Cryo-EM Reveals Integrin-Mediated TGF-β Activation without Release from Latent TGF-β.

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for reagents may be addressed to the Lead Contact, Stephen 5 Nishimura ( stephen.nishimura@ucsf.edu ). Antibodies, cell lines and plasmids used in this manuscript will be available from the Lead Contact upon execution of a materials transfer agreement.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Subjects

Adult airways were collected from first through fourth order bronchi from lobectomy specimens from resections performed for primary lung cancer. Informed consent was obtained from all surgical participants as part of an approved ongoing research protocol by the University of California San Francisco Committee on Human Research, in full accordance with the declaration of Helsinki principles. The specimens were de-indentified. Normal bronchial epithelial cells (HBEC) Normal human bronchial epithelial cells (HBEC) were isolated from human lung specimens, as previously described ( Araya et al., 2007 ). Specifically, airway epithelium was stripped freshly from bronchi from surgical specimens and after washing in PBS with dithiothreitol (5 mM) were digested overnight at 4°C with a protease XIV solution (0.4 mg/ml, Sigma). After washing with bronchial epithelial growth medium (BEGM, Lonza) with 2.5% FCS and tituration, HBEC were plated onto rat-tail Col I-coated (10 μg/ml, Corning, cat. no. 354236) dishes and incubated overnight; then the medium was changed to fresh BEGM. HBEC were passaged under conditional reprogramming conditions with BEGM with ROCK inhibitor (10 mM) and irradiated NIH3T3 fibroblasts, as described ( Liu et al., 2012 ). Human embryonic kidney cells (HEK293) HEK293 are an authenticated female line from a commercial vendor (ATCC). HEK293 cells were grown in DMEM + 10% FBS + penicillin-streptomycin + amphotericin B + 0.11mg/mL Sodium Pyruvate + 2mM L-glutamine + MEM nonessential amino acids, cultured at 37°C with 5%C O 2 . HEK293 and all other cell lines were routinely tested for mycoplasma contamination. For all established cell lines cell culture media and antibiotics were prepared by the University of California, San Francisco Cell Culture Facility using deionized water and analytical grade reagents. Fetal calf serum was obtained from Invitrogen (Carlsbad, CA). Chinese Hamster Ovary Cells (CHO) CHO-K1 are an authenticated female line from a commercial vendor (ATCC). CHOLec 3.2.8.1 have four independent mutations in the N- and O-glycosylation pathways producing proteins all of the high mannose glycosylation pattern ( Stanley, 1989 ). CHOLec 3.2.8.1 cells were grown in CHO-S-SFM II medium + 10% FBS + penicillin-streptomycin + amphotericin B, cultured at 37°C with 5%CO 2. CHOLec 3.2.8.1 cells were provided by Pamela Stanley ( Stanley, 1989 ). Authentication of CHOLec 3.2.8.1 cells was performed by changes in glycosylation patterns as estimated by migration of secreted proteins by SDS-PAGE. Transformed Mink Lung Epithelial Cells (TMLC) TMLC is a stable mink lung epithelial reporter cell line derived from mixed sex American Mink fetal lung epithelial cells that stably expresses a portion of the plasminogen activator inhibitor 1 promoter ( Abe et al., 1994 ). TMLC cells were grown in DMEM + 10% FBS + penicillin-streptomycin + amphotericin B, cultured at 37°C with 5%CO 2. TMLC are a gift from John Munger (NYU medical center, New York City, New York). Authentication was performed by increased luciferase activity in response to recombinant TGF-β. METHOD DETAILS Antibody isolation, characterization and production The C6D4, 68, 8B8, 3G9, 1D11 antibodies have been previously described ( Minagawa et al., 2014 ; Mu et al., 2002 ; Takasaka et al., 2018 ; Weinreb et al., 2004 ). Mouse anti-human αv, clone 11D12V2 was isolated as described, with the following modifications. Screening of hybridomas produced a polyclonal hybridoma 11D12 with reactivity against cell lines expressing the human αv-subunit (CHO-αvβ8, or SW480-β6). Subcloning produced two clones 11D12V1 and 11D12V2 both with reactivity against the αv-subunit. Variable (V) genes were isolated, sequenced, and the V H /V K genes cloned into mouse IgG 2a expression vectors and stably transfected into CHO-K1 cells. C6-RGD3 was created by splice overlap extension (SOE) polymerase chain reaction (PCR) into the VL CDRL1 region of a C6D4 IgG2a expression vector ( Minagawa et al., 2014 ) with the following oligonucleotide primers using the C6D4 IgG2a expression construct as a template: 5’-GATCTGGGGCGCCTCAAGAAGAACGCCTTGGCTTGGTACCAGCAG-3’; 5’-CTTGAGGCGCCCCAGATCTCCACGGCCGAGCAGACTCTGACTGGATTTG-3’. C6-RGD3 was transfected into CHO-K1 cells. Antibodies were produced and purified as described previously ( Minagawa et al., 2014 ). Briefly, stable transfected CHO-K1 cells were grown in spinner cultures in CHO SFMII media with antibiotics. Antibodies were purified from culture media using protein G sepharose columns (HiTrap, GE Healthcare). C6D4, C6-RGD3, and 11D12V2 fragments were generated by papain digestion (Pierce) of the IgG followed by Protein-A Agarose (Millipore) incubation and to remove Fc fragments and intact antibodies and final separation using Mono S ion-exchange chromatography (GE Healthcare). The 11D12V1 and 11D12V2 binding epitopes were estimated by negative staining electron microscopy (ns-EM), essentially as described below and in previous studies ( Minagawa et al., 2014 ; Takasaka et al., 2018 ). Clone 11D12V1 bound to the αv-head and 11D12V2 to the αv-thigh. Anti-β6 (MAB4155) was purchased (R&D Systems). A bioassay to measure cell-intrinsic TGF-β signaling To develop a cell intrinsic TGF-β activation system we used stable transfection of TMLC (Amaxa) with a vector containing either a WT human TGF-β1 IRES GFP, or a human TGF-β1 (R249A) IRES GFP cassette with puromycin resistance to obtain TMLC cells expressing WT L-TGF-β either capable of dissociating into LAP and mature TGF-β or not, due to the R249A mutation that normally allows furin cleavage of LAP from mature TGF-β ( Shi, et al, 2011 ).

