Plasmid construction
Plasmids were constructed using standard Gibson cloning methods. DNA fragments of APJ, BILF1, TfR1 scFv H7 (ref. 29), TfR1 VHH-A (ref. 30), APJ VHH JN241 (ref. 32), A2AR VH/VL domains Hu3F6 H5-L5 (ref. 33), CCR6 VH/VL domains from 4H11 (ref. 44) and Fc variants (knobs-into-holes Fc for heterobispecific constructs28, G236R/L328R/D356E/L358M and H435A variants to abolish FcγR and FcRn interactions61,62,63 and N297G variant for increased stability and developability64) were synthesized by Integrated DNA Technologies or Twist Bioscience. Binder sequences and/or Fc variants were subcloned into a pFuse-hIgG1 vector (InvivoGen) for mammalian expression.
The pcDNA-FLAG–AT1R and pCDNA-FLAG–RXFP1 plasmids were obtained as previously described31,65. AT1R VHH and RXFP1 VHH were developed as previously described31,66. ADORA2A-DuET was a gift from Thomas Sakmar (Addgene plasmid 213174; http://n2t.net/addgene:213174; RRID:Addgene_213174). The pCDH-TfR-mApple lentiviral vector was a gift from Angus Johnston (Addgene plasmid 179384; http://n2t.net/addgene:179384; RRID:Addgene_179384). GPCR sequences were fused with FLAG tag, HA tag and/or GFP and subcloned into pCDH or Tet-On lentiviral vectors for stable dox-inducible expression (Supplementary Fig. 8). The TfR1 sequence was fused with a v5 tag and/or BFP and subcloned into pCDH lentiviral vector for stable overexpression.
NFAT-luciferase reporter was a gift from Toren Finkel (Addgene plasmid 10959; http://n2t.net/addgene:10959; RRID:Addgene_10959). The pGloSensor cAMP plasmid was obtained from Promega. CCR6-Tango was a gift from Bryan Roth (Addgene plasmid 66243; http://n2t.net/addgene:66243; RRID:Addgene_66243). Notably, the C-terminal tail also included a V2 vasopressin receptor tail to enhance β-arrestin recruitment. The NF-κB GFP reporter was synthesized by Twist Bioscience and subcloned into the pCDH vector. The AT1R sequence was subcloned into the Tango vector for the β-arrestin recruitment assay60. RAB5, RAB7, RAB11, LAMP1, CCR7 and CXCR2 sequences were synthesized by Twist Bioscience and subcloned into pLLM lentiviral vectors.
Cell lines and culture
HEK293T, Lenti-X 293T, 293GP (Takara Bio), MC38 and HeLa cells and a stable pTetOn-FLAG–AT1R adherent Expi293 cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, incubated at 37 °C with 5% CO2. Jurkat, MDA-MB-231, OVCAR-8, PC9 and Raji cells were cultured in RPMI-1640 (Thermo Fisher Scientific) with 10% FBS and 1% penicillin/streptomycin, incubated at 37 °C with 5% CO2. All other Expi293 cells (Thermo Fisher) were cultured in Expi293 Expression Medium (Thermo Fisher Scientific), incubated with gentle shaking at 37 °C with 8% CO2. OVCAR-8 was generously provided by the Robert Rottapel lab (Princess Margaret Cancer Centre, University of Toronto). PC9 cells with doxycycline-inducible TfR1 knockdown were generously provided by the Pasi Jänne lab (Dana-Farber Cancer Institute). All other cell lines were originally sourced from ATCC and were authenticated by the supplier using standard genotyping methods. Cells were not tested for mycoplasma. Human iPSCs were obtained from Stanford Cardiovascular Institute Biobank and used per Institutional Review Board/Stem Cell Research Oversight Panel guidelines, adhering to federal, state and Stanford University human stem cell research policies (protocol no. SCRO-745). Human peripheral blood mononuclear cells were isolated from whole blood collars (Crimson Core, Brigham and Women’s Hospital).
Protein expression
Antibodies were expressed in Expi293 cells (Thermo Fisher Scientific) using FectoPRO (Thermo Fisher Scientific) with the manufacturer’s standard protocol. Antibodies were purified using Protein A affinity chromatography and buffer-exchanged into PBS. Proteins were kept at 4 °C for up to 1–2 weeks or flash frozen for storage at −80 °C. The purity and integrity of proteins were confirmed by SDS-PAGE electrophoresis (Supplementary Fig. 7).
Engineered cell generation
Lentivirus
Lenti-X 293T cells were transfected with the carrier plasmid and lentiviral helper plasmids (psPax2 and pMD2.G) using a standard Lipofectamine3000 protocol (Thermo Fisher Scientific). The following day, the medium was replaced. Viral supernatant was harvested 48–72 h post-transfection, centrifuged at 15,000g for 1 min to remove cell debris, and immediately used or stored at −80 °C. Cells were infected with lentivirus diluted 1:5 with 8 μg ml−1 polybrene. The medium was replaced the next day, and cells were subsequently cultured with selection antibiotics (2 μg ml−1 puromycin, 200 μg ml−1 hygromycin, or 100 μg ml−1 zeocin) or sorted.
