Dendritic cell Piezo1 directs the differentiation of TH1 and Treg cells in cancer
- Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, China
- Department of Immunology, School of Basic Medical Sciences, Fudan University, China
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, China
Figures
Figure 1 with 5 supplements
Dendritic cell (DC)-specific Piezo1 regulates T cell differentiation in cancer.
(A) MC38 tumor cells were implanted subcutaneously in WT and Piezo1-/- mice (n=10) and tumor size was measured every 5 days for 40 days. (B) Intracellular staining of IFNγ, IL-4, IL-17A, and Foxp3 …
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Figure 1—source data 1
Tumor size in WT and Piezo1-/- tumor-bearing mice.
- https://cdn.elifesciences.org/articles/79957/elife-79957-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Piezo1 expressions of dendritic cells (DCs) following innate stimuli.
Il12b and Tgfb1 mRNA (A and C) and Piezo1 mRNA (B) expression of splenic DCs from C57BL/6 or/and Piezo1-/- mice with the indicated treatment (lipopolysaccharide [LPS], 10 ng/ml or conditioned with 2 …
Figure 1—figure supplement 2
Dendritic cell (DC)-specific Piezo1 deficiency reduced T cell activities in aged mice.
(A) Body weights of WT and Piezo1-/- mice are shown (n=20). (B–C) Piezo1 deficiency in DCs resulted in less CD44highCD62Low cells in the CD4+ T cell population than WT DCs. A typical figure shown in …
Figure 1—figure supplement 3
Dendritic cell (DC)-specific Piezo1 deficiency alters T cell differentiation in the aged mice.
Intracellular IFNγ, IL-4, IL-17A, and Foxp3 expression by CD4+ T cells from the mesenteric lymph node (MLN), Peyer’s patche (PP), intraepithelial lymphocyte (IEL), and lamina propria lymphocyte …
Figure 1—figure supplement 4
Dendritic cell (DC)-specific Piezo1 regulates T cell differentiation in cancer.
MC38 tumor cells were implanted subcutaneously in WT and Piezo1-/- mice (n=10). (A) CD8+T cell number in tumor of WT and Piezo1-/- tumor-bearing mice at day 40. (B) Intracellular staining of IFNγ …
Figure 1—figure supplement 5
Dendritic cell (DC)-specific Piezo1 regulates T cell differentiation in cancer.
(A) B16.F10 tumor cells were implanted subcutaneously in WT and Piezo1-/- mice (n=10) and tumor size was measured every 5 days for 30 days. (B) Intracellular staining of IFNγ, IL-4, IL-17A, and …
Figure 2
Dendritic cell (DC)-specific Piezo1 expression directs the differentiation of TH1 and Treg cells in vivo.
(A–C) Naïve CD45.2+ T cells were transferred into CD45.1+ C57BL/6 WT mice, and the mice were immunized with WT and Piezo1-/- splenic DCs and lipopolysaccharide (LPS). DLN cells were analyzed at day …
Figure 3 with 4 supplements
Piezo1 regulates IL-12 and TGFβ1 production by dendritic cells (DCs) to direct TH1 and Treg cell differentiation.
(A–B) Intracellular staining of IL-12p40 and TGFβ1 expression in WT and Piezo1-/- splenic DCs after 5 hr of treatment with lipopolysaccharide (LPS) (A; 10 ng/ml) or culture on 2 and 50 kPa hydrogels …
Figure 3—figure supplement 1
Dendritic cell (DC)-specific Piezo1 expression directs TH1 and Treg differentiation in vitro.
(A–B) C57BL/6 naïve CD4+ T cells were cocultured with lipopolysaccharide (LPS)-pulsed splenic CD11b+ DCs (A) or CD8+ DCs (B) from WT and Piezo1-/- mice for 5 days. Intracellular staining of IFNγ and …
Figure 3—figure supplement 2
Dendritic cell (DC)-specific Piezo1 expression directs TH1 and Treg differentiation in vitro.
(A) Naïve T cells from C57BL/6 mice were cocultured with splenic DCs from WT and Piezo1-/- mice for 5 days in the presence of anti-CD3 (2 ng/ml) and lipopolysaccharide (LPS) (10 ng/ml). The relative …
Figure 3—figure supplement 3
Piezo1 regulates dendritic cell (DC) homeostasis and function.
(A) 7-AAD and Annexin V staining in WT and Piezo1-/- splenic DCs. The representative data from flow cytometry analysis shown (left), and data summarized (right). (B–C) Expression of indicated …
Figure 3—figure supplement 4
Piezo1 regulates dendritic cell (DC) cytokine production.