Human TGF-β1 IRES

GFP or human TGF-β1 (R249A) IRES GFP TMLC cells were sorted for equal expression using GFP fluorescence and did not present any L-TGF-β on their cell surface ( Fig. S7 ). In contrast, stable transfection of these lines with a HA-GARP construct with a blastacidin resistance cassette followed by selection and sorting resulted in high surface expression of TGF-β1/GARP or TGF-β1 (R249A)/GARP, as measured by anti-HA (clone 5E11D8, GenScript, Piscataway, NJ) or anti LAP (R&D Systems, AF426). To confirm lack of releasable of TGF-β from the L-TGF-β R249A/GARP cell surface complex, L-TGF-β (R249A)/GARP expressing or WT L-TGF-β/GARP TMLC cells were surface biotinylated using EZ-link sulfo-NHS biotin (Thermo Fisher Scientific) and immunoprecipitated using anti-HA, resolved by 4–12% gradient SDS-PAGE, under non-reducing conditions, immunoblotted, probed with streptavidin-HRP and detected by chemiluminescence, essentially as described ( Mu, et al, 2002 ). Immunoprecipitations confirmed association of GARP with WT L-TGF-β, L-TGF-β (R249A), and absence of cleavage of LAP from mature TGF-β in the L-TGF-β (R249A)/GARP TMLC cells ( Fig. S7 ). A TMLC assay to measure integrin-mediated cell-intrinsic TGF-β signaling The αvβ8 ectodomain was coated along with the controls αvβ3 (R&D Systems), BSA (Sigma-Aldrich) or anti-LAP (R&D AF426, 1 μg/ml) onto ELISA plates in PBS (1mM Ca 2+ and 1mM Mg 2+ ) 1 hour at RT. Wells were subsequently washed in PBS and blocked in PBS with 1% BSA WT L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP expressing TMLC cells were plated at a density of 1×10 5 cells/ml in basal media. After 16 hrs, media was removed and applied to wells containing TMLC reporter cells to measure diffusible mature TGF-β, which were incubated overnight prior to lysis and determination of luciferase activity (Promega) as reported ( Mu, et al, 2002 ). To measure cell-intrinsic TGF-β1 activation, the attached L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP or parental TMLC cells were lysed and assayed for luciferase activity. To facilitate comparison between different TMLC lines expressing WT L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP (or parental TMLC cells) normalized luciferase activity was expressed as activated TGF-β in pg/ml. Normalization was performed by interpolating luciferase activity against standard curves generated using each TMLC line with varying doses of recombinant human TGF-β1 (R&D Systems).