AT1R NFAT activation
Adherent Expi293 cells with stable dox-inducible FLAG–AT1R were obtained as previously described31 and were further transduced with BFP-TfR1-v5. For AT1R degradation and imaging, adherent Expi293 cells were generated by transitioning from shaking at 37 °C with 5% CO2 to shelf incubation at 37 °C with 5% CO2, while gradually adapting cells from Expi293 medium to DMEM with 10% FBS + 1% Pen-Strep over 2–3 weeks. Adherent Expi293 cells were transduced with FLAG–AT1R–GFP and BFP–TfR1–v5 using lentivirus and sorted to obtain homogeneous AT1R and TfR1 expression levels.
Other AT1R variants
Cells were transduced with TfR1 constructs, sorted and transfected with AT1R variants using Lipofectamine3000.
β-Arrestin recruitment
HTLA cells were transduced with TfR1 using lentivirus as described above, then transfected with AT1R or CCR6 Tango constructs.
APJ
HEK293T cells were transduced with TetOn-FLAG–APJ and TfR1–v5 using lentivirus and cultured with puromycin and zeocin to obtain a stable cell line.
BILF1
Raji cells were seeded in a six-well plate and transduced with TetOn-FLAG–BILF1 through lentiviral spinfection (centrifugation at 750g, 37 °C, for 2 h). Cells were expanded and subsequently sorted to obtain a homogeneous stable cell population with high BILF1 expression.
RXFP1 and CCR6 imaging
HeLa cells were transduced with FLAG–RXFP1–GFP or FLAG–CCR6–GFP, BFP–TfR1–v5 and endosome/lysosome markers (RAB5, RAB7, RAB11 and LAMP1) fused to mCherry with lentivirus. PC9 cells with doxycycline-inducible TfR1 knockdown were transduced with FLAG–CCR6–GFP. Cells were sorted to obtain homogeneous GFP or mCherry fluorescence levels.
CCR6
Jurkat cells were infected with doxycycline-inducible FLAG–CCR6 lentivirus diluted 1:7 with 8 μg ml−1 polybrene, spinning at 890g at 30 °C for 2 h. The medium was changed the next day with 20% FBS RPMI-1640, and cells were further cultured with 2 μg ml−1 puromycin.
CCR6 Fab binding to CCR6 compared with CCR7 and CXCR2
Lenti-X 293 T cells transfected in a 10 cm plate with 15 μg of the CCR6-TANGO plasmid using Lipofectamine3000. Retrovirus 6–7 × 106 293GP cells were plated in a 10 cm plate. The following day, cells were co-transfected with 9 μg MMLV Firefly Luciferase-mNeonGreen and 4.5 μg RD114 packaging plasmid. Media was replaced after 18–24 h, and retroviral supernatant was collected twice at 48 h and 72 h after transfection. A non-tissue culture-treated six-well plate was coated with 25 μg ml−1 retronectin (Takara) in PBS overnight. After blocking with 2% BSA in PBS, 1.5 ml of Firefly Luciferase-mNeonGreen retroviral supernatant was added. The plate was then centrifuged at 2,000g at 32 °C for at least 2 h. Viral supernatant was then removed, and 0.5 × 106 doxycycline-inducible FLAG–CCR6 cells were plated in each well for transduction before expansion and cell sorting for mNeonGreen expression.
Western blots
M3R
BFP–TfR1-v5+ Expi293 cells were transfected with FLAG–M3R in a six-well plate at 70–80% confluency with Lipofectamine3000 (described above). The next day, cells were trypsinized and seeded in 12-well plates. After specified treatments, cells were harvested in 2 mM EDTA in PBS, pelleted by spinning at 500–800g for 1–5 min, and incubated with RIPA lysis buffer (Thermo Fisher) on ice for 30–60 min. The lysates were centrifuged at 20,000g, and the clarified supernatant was sonicated. Samples were loaded into SDS-PAGE gels (Bio-Rad) in equal amounts with 4× Laemmli sample buffer (Bio-Rad) with BME.
CCR6
Doxycycline-inducible CCR6+ Jurkat cells were treated with 250 ng μl−1 doxycycline. After 24 h, 9 × 105 cells were seeded in each well of a 12-well plate. After specified treatments, cells were harvested as described above. Cell pellets were resuspended in 1% digitonin, 150 mM NaCl and 100 mM Tris pH 7.5 and 1× protease inhibitor (Thermo Fisher) and incubated on ice for 45 min. Lysates were clarified, sonicated and loaded onto a gel as described above.
All gels were run at 150 V for 60 min and transferred onto LF-PVDF membranes using Trans-Blot Turbo Western Blotting Transfer System (Bio-Rad). Membranes were blocked for 15–30 min with 5% milk in TBST at room temperature, washed with TBST, stained with primary antibody overnight at 4 °C, washed with TBST, stained with secondary antibody (anti-mouse IgG or anti-rabbit IgG) for 1 h at room temperature, washed with TBST and imaged with a LICOR imager and Image Studio software (v.5.2.5). Bands were quantified with ImageJ software (v.2.16). Uncropped blots are shown in Supplementary Fig. 1. Western blot biological replicates are shown in Supplementary Fig. 9. Antibodies used were rabbit anti-β-actin (Cell Signaling Technology, 4970S, 1:1,000), mouse M1 anti-FLAG (expressed from hybridoma and purified in-house, 1:500, requires 2 mM CaCl2), anti-CCR6 (Thermo Fisher, 14-1969-82, 1:300), anti-TfR1 (Thermo Fisher, 13-6800, 1:1,000), IRDye 800CW goat anti-mouse IgG (LICOR, 926-32210, 1:10,000), IRDye 680RD goat anti-rabbit IgG (LICOR, 926-68071, 1:10,000).
