric analysis of neuropilin, GARP, TIGIT, PGLYRP1 and ST2 expression in human ovarian cancer Th17–Treg CD4+ TAL subsets (n=4, refer to Table 1a for the details on patient data). All data are mean±s.d. *P<0.05 and **P<0.01 by paired t-test. (d) Linear regression of Il17a and Foxp3 expression in ovarian cancer TALs (n=15, refer to Table 1b for the details on patient data). R2=0.2927, P=0.0373. (e) Linear regression of Rorc and Foxp3 expression in colorectal cancer tissue (n=20, refer to Table 1c for the details on patient data). R2=0.2475, P=0.0256. (f,g) Tumour-associated microenvironment induces Foxp3 expression and IL-10 production in human IL-17-producing CD4+ cells. Expression of Foxp3 (n=10) and Il17a (n=8) and IL-10 production (n=6) by IL-17A-producing CD4+ cells cultured in control, Th17- or Treg-driving (TGF-β, ovarian cancer conditioned medium) conditions for 6 days. All data are mean±s.d. *P<0.05, **P<0.01 and ***P<0.001 by one-way analysis of variance (ANOVA).
See this image and copyright information in PMC

References

    1. Pardoll D. M. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12, 252–264 (2012). - PMC - PubMed
    1. von Boehmer H. & Daniel C. Therapeutic opportunities for manipulating T(Reg) cells in autoimmunity and cancer. Nat. Rev. Drug Discov. 12, 51–63 (2013). - PubMed
    1. Rubtsov Y. P. et al. Stability of the regulatory T cell lineage in vivo. Science 329, 1667–1671 (2010). - PMC - PubMed
    1. Zhou X. Y. et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat. Immunol. 10, 1000–U1104 (2009). - PMC - PubMed
    1. Sakaguchi S., Vignali D. A. A., Rudensky A. Y., Niec R. E. & Waldmann H. The plasticity and stability of regulatory T cells. Nat. Rev. Immunol. 13, 461–467 (2013). - PubMed

Publication types

MeSH terms

Substances