Integrin DNA constructs

Wild-type and mutant recombinant human integrin αvβ8 and αvβ6 truncated at the junction of the ectodomains and transmembrane domains in pcDNA1neo have been described ( Nishimura et al., 1994 ; Weinreb et al., 2004 ). β8 Δ SDL was prepared as described ( Takasaka et al., 2018 ). To create mutant β8 subunits, SOE was performed using PCR with WT β8 as a template to create mutant constructs all in pcDNA6 V5HisA (Invitrogen) with a stop codon inserted before the V5/His tag. The following mutagenic primers (all in 5 ‘to 3’ orientation) were used: β8 I208R5’-CAGAAGATCTCTGGAAACAGAGATACACC-3’; 5’-GAAGTTTGGTCGACATAATGC-3’ β8 Y172N: 5’-CAATGCAGTGACAACAATTTAGACTGC-3’, 5’-GCAGTCTAAATTGTTGTCACTGCATTG-3’; β8 Y172M: 5’-GATTCATAATCAATGCAGTGACATGAATTTAGACTGCATGCC-3’, 5’-GGCATGCAGTCTAAATTCATGTCACTGCATTGATTATGAATC-3’; β8 Y172A: 5’-GATTCATAATCAATGCAGTGACGCCAATTTAGACTGCATGCC-3’, 5’-GGCATGCAGTCTAAATTGGCGTCACTGCATTGATTATGAATC-3’; β6 I183N: 5’-CCCTTGCAGTAGTAATCCATACTTCTG-3’, 5’-CAGAAGTATGGATTACTACTGCAAGGG-3’. TGF-β DNA constructs Porcine L-TGF-β1 with a C4S mutation, to improve secretion and prevent association with L-TGF-β binding proteins, and an N-terminal cleavable (HRV 3C) 7x Histidine-streptavidin binding protein tag to facilitate purification ( Shi et al., 2011 ), were joined by SOE PCR from pcDNA-GS-TGF-β1 (gift from Dr. Sun, National Institutes of Health, Bethesda, MD ( Zou and Sun, 2004 )) and subcloned into pcDNA6 (Invitrogen). Porcine L-TGF-β1 C4S pcDNA6 was modified using the following mutagenic primers, RGE (a mutation that disrupts the integrin binding recognition sequence on L-TGF-β and therefore minimizes the number of L-TGF-βs that bind two integrins simultaneously): 5’-CCGCCGGGGTGAACTGGCCAC-3’; 5’-GTGGCCAGTTCACCCCGGCGG-3’; R249A: 5’-ACCTGCACAGCTCCCGGCACCGCGCAGCCCTGG-3’. Wild type human TGFβ1 was derived from human TGFβ1_pLX307 ( Rosenbluh et al., 2016 ) by removing the C-terminal V5 tag using PCR with the following primers; 5’-ATGGCCACCCCGCTGG-3’, 5’-CTCTACTAGTCTCGAGTTATCAGCTGCACTTGCAGGAGCGCAC-3’. The WT human TGF-β1 IRES GFP cassette was then subcloned into a version of pcDNA6 (Invitrogen) with a puromycin resistance cassette which was amplified from TGFβ1_pLX307 using 5’-ATCGTTTCAGACCCACCTCCC-3’ and 5’-CTCTGCTTAGCGAATTCGTTAACTGGCACCGGG-3’. A R249A mutant of this construct was produced using SOE PCR employing the following mutagenic primers; 5’-CACCGCGCAGCCCTGGACACCAAC-3’, 5’-CCAGGGCTGCGCGGTGCCGGGAG-3’. GARP DNA constructs N-terminal HA tagged human GARP pcDNA3 ( Cuende et al., 2015 ) was provided by Sophie Lucas (de Duve Institute, UCLouvain, Brussels, Belgium) and the entire HA-GARP reading frame transferred into pcDNA6 (Invitrogen). All constructs were verified by sequencing.