Flow cytometric receptor depletion assays
AT1R
Stable FLAG–AT1R–GFP+ BFP–TfR1–v5+ Expi293 cells were seeded in 24-well plates and induced with 1 μg ml−1 dox for 24 h. After specified treatments at 70–80% confluency, cells were collected with 300 μl cold EDTA (1 mM in PBS) for flow cytometry analysis.
AT1R mutant comparisons
BFP–TfR1–v5+ Expi293 cells were transfected with FLAG–HA–AT1R–GFP, AT1R WT, T282M or T332A/S335A/T336A/S338A using Lipofectamine3000 and seeded for the assay as described above.
RXFP1, A2AR and AT1R variant comparisons
Expi293 cells were seeded in a 125 ml flask at 3 × 106 cells per ml and transfected with FLAG-tagged and/or GFP-fused GPCR and TfR1 using FectoPRO (described above). The next day, cells were enhanced with 360 μl valproic acid (300 mM) and 720 μl glucose (20%). Two days after transfection, cells were seeded in 24-well plates.
APJ
TetOn-FLAG–APJ+ TfR1–v5+ HEK293T stable cells were seeded in a 24-well plate and induced with 50 ng ml−1 dox for 24 h. After specified treatments, cells were harvested for flow cytometry analysis as described above.
BILF1
HEK293T cells were transfected with TetOn-FLAG–HA–BILF1, TfR1–v5 and NF-κB–GFP reporter (internalization and signalling assays were performed on the same cells) in a six-well plate. The next day, cells were trypsinized and seeded into 12-well plates, induced with doxycycline, treated with GTAC or controls for the specified duration and harvested for flow cytometry analysis.
Temperature comparisons
TetTon-FLAG–BILF1+ Raji cells were seeded at 50,000 per well in a 96-well plate, induced with 1 µg ml−1 doxycycline for 24 h. After specified treatments on ice or at 37 °C, cells were harvested for flow cytometry.
CCR6
Doxycycline-inducible CCR6+ Jurkat cells were induced with 25 ng µl−1 doxycycline for 24 h. A total of 2.5 × 105 cells were seeded in each well of a 96-well flat-bottom plate and were treated with GTAC for 2 h or 24 h before being analysed using flow cytometry. A total of 6 × 104 PC9 cells with constitutive CCR6 expression and doxycycline-inducible TfR1 knockdown were seeded in a 24-well plate and were induced with varying doxycycline concentrations for 48 h. After specified GTAC treatments, cells were harvested for flow cytometry. For assessing degradation, 2 × 105 CCR6-GFP+ PC9 cells were seeded in a 12-well plate, treated with GTAC the next day for 18 h and harvested for flow cytometry.
Flow cytometry and fluorescence-activated cell sorting
Harvested cells were centrifuged at 300g for 5 min. Pellets were washed 1× with fluorescence-activated cell sorting (FACS) buffer (ice-cold PBS + 2% BSA). Cells were either analysed (flow cytometry) or sorted (FACS) directly, or incubated with primary antibody in FACS buffer for 20–30 min on ice. Cells were washed 3× and resuspended in FACS buffer for flow analysis.
On-cell binding
Cells were treated with Ab/GTAC diluted in PBS and were incubated on ice for 10–20 min, stained with secondary antibody for 10–20 min and washed 3× before being resuspended for flow analysis. Flow cytometry was performed using a CytoFLEX cytometer (Beckman Coulter, V0-B2-R2) and CytExpert software (v.2.3.1.22). FACS was performed using a CytoFLEX SRT cell sorter (Beckman Coulter, V0-B2-Y5-R3) and CytExpert SRT software (v.1.1.0.10007). Data were analysed with FlowJo (v.10.8.0). Representative gating strategies for live cells and single cells are shown in Supplementary Fig. 6.
Primary antibodies
The primary antibodies were Alexa Fluor 647 anti-FLAG Tag (M1) (1:500 dilution, purified from hybridoma supernatant and conjugated in-house), Alexa Fluor 647 anti-HA.11 Epitope Tag (1:1,000 dilution, BioLegend 682404), Alexa Fluor 488 anti-V5 Tag (1:1,000 dilution, Thermo Fisher 37-7500-A488), FITC anti-human CD71 (1:500 dilution, BioLegend 334104), and Alexa Fluor 647 anti-human CD71 (1:500 dilution, BioLegend 334118).
Secondary antibodies
The secondary antibodies were Alexa Fluor 647 Protein A (1:500 dilution, Thermo Fisher P21462) and Alexa Fluor 488 Protein A (1:500 dilution, Thermo Fisher P11047).
BILF1 signalling and functional assays
NF-κB activation
HEK293T cells were transfected with TetOn-FLAG–HA–BILF1, TfR1–v5 and NF-κB–GFP reporter in a six-well plate. The next day, cells were trypsinized and seeded into 12-well or 24-well plates, induced with doxycycline, subjected to specified treatments and harvested for flow cytometry analysis.