(A) mRNA expressions of Il12 (p70) and Tgfb1 expression in WT and Piezo1-/- mesenteric lymph node (MLN) DCs. (B) mRNA expressions of Il12 (p70), Il23, and Tgfb1 expression in WT and Piezo1-/- …
Figure 4 with 2 supplements
TGFβR2-pSmad3 and IL-12Rβ2-pSTAT4 are required for the T cell differentiation induced by dendritic cell (DC)-specific Piezo1 expression.
(A) Expression of TGFβR2 and IL-12Rβ2 in T cells cocultured with WT or Piezo1-/- splenic DCs for 5 days. A representative figure shown on the left, and the data summarized on the right. (B) …
Figure 4—figure supplement 1
Expressions of TGFβR1/2 and IL-12Rβ1 in T cells induced by Piezo1-/- dendritic cells (DCs).
Expression of TGFβR1, TGFβR3 (A), and IL-12Rβ1 (B) in T cells cocultured with WT or Piezo1-/- splenic DCs for 5 days. Data are representative of three independent experiments (mean ± s.d.; n=4).
Figure 4—figure supplement 2
Knockdown of Tgfbr2 short hairpin RNA (shRNA) and Il12rb2 shRNA in T cells.
Sorted CD4+ T cells were transfected with control or Tgfbr2 shRNA vector (A) or Il12rb2 shRNA vector (B) and stimulated with WT or Piezo1-/- splenic dendritic cells (DCs) for 5 days in the presence …
Figure 5 with 5 supplements
Piezo1 regulates TGFβ1 and IL-12 production through the SIRT1-HIF1α-glycolysis pathway.
(A–B) Sorted splenic dendritic cells (DCs) from WT or Piezo1-/- mice were stimulated with lipopolysaccharide (LPS) (10 ng/ml; A) or with 2 or 50 kPa hydrogels (B) for 24 hr in the presence or …
Figure 5—figure supplement 1
Piezo1 alters glycolytic metabolic signaling activities of dendritic cells (DCs).
Sorted splenic DCs from WT mice were stimulated with lipopolysaccharide (LPS) (10 ng/ml) or with 2 or 50 kPa hydrogels conditions for 24 hr in the presence or absence of 2-deoxy-D-glucose (2-DG) (1 …
Figure 5—figure supplement 2
Glycolysis activities are required for IL-12 and TGFβ1 production in dendritic cells (DCs) induced by Yoda1 treatment.
(A–B) Sorted splenic DCs from WT mice were stimulated with lipopolysaccharide (LPS) (10 ng/ml) or with 2 or 50 kPa hydrogels conditions for 24 hr in the presence or absence of 2-deoxy-D-glucose …
Figure 5—figure supplement 3
Glycolytic metabolism activities are required for IL-12 and TGFβ1 production in dendritic cells (DCs) induced by Piezo1 deficiency.
(A) Sorted splenic DCs from WT or Piezo1-/- mice were stimulated with lipopolysaccharide (LPS) (10 ng/ml) or 2 or 50 kPa hydrogels for 24 hr in the presence or absence of succinate (5 mM). The …
Figure 5—figure supplement 4
TGFβ1, HIF1α, and glycolysis activity alteration in dendritic cells (DCs) induced by Piezo1 and SIRT1 deficiency.
Splenic DCs from WT, Piezo1-/-, Sirt1-/-, and Piezo1/Sirt1 double knockout (DKO) mice were stimulated with lipopolysaccharide (LPS) (10 ng/ml). (A) Intracellular staining of TGFβ1. (B) Intracellular …
Figure 5—figure supplement 5
TGFβ1, SIRT1, and glycolysis activity alteration in dendritic cells (DCs) induced by Piezo1-HIF1α signaling.
Splenic DCs from WT or Hif1a-/- mice were stimulated with lipopolysaccharide (LPS) (10 ng/ml) in the presence or absence of Yoda1 (25 μM). (A) Intracellular staining of TGFβ1. (B) Intracellular …
Figure 6 with 2 supplements
Piezo1 regulates TGFβ1 and IL-12 production through the calcium-calcineurin-NFAT axis.
(A) Measurement of intracellular Ca2+ concentrations with Fura2 dye in splenic dendritic cells (DCs) from WT or Piezo1-/- mice treated with lipopolysaccharide (LPS) (10 ng/ml) or cultured on plates …
Figure 6—figure supplement 1
Piezo1 regulates TGFβ1 and IL-12 production through calcium signaling.
(A) Intracellular Ca2+ using Fura2 in splenic dendritic cells (DCs) from WT or Piezo1-/- mice with indicated treatment (Yoda1, 25 μM; ruthenium red, 30 μM; gadolinium chloride, 10 μM). (B–C) …
Figure 6—figure supplement 2
Piezo1 regulates TGFβ1 production through calcium-calcineurin-NFAT axis.