Secreted protein expression and purification

Integrin constructs were expressed using stably expressing CHOLec 3.2.8.1 cells grown in spinner cultures in CHO SFMII (Thermo Fisher) with antibiotics, for structural studies, or transiently transfected in HEK293 cells using 293 SFMII (Thermo Fisher) for biochemical studies. Integrin purification was carried out by affinity chromatography using a Protein G-clone 8B8 column followed by size exclusion chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare) in 20mM Tris-HCL pH 7.5, 150 mM NaCl, 1mM CaCl 2 and 1mM MgCl 2 . To produce L-TGF-β for structural studies, 293 cells were transiently transfected with equal amounts of porcine L-TGF-β1 C4S R249A RGD and C4S R249A RGE plasmids, to favor formation of L-TGF-β1 with a single intact RGD binding site to favor 1:1 binding stoichiometry to reduce sample heterogeneity, and purified as described ( Shi et al., 2011 ). Briefly, supernatants were collected from spinner cultures, clarified by centrifugation, filtered though a PES (polyethersulfone) membrane, 0.2 mm pore size (Millipore), concentrated, and purified using Ni-NTA agarose (Qiagen), washed with three column volumes of 0.6 M NaCl, 0.01 M Tris (pH 8.0) and eluted with 0.25 M imidazole in TBS. The was adjusted to pH 7.4 then applied to Strep-tactin agarose (IBA) (1 ml per 1 L of culture supernatant) and washed with TBS (pH 7.4). Tag was cleaved with recombinant His-tagged HRV-3C protease (Novagen, 100 U mg–1, 1 mg ml–1), diluted 20-fold in TBS (pH 7.4) with 10% glycerol, applied to the column, and incubated at 4 °C for 16 h. The flow-through was washed with two column volumes of TBS (pH 7.4), containing untagged proTGF-β1, then concentrated using centrifugal concentrators (Millipore) to about 1 mg ml–1 in 10 mM Tris (pH 7.5), 75 mM NaCl. The homogeneity and purity of all protein preparations were verified by SDS-PAGE stained with Coomassie blue and protein concentrations were measured by bicinchoninic acid assay (Pierce).

Cryo-EM sample preparation

To prepare integrin-Fab or integrin-L-TGF-β complexes, 100 mg of recombinant αvβ8 was incubated in a 2-fold molar excess of each Fab or L-TGF-β, incubated at room temperature for 30 min, subjected to size exclusion chromatography and concentrated to 6 to 9 mg/ml. For cryo-EM grid preparation, 2.5 μL of purified αvβ8 complex were deposited onto Quantifoil grids. For the αvβ8/L-TGF-β-R249A at 0.25 mg/ml, a 400 mesh 1.2/1/3 holey carbon gold grid was used. For the αvβ8/L-TGF-β/RGD-RGE complex at 0.075 mg/mL, a 400 mesh 2/2 holey carbon copper grid that had been covered with a thin layer of graphene oxide was used. For the αvβ8/C6D4/11d12v2 and the αvβ8/C6-RGD3/11d12v2 complexes, both at 6.8 mg/mL, 300 mesh 1.2/1.3 holey carbon gold grids were used. Except for the graphene-coated grid, grids were glow-discharged for 60s at 15 mA prior to sample application and freezing. The αvβ8/L-TGF-β C4S R249A RGD and αvβ8/L-TGF-β C4S R249A RGD/RGE complexes were frozen using a FEI Vitrobot Mark IV using a 4 second blot time. The αvβ8/C6D4/11d12v2 and αvβ8/C6-RGD3/11d12v2 complexes were frozen using a FEI Vitrobot Mark III using a 3–4 second blotting time. All grids were frozen with 100% humidity at 20°C and plunge-frozen in liquid ethane cooled by liquid nitrogen.

Cryo-EM data acquisition

Four datasets were acquired on a FEI Titan Krios transmission electron microscope operated in nano-probe mode at 300 kV equipped with a Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV and a Gatan K2 Summit direct detector. Automated data collection was carried out using the SerialEM software ( Mastronarde, 2005 ). Movies were recorded in super-resolution mode with a super-resolution pixel size of 0.673 Å/px and a nominal magnification of 105kx at a dose rate of ~8 e − /px/s. For the αvβ8/L-TGF-β-R249A complex and the αvβ8/LTGF-β/RGD-RGE, each 16 second movie contained 80 frames of 200 ms each, which corresponds to a total dose of ~70 e − /Å 2 . For the αvβ8/C6D4/11d12v2 complex and the αvβ8/C6RGD3/11d12v2 complex, each 12 second movie contained 60 frames of 200 ms, which corresponds to a total dose of ~50 e − /Å 2 . Each dataset was collected in a single session with a nominal defocus range of 1.0 – 2.5 μm under focus. Total micrographs collected for each dataset are as follows: αvβ8/L-TGF-β-R249A complex: 1684 micrographs; αvβ8/L-TGF-β-RGDRGE complex: 2682 micrographs; αvβ8/C6D4/11d12v2 complex: 1644 micrographs; αvβ8/C6RGD3/11d12v2 complex: 4033 micrographs.