MHC-I expression
TetOn-FLAG–BILF1+ Raji cells were seeded at 25,000 per well in a 96-well plate, induced with 1 µg ml−1 doxycycline for 24 h, treated with GTAC or controls for 24 h, and harvested for flow cytometry analysis.
RXFP1 cAMP assays
HEK293T cells were transfected with FLAG–RXFP1, TfR1–v5 and Promega GloSensor plasmid (1 μg each per well) using Lipofectamine3000. The next day, cells were trypsinized, seeded in a 96-well plate (50,000 cells per well) and incubated 24 h. Cell culture medium was removed and replaced with Ab or GTAC diluted in CO2-independent medium with 10% FBS + 5% d-luciferin reagent: 40 mg ml−1 d-luciferin potassium salt (GoldBio eLUCK-500) in 10 mM HEPES buffer. Cells were incubated for 2 h (unless otherwise specified) in the dark. Luminescence was measured with a CLARIOstar PLUS microplate reader (BMG Labtech, used for all plate-reader assays unless otherwise stated) to obtain the pre-read; recombinant human RLN2 (Fisher Scientific 3596RN025) was added immediately, and another measurement was taken after 30 min. Unless otherwise specified, Ab or GTAC dose–response curves were generated, and IC50 values were obtained using 1 nM RLN2 to activate signalling; RLN2 dose–response curves and EC50 values were obtained after treating cells with 10 nM Ab/GTAC.
Time course
HEK29T cells were transfected with FLAG–RXFP1, TfR1–v5 and GloSensor as described above. After specified treatments, cell culture medium was then removed and replaced with d-luciferin reagent diluted in CO2-independent medium, and the assay proceeded as described above, using 1 nM RLN2 to activate signalling.
RXFP1 expression compared with cAMP correlation curve
A FLAG–RXFP1+ TfR1+ HEK293T stable cell line was generated with lentivirus. Cells were seeded in a 24-well plate (50% confluency) and induced with varying doxycycline concentrations for 24 h before harvesting for flow cytometry. In parallel, cells were transfected with GloSensor plasmid as described above. The next morning, cells were trypsinized, seeded in a 96-well plate (50,000 cells per well) and induced with varying doxycycline concentrations for 24 h before proceeding with the cAMP assay as described above, using 1 nM RLN2 to activate signalling.
Affinity-matured RXFP1 Nb (RX002i4)
HEK293T cells were seeded in white, clear-bottomed 96-well plates, coated with poly-d-lysine at 40,000 cells per well. The cells were transfected with FLAG–RXFP1 and Promega GloSensor plasmids for at least 24 h (20 ng each per well) using FuGENE according to the manufacturer’s protocol. Post-transfection, the cells were incubated in CO2-independent medium with 10% (v/v) FBS and 2 mg ml−1 of d-luciferin (40 μl per well) and incubated for 2 h at room temperature in the dark. Luminescence was measured as a pre-read using a SpectraMax M5 microplate reader before cells were treated with RX002i4 (at 1 mM per well) or PBS for 20 min. Following this, RLN2 was added to the wells in a dose-dependent manner for 30 min, and luminescence was measured.
RXFP1 Nb binding and competition with RLN2
Expi293F TetR cells were transfected with FLAG–RXFP1 or empty pcDNA-Zeo-tetO vector using FectoPro according to the manufacturer’s instructions. Cells were enhanced with 0.4% glucose and 3 mM sodium valproic acid 24 h post-transfection. Protein expression was induced with 4 μg ml−1 doxycycline 48 h post-transfection. Cells were aliquoted into a V-bottomed, 96-well plate (100,000 cells per well) 24 h post-induction and harvested by spinning at 200g for 5 min at 4 °C. Cells were washed once and blocked for 20 min in HEPES-buffered saline containing 1% (v/v) FBS and 2 mM calcium chloride (flow cytometry buffer) at 4 °C, with shaking.
Direct binding
Cells were incubated with a dilution series of RX002i4 Nb for 60 min at 4 °C, with shaking. For competition, cells were incubated with 200 nM SE301 (Fc-tagged RLN-2) and a dilution series of RX002i4 Nb for 60 min at 4 °C, with shaking. Cells were washed once and incubated with M1 anti-FLAG antibody labelled with Alexa Fluor-488 (1:200) and Alexa Fluor-647 anti-V5 (1:1,000) or Alexa Fluor-647 anti-human IgG Fc (1:1,000) for 30 min. Cells were washed once and analysed on a CytoFLEX flow cytometer (Beckman Coulter) using CytExpert software.
RXFP1 signalling qPCR analysis
OVCAR-8 (500,000 cells per well) were seeded in a six-well plate and incubated overnight. Cells were starved for 6 h and subsequently treated with Ab or GTAC (100 nM) for 18 h. Cells were induced with 60 ng ml−1 rhRLN2 and incubated for 8 h. Cell lysate was harvested with Trizol (Thermo Fisher 15596026) (700 μl per well). RNA extraction was performed with Qiagen kits (Qiagen 217084, 79254, 79656), RIN scores >8.0 were confirmed for quality control, and cDNA was synthesized from 1 μg RNA per sample with Superscript IV (Life Technologies 18090010) following standard protocol. qPCR was run with Taq polymerase, dNTPs and SYBR Safe Green in-house master mix. GAPDH and ACTB primers were designed and previously validated in-house. MMP9, MMP23, NOTCH1, NOTCH3 and DVL1 primer sequences were generously provided by the Robert Rottapel Lab (Princess Margaret Cancer Centre, University of Toronto), with sequences listed in Supplementary Table 6.