(A or C) Intracellular staining of TGFβ1 in splenic dendritic cells (DCs) from WT mice with indicated treatments. (B) Intracellular staining of TGFβ1 in splenic DCs from WT or Piezo1-/- mice with …
Figure 7 with 1 supplement
IL-12 and TGFβ1 are critical for dendritic cell (DC) piezo1-dependent T cell differentiation in promoting cancer growth.
(A) MC38 tumor cells were implanted subcutaneously in WT and Piezo1-/- mice (n=10), IL-12 100 ng or anti-TGFβ1 mAb 200 ng per mouse in 50 µl volume or vehicle (PBS) was locally injected into tumor …
Figure 7—figure supplement 1
Dendritic cell (DC) Piezo1 controls the differentiation of TH1 and Treg cells in cancer.
Proposed model of how Piezo1 in DCs responses to inflammatory stimuli or stiffness signals to regulate the differentiation of TH1 and Treg cell populations in regulating cancer growth.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Mouse: C57BL/6J (CD45.1 and CD45.2) | Jackson Laboratory | RRID:IMSR_JAX:000664 | |
| Genetic reagent (Mus musculus) | Mouse: Piezo1flox/flox | Jicuiyaokang company of China | ||
| Genetic reagent (Mus musculus) | Mouse: Cd11c-Cre | Nanfangmoshi biological company of China | ||
| Genetic reagent (Mus musculus) | Mouse: Piezo1-/- | Jicuiyaokang company of China | ||
| Genetic reagent (Mus musculus) | Mouse: Sirt1flox/flox | Jackson Laboratory | ||
| Genetic reagent (Mus musculus) | Mouse: Hif1aflox/flox | Jackson Laboratory | ||
| Genetic reagent (Mus musculus) | Mouse: Cd4-Cre | Jackson Laboratory | ||
| Genetic reagent (Mus musculus) | Mouse: OTII TCR-transgenic mice | Jicuiyaokang company of China | ||
| Biological sample (Mus musculus) | Primary mouse splenocyte cells | Beijing Normal University | Freshly isolated from mice | |
| Biological sample (Mus musculus) | Primary mouse bone marrow cells | Beijing Normal University | Freshly isolated from mice | |
| Biological sample (Mus musculus) | Primary mouse serum | Beijing Normal University | Freshly isolated from mice | |
| Cell line | Mouse colon cancer cell line MC-38 | China cell bank ATCC Center | MC38 | |
| Cell line | Mouse melanoma cell line B16.F10 | China cell bank ATCC Center | B16.F10 | |
| Cell line | Human DC | Lonza | CC-2701 | |
| Cell line | Human cord blood CD4+ T cells | Lonza | 2C-200 | |
| Antibody | Rat monoclonal anti-mouse CD11c FITC | Thermo Fisher Scientific | Cat # MHCD11C01; RRID: AB_10373970 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD11c APC eFluor780 | Thermo Fisher Scientific | Cat# 47-0116-42 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD11c PE | Thermo Fisher Scientific | Cat# 12-0114-81 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD4 APC-Cy7 | BD Bioscience | Cat#130-109-536 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD8α FITC | Thermo Fisher Scientific | Cat# 11-0081-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD11b FITC | Thermo Fisher Scientific | Cat# 11-0112-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse Ly6G PE | Thermo Fisher Scientific | Cat# 12-9668-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse F4/80 PE | Thermo Fisher Scientific | Cat# 12-4801-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD19 PE | Thermo Fisher Scientific | Cat# 12-0199-42 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse TCR FITC | Thermo Fisher Scientific | Cat# TCR2730 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD44 FITC | Thermo Fisher Scientific | Cat# 11-0441-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD62L APC | Thermo Fisher Scientific | Cat# 11-0621-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD80 APC | Thermo Fisher Scientific | Cat# 17-0801-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD54 FITC | Thermo Fisher Scientific | Cat# 17-0549-42 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse MHCII APC | Thermo Fisher Scientific | Cat# 17-5320-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse CD45 APC | Thermo Fisher Scientific | Cat# 17-0459-42 | FCS (1:200) |