Imaging Processing

Dose fractionated super-resolution image stacks were motion corrected and binned 2 × 2 by Fourier cropping using MotionCor2 ( Zheng et al., 2017 ). Motion corrected sums without dose-weighting were used for contrast transfer function (CTF) determination using GCTF ( Zhang, 2016 ) or CTFFIND4 ( Rohou and Grigorieff, 2015 ). Particles were picked using the reference-free method using Gautomatch ( http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch ) and boxed out using Relion 3.0 ( Zivanov et al., 2018 ) with a box size of 300 pixels and binned to 64 pixels. After 2D alignment and classification was carried out using cryoSPARC ( Punjani et al., 2017 ), selected particles were re-extracted in Relion 3.0 and binned to 128 pixels to generate ab initio initial models using cryoSPARC. 3D classification schemes are outlined for the αvβ8/LTGFB-R249A complex and the αvβ8/L-TGF-β-RGD-RGE complex in Fig. S5 and for the αvβ8/C6RGD3/11d12v2 complex in Fig. S4 . No 3D classification was used for the αvβ8/C6D4/11d12v2 complex dataset. For all final maps, non-uniform refinement, local resolution refinement, local resolution estimation, sharpening, and local filtering was carried out using cryoSPARC to yield maps with a final pixel size of 1.345Å/px. The number of particles contributing to the final maps are as follows: αvβ8/L-TGF-β (conformation iv): 43,600 particles; αvβ8/C6D4/11d12v: 84,266 particles; αvβ8/C6-RGD3/11d12v2: 221,159 particles. Images were rendered using UCSF Chimera ( Pettersen et al., 2004 ) and PyMol ( DeLano, 2002 ).

Model Building and Refinement

The atomic model of the αv headpiece from the crystal structure of αvβ6 (PDB: 4UM8) with glycans removed was fitted to the cryo-EM map as a rigid body. An atomic model of the β8 headpiece was generated by rigid body fitting of a homology model based on the same crystal structure (4UM8) using Modeller ( Webb and Sali, 2014 ) and adjusted around the SyMBS cation area using the crystal structure of αvβ3 (PDB: 3IJE), then fitted into the cryo-EM density map as a rigid body. Atomic models of Fabs were generated with RosettaAntibody using multiple-template grafting and H3 loop modelization ( Lyskov et al., 2013 ) based on the primary sequence of their V h /V k . An atomic model of the arm domain of L-TGF-β1 was generated from the crystal structure of L-TGF-β1 bound to αvβ6 (PDB: 5FFO). The models for Fab C6D4 and Fab C6-RGD3 or L-TGF-β1 were then fitted as a rigid body to the map. Prototypical CHOLec3.2.8.1 glycans were added back to the model at the solvent exposed N-glycosylation consensus sites using GLYCAM ( Singh et al., 2016 ). The sugar base of glycans were trimmed to fit into the corresponding densities and further refined in Rosetta using a dedicated protocol that uses physically realistic geometries based on prior knowledge of saccharide chemical properties ( Frenz et al., 2019 ). After rigid body fitting, all models were manually adjusted to fit the cryo-EM density maps in COOT ( Emsley et al., 2010 ), followed by real space refinement using Phenix ( Adams et al., 2010 ), and Rosetta ( Wang et al., 2016 ). All modeling was aided by using EM maps that were focused on specific regions, as well as sharpened and unsharpened maps. All maps used for modeling have been deposited.

Antibody binding assays

ELISA plates were coated with recombinant αv-integrins (1 mg/ml coating concentration, all from R&D systems) blocked with 5% BSA in PBS for 1 hour, and antibodies allowed to bind for 2 hours at RT and detected with anti-mouse-HRP.