AT1R NFAT assays
A stable adherent Expi293 cell line with inducible FLAG–AT1R and constitutive TfR1 was seeded in a six-well plate and incubated overnight. Cells were transfected at 70–80% confluency with NFAT-luciferase (1 μg per well using Lipofectamine3000) and incubated 16 h. Cells were trypsinized, seeded in a 96-well plate (50,000 cells per well) and induced with 1 μg ml−1 dox for 24 h. Cells were treated with Ab or GTAC for 2 h, and subsequently ANGII (MedChemExpress HY-13948) was added (9 nM ANGII was used to generate Ab or GTAC dose–response curves and obtain IC50 values). Four hours later, cell culture medium was removed and replaced with Promega Bright-Glo reagent diluted 1:1 in 10 mM HEPES buffer (40 μl per well). Luminescence was measured with a plate reader.
β-Arrestin recruitment assay
AT1R
TfR1+ HTLA cells were transfected with AT1R–Tango (1 μg per well using Lipofectamine3000). The next day, cells were trypsinized, seeded in a 96-well plate (50,000 cells per well) and induced with 1 μg ml−1 dox. After 24 h of transfection, cells were starved and treated with Ab or GTAC for 2 h before adding ANGII (200 nM ANGII was used to generate Ab or GTAC dose–response curves and obtain IC50 values). Then, 14 h later, cell culture medium was removed and replaced with Promega Bright-Glo reagent diluted 1:1 in 10 mM HEPES buffer (40 μl per well). Luminescence was measured using the Bright-Glo Luciferase Assay System (Promega) on a plate reader.
CCR6
TfR1+ HTLA cells were transfected in a 10-cm plate with 15 μg of the CCR6–TANGO plasmid using Lipofectamine3000 (Thermo Fisher Scientific). Cells were trypsinized 18 h after transfection and were seeded in serum-free DMEM into poly-d-lysine-coated 96-well clear-bottom plates with 4.5 × 104 cells per well. After 6 h, cells were pre-treated with 10 nM of CCR6 GTACs for 1 h followed by incubation with both the GTACs and CCL20 for 18 h. Luminescence was measured as described above.
F-actin polymerization
Doxycycline-inducible CCR6+ Jurkat cells were treated with 25 ng μl doxycycline. After 24 h, 2.5 × 105 cells were seeded in a 96-well flat-bottom plate and were incubated with GTACs for 2 h. A total of 10 nM of GTACs were used for experiments with only one dose. Cells were then resuspended in a V-bottom plate with 50 nM CCL20 followed by staining and fixation with phalloidin staining buffer after the indicated time. Phalloidin staining buffer consists of 1% BSA, 0.2% Triton X-100, 4% paraformaldehyde and 1 U of Flash Phalloidin Green 488 (BioLegend). Cells were stimulated for 1 min for experiments with only one time point. Cells were washed 3× and resuspended in cold 0.1% Triton X-100 in PBS for flow cytometry analysis.
Chemotaxis assay
Doxycycline-inducible CCR6+ luciferase+ Jurkat cells were treated with 25 ng μl−1 doxycycline. After 24 h, 4 × 105 cells were seeded in a 96-well flat-bottom plate and were incubated with GTACs for 2 h in 100 μl of serum-free RPMI-1640. Each 100 μl cell solution was then transferred to hanging cell culture inserts with 5 µm pores (Millipore Sigma). These inserts were placed within a 24-well plate that contained 600 μl of 100 nM CCL20, and cells were allowed to incubate for 2 h. Then, migrated cells were collected from the outer chambers of the 24-well plate and were transferred to a white 96-well flat-bottomed plate. A total of 150 µg ml−1 d-luciferin (GoldBio) was added to the cells to allow for quantification using luminescent readout with a plate reader. For chemotaxis of TH17 cells, 50 μl of migrated cells were mixed with 50 μl of Precision Count Beads (BioLegend 424902) to enable quantification of cell number using flow cytometry.
Imaging and colocalization
AT1R live imaging
FLAG–AT1R–GFP+ BFP–TfR1–v5+ Expi293 cells were used. For RXFP1 live imaging assays, FLAG–RXFP1–GFP+ BFP–TfR1–v5+ RAB11–mCherry+ HeLa cells were used. For both assays, 24-well glass-bottom optical imaging plates were pre-coated with poly-l-lysine (30 min at room temperature) and washed 3× with PBS; cells were subsequently seeded in coated plates (100,000 cells per well, 500 μl total culture volume), induced with 1 µg ml−1 dox, and incubated for 24 h. Cells were treated with Ab, GTAC or ligand for the specified time before removing the cell culture medium and adding imaging solution (500 μl PBS + 10% FBS + 2% Pen-Strep).