| Antibody | Rat monoclonal anti-mouse NK1.1 PE | Thermo Fisher Scientific | Cat# 25-5941-82 | FCS (1:200) |
| Antibody | Rat monoclonal Anti-mouse CCR7 APC | Thermo Fisher Scientific | Cat# A18389 | FCS (1:200) |
| Antibody | Mouse monoclonal anti-mouse PDL1 PE | Thermo Fisher Scientific | Cat# 12-5982-82 | FCS (1:200) |
| Antibody | Mouse monoclonal anti-mouse PDL2 PE | Thermo Fisher Scientific | Cat# 12-5986-82 | FCS (1:200) |
| Antibody | Mouse monoclonal anti-mouse IFNγ PE | Thermo Fisher Scientific | Cat# 12-7311-82 | FCS (1:100) |
| Antibody | Mouse monoclonal anti-mouse IL-4 PE | Thermo Fisher Scientific | Cat# 12-7041-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse Foxp3 PE | Thermo Fisher Scientific | Cat# 12-5773-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse IL-12p40 | Thermo Fisher Scientific | Cat# 12-7123-82 | FCS (1:100) |
| Antibody | Rat monoclonal anti-mouse TGFβ1 | Thermo Fisher Scientific | Cat# 12-9829-42 | FCS (1:100) |
| Commercial assay or kit | Fixation/Permeabilization Solution Kit | BD Bioscience | Cat# 554714 | |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | Cat# 74106 | |
| Commercial assay or kit | FastQuant RT Kit | Tiangen | Cat# KR106-02 | |
| Commercial assay or kit | Mouse IL-12 p70 Quantikine ELISA Kit | R&D Systems | Cat# M1270 | |
| Commercial assay or kit | Mouse TGF-beta 1 DuoSet ELISA | R&D Systems | Cat# DY1679 | |
| Commercial assay or kit | SuperReal PreMix Plus SYBR Green | Tiangen | Cat# FP205-02 | |
| Chemical compound, drug | Cyclosporin A | Signa-Aldrich | Cat# 59865-13-3 | |
| Chemical compound, drug | Yoda1 | MCE | Cat# HY-18723 | |
| Chemical compound, drug | 11R-VIVIT | MCE | Cat# HY-P1430 | |
| Chemical compound, drug | Gadolinium chloride | Sigma-Aldrich | Cat# 19423-81-5 | |
| Chemical compound, drug | Ruthenium red | Sigma-Aldrich | Cat# 12790-48-6 | |
| Chemical compound, drug | 2-Deoxy-D-glucose | Sigma-Aldrich | Cat# 29702-43-0 | |
| Chemical compound, drug | Diethyl succinate | Sigma-Aldrich | Cat# 123-25-1 | |
| Chemical compound, drug | IL-4 | R&D Systems | Cat# 204-IL | |
| Chemical compound, drug | IL-12 | Peprotech | Cat# 210–12 | |
| Chemical compound, drug | Fibronectin | Sigma-Aldrich | Cat# ECM001 | |
| Chemical compound, drug | Carbonyl cyanide-4-[trifluoromethoxy] phenylhydrazone | Sigma-Aldrich | Cat# SML2959 | |
| Chemical compound, drug | Rotenone | Sigma-Aldrich | Cat# R8875 | |
| Chemical compound, drug | Fura2 AM | Sigma-Aldrich | Cat# 47989 | |
| Chemical compound, drug | LPS | Sigma-Aldrich | Cat# 2630 | |
| Chemical compound, drug | Freund’s Adjuvant, Complete | Sigma–Aldrich | Cat# F5881 | |
| Chemical compound, drug | PMA | Sigma-ALdrich | Cat# P8139 | |
| Chemical compound, drug | Ionomycin | Sigma-Aldrich | Cat# I0634 | |
| Chemical compound, drug | GM-CSF | R&D Systems | Cat# 7954GM-010/CF | |
| Chemical compound, drug | Collagenase D | Worthington | Cat# LS005273 | |
| Chemical compound, drug | Deoxyribonuclease I | Beyotime Biotechnology | Cat# D7076 | |
| Chemical compound, drug | Percoll | GE Health | Cat# 17-0891-01 | |
| Software, algorithm | Adobe Illustrator | Adobe | RRID:SCR_010279 | |
| Other | One-micrometer latex microspheres | Polysciences, Inc | Cat# 19821 | |
| Other | 7-AAD | BD Bioscience | Cat# 559925 RRID: AB_2869266 | A dye for identifying cell death |
| Other | Streptavidin-APC/eFluor 780 | Thermo Fisher Scientific | Cat# 47-4317-82; RRID:AB_10366688 | A secondary antibody for indirect staining to detect biotinylated primary antibody; FCS (1:500) |
| Other | Streptavidin-eFluor 450 | Streptavidin-eFluor 450 | Cat# 48-4317-82; RRID:AB_10359737 | A secondary antibody for indirect staining to detect biotinylated primary antibody; FCS (1:500) |
| Other | SYLGARD 527 A&B Silicone Dielectric Gel | Dow | Silicone Dielectric Gel |