Cell adhesion assays

ELISA plates were coated with integrins (2 mg/ml coating concentration) and blocked with PBS with 5% BSA for 1 hour and then CHO-GARP/L-TGF-β, or CHO mock (5 ×10 5 ) transfectants in the presence of various concentrations of C6D4, C6-RGD3 or 3G9 were centrifuged onto integrin coated wells (10 × g) for 5 min allowed to adhere for 30 min at RT after which the plates were inverted and centrifuged (10 × g) for 5 min and then immediated fixed and stained (1% formaldehyde, 20% MeOH, 0.5% crystal violet) and after extensive washing, dye was solubilized in PBS with 1% T-X100 for 1 hr at RT and attached cells estimated by absorbance (A 595 ).

L-TGF-β1 binding assays

ELISA plates were coated with recombinant porcine L-TGF-β1 (0.5 μg/ml coating concentration), blocked with 5% BSA in PBS for 1 hour, and integrins at various concentrations were allowed to bind for 2 hours at RT, and detected with 8B8 antibody and anti-mouse-HRP. Cell staining, flow cytometry and cell sorting Cell staining for L-TGF-β1 was confirmed by flow cytometry using anti-LAP (R&D biotinylated anti-LAP BAF246 and streptavidin APC) and GARP cell surface expression was confirmed by anti-HA staining (clone 5E11D8 (Genscript, Piscataway, NJ)). EGFP expression was also used as a surrogate marker for L-TGF-β1 expression. High-expressing pools of expressing cells were established by sorting (BD FACSAria, BD Biosciences, US).

Peptide competition assays

For peptide competition assays, ELISA plates were coated with recombinant porcine L-TGF-β1 (0.5 μg/ml coating concentration), blocked with 5% BSA in PBS for 1 hour, and integrins at 0.5 μg/ml were pre-incubated with peptides at various concentrations for 20 minutes, allowed to bind for 2 hours at RT, and detected with 8B8 antibody and anti-mouse-HRP. Sequence alignments Multiple protein sequence alignments for integrins were generated using Clustal Omega ( Madeira et al., 2019 ).

QUANTIFICATION AND STATISTICAL ANALYSIS

ELISA and TMLC assays are reported as means ± s.e.m. All assays were repeated a minimum of 3 times. All statistical analyses were performed using the software package Prism 7 (GraphPad Software, San Diego, CA).

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for reagents may be addressed to the Lead Contact, Stephen 5 Nishimura ( stephen.nishimura@ucsf.edu ). Antibodies, cell lines and plasmids used in this manuscript will be available from the Lead Contact upon execution of a materials transfer agreement.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Subjects

Adult airways were collected from first through fourth order bronchi from lobectomy specimens from resections performed for primary lung cancer. Informed consent was obtained from all surgical participants as part of an approved ongoing research protocol by the University of California San Francisco Committee on Human Research, in full accordance with the declaration of Helsinki principles. The specimens were de-indentified. Normal bronchial epithelial cells (HBEC) Normal human bronchial epithelial cells (HBEC) were isolated from human lung specimens, as previously described ( Araya et al., 2007 ). Specifically, airway epithelium was stripped freshly from bronchi from surgical specimens and after washing in PBS with dithiothreitol (5 mM) were digested overnight at 4°C with a protease XIV solution (0.4 mg/ml, Sigma). After washing with bronchial epithelial growth medium (BEGM, Lonza) with 2.5% FCS and tituration, HBEC were plated onto rat-tail Col I-coated (10 μg/ml, Corning, cat. no. 354236) dishes and incubated overnight; then the medium was changed to fresh BEGM. HBEC were passaged under conditional reprogramming conditions with BEGM with ROCK inhibitor (10 mM) and irradiated NIH3T3 fibroblasts, as described ( Liu et al., 2012 ). Human embryonic kidney cells (HEK293) HEK293 are an authenticated female line from a commercial vendor (ATCC). HEK293 cells were grown in DMEM + 10% FBS + penicillin-streptomycin + amphotericin B + 0.11mg/mL Sodium Pyruvate + 2mM L-glutamine + MEM nonessential amino acids, cultured at 37°C with 5%C O 2 . HEK293 and all other cell lines were routinely tested for mycoplasma contamination. For all established cell lines cell culture media and antibiotics were prepared by the University of California, San Francisco Cell Culture Facility using deionized water and analytical grade reagents. Fetal calf serum was obtained from Invitrogen (Carlsbad, CA). Chinese Hamster Ovary Cells (CHO) CHO-K1 are an authenticated female line from a commercial vendor (ATCC). CHOLec 3.2.8.1 have four independent mutations in the N- and O-glycosylation pathways producing proteins all of the high mannose glycosylation pattern ( Stanley, 1989 ). CHOLec 3.2.8.1 cells were grown in CHO-S-SFM II medium + 10% FBS + penicillin-streptomycin + amphotericin B, cultured at 37°C with 5%CO 2. CHOLec 3.2.8.1 cells were provided by Pamela Stanley ( Stanley, 1989 ). Authentication of CHOLec 3.2.8.1 cells was performed by changes in glycosylation patterns as estimated by migration of secreted proteins by SDS-PAGE. Transformed Mink Lung Epithelial Cells (TMLC) TMLC is a stable mink lung epithelial reporter cell line derived from mixed sex American Mink fetal lung epithelial cells that stably expresses a portion of the plasminogen activator inhibitor 1 promoter ( Abe et al., 1994 ). TMLC cells were grown in DMEM + 10% FBS + penicillin-streptomycin + amphotericin B, cultured at 37°C with 5%CO 2. TMLC are a gift from John Munger (NYU medical center, New York City, New York). Authentication was performed by increased luciferase activity in response to recombinant TGF-β.