BILF1 immunofluorescence
FLAG–HA–BILF1+ TfR1–v5+ HEK293T cells were used. For CCR6 immunofluorescence assays, FLAG–CCR6–GFP+ BFP–TfR1–v5+ HeLa cells were used. For both assays, cells were seeded in 24-well glass-bottom optical imaging plates pre-coated with poly-l-lysine as previously described. The next day, cells were treated with GTAC or controls for the specified time. Cells were then fixed with ice-cold methanol for 10 min, washed with 70% ethanol and PBS, stained with primary antibody diluted in PBS with 3–5% BSA at 4 °C overnight, washed 3×, and stained with secondary antibody for 1–2 h at room temperature. The staining solution was washed and replaced with PBS before imaging.
Antibodies
The antibodies used were anti-RAB5 rabbit mAb C8B1 (CST 3547, 1:200); anti-RAB7 rabbit mAb D95F2 (CST 9367, 1:200); anti-RAB11 rabbit mAb D4F5 (CST 5589, 1:200); anti-LAMP1 rabbit mAb D2D11 (CST 9091, 1:200); and Alexa Fluor 647 donkey anti-rabbit IgG antibody (BioLegend 406414, 1:1,000).
CCR6 live imaging
GTACs were conjugated to Alexa Fluor 647 using Zip Alexa Fluor Rapid Antibody Labelling Kits (Thermo Fisher Scientific). HeLa cells (4 × 104) were plated in poly-d-lysine-coated 24-well glass-bottom plates 2 days before imaging. Cells were treated with GTAC for specified durations and washed 3× with PBS before imaging. For inhibitor studies, cells were pre-treated for 1 h before GTAC treatment with MG132 (MedChemExpress), Chloroquine (MedChemExpress) or Bafilomycin (MedChemExpress).
CCR6 immunofluorescence
Cells were plated in glass-bottom plates and treated with GTACs similarly to previously described before fixation and staining (as described for BILF1). All imaging was performed using a Zeiss 980 confocal/Spectral/Airyscan Microscope (Zen v.3.4, Blue edition) through the DFCI Molecular Imaging Core. Images were acquired using 405 nm, 488 nm and 594 nm lasers, processed and analysed using ImageJ (v.2.16.0) and Fiji (v.2.9.0).
Jurkat cell split-luciferase assay
Doxycycline-inducible CCR6+ Jurkat cells were either treated with 25 ng μl−1 doxycycline for 24 h or left untreated. Jurkat cells (1 × 105) were seeded in 96-well V-bottom plates and were spun down at 300g for 5 min. Cells were washed with PBS and were then resuspended with 10 nM Fc-H7-LgBit for 10 min on ice. After washing in PBS 3×, cells were resuspended with 10 nM GTAC-smBit for 10 min on ice and were washed another 3× in PBS. Cells were then resuspended in Nano-Glo Luciferase Assay Substrate (Promega, N1110, 1:100) in PBS and moved to a white-bottom 384-well plate, and luminescence was measured with a plate reader.
ECORE with iPSC-cardiomyocytes
ECORE sample fabrication
Samples were prepared as described previously67. In brief, a 3D-printed well was glued to a UV–ozone-cleaned, ITO-coated glass slide (resistance 70–100 Ω). A PEDOT:PSS film was electropolymerized onto the ITO from a 10 mM 3,4-ethylenedioxythiophene (EDOT) (483028, Sigma-Aldrich) with 2 w/w% poly(sodium 4-styrenesulfonate) (PSS) (molecular weight 70,000, Sigma-Aldrich) aqueous solution by applying a constant 100 µA for 40 s, producing a 28 mm2, about 60 nm-thick film.
ECORE optical setup
The ECORE optical hardware was identical to our previous report67 except for the laser source. A 200-mW, 532-nm diode-pumped solid-state laser (Spectra-Physics) was single-mode-fibre-coupled and split by polarizing beam-splitter cubes into a sample and a reference beam; half-wave plates set their polarization (sample beam s-polarized) and intensity ratio. The sample beam was focused to an approximately 30 µm spot onto a BK-7 equilateral prism and the laser incident angle set to 68°. The reflected sample and reference beams were combined on a differential photodetector, suppressing common-mode noise. Clampfit 11 and Python (v.3.12) were used for electrochromic optical recording data analysis.
hiPSC cardiomyocyte culture
The ECORE samples were disinfected with 70% EtOH for 2 h. After rinsing with water three times, the polymer film was coated with 1 mg ml−1 poly-l-lysine in 1× phosphate-buffered saline (PBS) solution (21-040-CM, Corning) for 30 min, followed by 3× PBS rinsing. The film was then coated with 0.5% glutaraldehyde in PBS for 15 min, followed by 3× PBS rinsing. The film was coated with 1:200 Matrigel (REF 35623, Corning) in DMEM/F12 (REF 10565-018, Gibco) at 37 °C for 1 h. hiPSC-derived cardiomyocytes were maintained in Matrigel pre-coated 24-well plates in RPMI 1640 medium (REF 11875-093, 500 ml, Gibco) with 1× B-27 supplement (REF 17504-044, 10 ml, Gibco). The culture medium was refreshed every 2–3 days. Five days before the experiment, cells were transferred onto the polymer films. Cardiomyocytes were dissociated with TryPLE select 10× (REF A12177-01, Gibco) at 37 °C for 7 min, then 4 × 104 cells were plated to each coated polymer sample in RPMI 1640 medium with 1× B-27 supplement and 10% KnockOut Serum Replacement (KSR) (REF A31815-01, Gibco). The samples with cells were cultured in a 5% CO2 incubator at 37 °C. The medium was changed to RPMI 1640 medium with 1× B-27 supplement 24 h after cell seeding and refreshed every 2–3 days.
AT1R pharmacology experiments with iPSC-cardiomyocytes
ANGII was dissolved in PBS with 10% DMSO to make 10 mg ml−1 stock solutions and kept at −80 °C. It was diluted in HEPES-buffered Tyrode’s solution to 5 μM on the day of experiment. During ECORE recording, 200 μl of 5 μM ANGII was added to the sample well with 800 μl of HEPES-buffered Tyrode’s solution, yielding a final drug concentration of 1 μM. DMSO (10%) in PBS is diluted and added to samples in the same way as ANGII as a control experiment.
TH17 cell differentiation
Human peripheral blood mononuclear cells were isolated from whole blood collars (Crimson Core, Brigham and Women’s Hospital) by Ficoll gradient centrifugation and subsequent lysis of red blood cells with ACK Lysis Buffer (Thermo Fisher A1049201). MojoSort Human CD4 Naïve T Cell Isolation Kits (BioLegend 480041) were then used to isolate naive CD4+ T cells from peripheral blood mononuclear cells following the manufacturer’s protocol. Isolated cells were analysed for purity by flow cytometry using Alexa Fluor 647 anti-CD45 (BioLegend 304056, 1:50), Alexa Fluor 488 anti-CD4 (BioLegend 317408, 1:50). Primary human naive CD4+ T cells were cultured in X-VIVO 15 medium (Lonza Bioscience, 04-418Q) supplemented with 5% FBS, 4 μM N-acetyl-l-cysteine, 0.05 mM 2-mercaptoethanol, and CD3/CD28 activation beads (BioLegend 422604) at a 1:1 bead:cell ratio. For differentiation to TH17 cells, 25 ng ml−1 IL-6 (BRB Preclinical Biologics Repository L0406011), 10 ng ml−1 IL-1β (GoldBio 1110-01B-10), 15 ng ml−1 IL-23 (Thermo Fisher 200-23-10UG), 5 ng ml−1 TGF-β1 (Thermo Fisher 100-21-10UG), 10 µg ml−1 anti-IFN-γ (BioLegend 506502) and 10 µg ml−1 anti-IL-4 (BioLegend 500802) were added to culture for 5 days. TH17 cells were analysed for purity by flow cytometry using PE/Cyanine7 anti-CXCR3 (BioLegend 353720, 1:50), Alexa Fluor 647 anti-CCR6 (BioLegend 353415, 1:50) and Alexa Fluor 488 anti-CD4 (BioLegend 317408, 1:50).
Biolayer interferometry
Biolayer interferometry measurements were performed through the Harvard Medical School Center for Macromolecular Interactions Core using an Octet RED384 (ForteBio) instrument with Octet BLI Discovery, v.13.0 software. A total of 4 µg ml−1 biotinylated human TfR1 (Sino Biological, 11020-H07H-B-100) or mouse TfR1 (ACROBiosystems, TFRM824925UG) extracellular domains were immobilized on optically transparent streptavidin biosensors (ForteBio) and loaded for roughly 120 s until a 1–2 nm signal was achieved. After washing with buffer, monomeric TfR1 VHH variant binders fused to an Fc region were used as the analyte for association and dissociation measurements. Tris-buffered saline with 0.1% Tween-20 detergent was used as the buffer. Data were analysed using the ForteBio Octet Analysis Studio v.13.0 software, and kinetic parameters were determined using a 1:1 monovalent binding model.
Intracellular iron measurements
MDA-MB-231 cells were seeded in a 96-well flat-bottom plate. The next day, they were treated with 10 nM of TfR1-targeting constructs or 100 µM of ammonium iron (II) sulfate (Sigma, 215406) for 72 h. Cells were washed 3× with PBS, then treated with 1 μmol l−1 FerroOrange working solution (Dojindo Laboratories, F374) and were incubated at 37 °C for 30 min. Fluorescence intensity was then measured by a plate reader.
Animal experiments
All experiments were performed according to the Guide for the Care and Use of Laboratory Animals and were approved by the National Institutes of Health and the Committee on Animal Care at Harvard Medical School (IS-00003882). Male C57BL/6N-Ccr6tm1(CCR6)Bcgen/Bcgen mice were purchased from Biocytogen. All mice were maintained under specific-pathogen-free conditions. All mice were kept in a 12–12 h light–dark cycle. The main room temperature was set at 20–26 °C, and humidity was 40–65%. Mice had ad libitum access to food and water. Sample size was ensured to meet a minimum of n = 3 mice per group. Randomization and blinding were not included for this study.
IMQ-induced mouse psoriasis model and antibody administration
A daily dose of 10 mg of IMQ-containing cream (5%, Taro Pharmaceutical Industries) was applied to the ears of mice aged 10 weeks for 6 consecutive days. Ear thickness was measured daily with a thickness gauge (Mitsutoyo), and the difference from day 0 was expressed as a change in ear swelling. Psoriasis area severity index (PASI) scores were also evaluated daily for each mouse as previously reported54. CCR6 Fab–Fc or TfR1HIGH GTAC was administered daily at a dose of 10 mg kg−1 for 6 consecutive days, and IMQ-containing cream was applied after 5–10 min of antibody administration.
Flow cytometry of mouse immune cells
Ear skin was cut and split into dorsal and abdominal sides, and the dorsal side was incubated for 55 min at 37 °C with 500 μl of digestion buffer (13 ml of complete RPMI 1640, 5 mg Liberase TL (Roche), 3.3 mg DNase I (Sigma) and 10 μg ml−1 brefeldin A (Sigma)). Ear skin was then incubated with 10 mmol EDTA-2Na (Corning) for 5 min at 37 °C. The cell suspensions were filtered using a 40-μm cell strainer (MTC Bio), stained with indicated antibodies, and then subjected to flow cytometric analysis. The gating strategy is shown in Supplementary Fig. 6. Flow cytometry was performed using a Symphony A5 (BD) and analysed with FlowJo (Treestar).
RNA extraction and quantitative PCR analysis
Total RNA was isolated from the ear skin, 24 h after the last IMQ application, using Trizol (Invitrogen) and RNeasy kits (Qiagen). cDNA was synthesized using oligo dT (iScript cDNA synthesis kit, Bio-Rad). Quantitative real-time reverse transcriptase PCR was performed with SYBR Green I (Roche) and an ABI QuantStudio5 (Thermo Fisher Scientific) according to the manufacturer’s instructions. All primers were obtained from Integrated DNA Technologies (Supplementary Table 6). For each sample, reactions were performed in technical triplicate and validated against a control reaction lacking reverse transcriptase. Results were normalized to levels of GAPDH mRNA.
Histology and epidermal measurements
Ear skin samples were fixed with 10% formalin (Sigma) and embedded in paraffin. Sections of 5 μm thickness were prepared and stained with haematoxylin and eosin. Images of stained sections were obtained with an Olympus VS200 Widefield Slide Scanner. Epidermal thickness was calculated as a measure of the total area of the epidermis divided by the total length of the epidermis using OlyVIA 3.8 (Olympus).
Plasma half-life measurements
Male C57BL/6NJ mice (Taconic Biosciences) aged 6 weeks were injected with either 10 mg kg−1 (body weight) CCR6 GTAC or 10 mg kg−1 (body weight) CCR6-Fab-Fc control (n = 3 for each group). Blood samples were collected from the facial vein using ethylenediaminetetraacetic acid capillary tubes on days 1, 3, 7 and 10. Plasma was obtained by centrifuging the blood at 2,000g at 4 °C for 15 min. Then, 2 µl of plasma was diluted in 10 µl of PBS and subjected to standard western blot analysis as previously mentioned with an anti-human IgG antibody (LICOR 926-32232, 1:5,000). With the same samples, a stain-free gel was also run and imaged to determine total protein levels.
Tissue preparation and cryostat sectioning
Organs were removed and fixed by immersion in 4% paraformaldehyde (in PBS). Tissue samples were then transferred into sucrose gradient solutions and incubated overnight at each concentration (15%, 30%: w/v = g ml−1). Samples were then sealed in OCT compound (Optimal Cutting Temperature compound, Scigen) and frozen quickly at −80 °C. Frozen 30 μm sections were made on the cryostat (Leica CM1950), and the slices were stored free-floating in an ethylene-glycol-based cryoprotectant solution at −20 °C until used for downstream immunofluorescence experiments.
Immunofluorescence staining
Tissue slices were washed briefly in PBS-X (pH 7.4, supplemented with 0.05% Triton X) and blocked for 1 h with 5% normal donkey serum (AB7475, Abcam) diluted in PBS-X at room temperature. Samples were then incubated overnight at 4 °C with primary TfR1 antibody (13-6800, Invitrogen, 2.5 μg ml−1) diluted to 2.5 μg ml−1 in blocking buffer. After washing three times in PBS-X, samples were incubated with a secondary antibody cocktail containing PBS-X, 1% normal donkey serum, 1,000× diluted donkey anti-mouse 647 (A32787, Invitrogen) and 5,000× diluted DAPI stain (AB228549, Abcam) for 2 h at room temperature. Stained slices were washed three times in PBS-X and once in PBS for 5 min, then mounted onto slides and cover-slipped for imaging on the Zeiss AxioScan7.
Data and statistical analysis
All graphing and statistical analyses were performed in GraphPad Prism (v.10.4.2) or Microsoft Excel (v.16.98). Unpaired two-tailed t-tests were used for statistical analyses unless otherwise noted. A two-tailed P value was used to determine statistical significance for all analyses. The following convention was used to assign significance: not significant (NS), P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Sample sizes followed the common standard of biological replicates of n = 3 or more, with n = 2 in a few cases, and were not determined using statistical methods. All schematics were created with BioRender.com, Affinity Designer (v.2.6.3), and Adobe Illustrator (v.30.x). Raw flow cytometry plots were created with FlowJo (v.10.8.0). All figures were assembled in Affinity Designer (v.2.6.3).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