METHOD DETAILS Antibody isolation, characterization and production The C6D4, 68, 8B8, 3G9, 1D11 antibodies have been previously described ( Minagawa et al., 2014 ; Mu et al., 2002 ; Takasaka et al., 2018 ; Weinreb et al., 2004 ). Mouse anti-human αv, clone 11D12V2 was isolated as described, with the following modifications. Screening of hybridomas produced a polyclonal hybridoma 11D12 with reactivity against cell lines expressing the human αv-subunit (CHO-αvβ8, or SW480-β6). Subcloning produced two clones 11D12V1 and 11D12V2 both with reactivity against the αv-subunit. Variable (V) genes were isolated, sequenced, and the V H /V K genes cloned into mouse IgG 2a expression vectors and stably transfected into CHO-K1 cells. C6-RGD3 was created by splice overlap extension (SOE) polymerase chain reaction (PCR) into the VL CDRL1 region of a C6D4 IgG2a expression vector ( Minagawa et al., 2014 ) with the following oligonucleotide primers using the C6D4 IgG2a expression construct as a template: 5’-GATCTGGGGCGCCTCAAGAAGAACGCCTTGGCTTGGTACCAGCAG-3’; 5’-CTTGAGGCGCCCCAGATCTCCACGGCCGAGCAGACTCTGACTGGATTTG-3’. C6-RGD3 was transfected into CHO-K1 cells. Antibodies were produced and purified as described previously ( Minagawa et al., 2014 ). Briefly, stable transfected CHO-K1 cells were grown in spinner cultures in CHO SFMII media with antibiotics. Antibodies were purified from culture media using protein G sepharose columns (HiTrap, GE Healthcare). C6D4, C6-RGD3, and 11D12V2 fragments were generated by papain digestion (Pierce) of the IgG followed by Protein-A Agarose (Millipore) incubation and to remove Fc fragments and intact antibodies and final separation using Mono S ion-exchange chromatography (GE Healthcare). The 11D12V1 and 11D12V2 binding epitopes were estimated by negative staining electron microscopy (ns-EM), essentially as described below and in previous studies ( Minagawa et al., 2014 ; Takasaka et al., 2018 ). Clone 11D12V1 bound to the αv-head and 11D12V2 to the αv-thigh. Anti-β6 (MAB4155) was purchased (R&D Systems). A bioassay to measure cell-intrinsic TGF-β signaling To develop a cell intrinsic TGF-β activation system we used stable transfection of TMLC (Amaxa) with a vector containing either a WT human TGF-β1 IRES GFP, or a human TGF-β1 (R249A) IRES GFP cassette with puromycin resistance to obtain TMLC cells expressing WT L-TGF-β either capable of dissociating into LAP and mature TGF-β or not, due to the R249A mutation that normally allows furin cleavage of LAP from mature TGF-β ( Shi, et al, 2011 ).

Human TGF-β1 IRES

GFP or human TGF-β1 (R249A) IRES GFP TMLC cells were sorted for equal expression using GFP fluorescence and did not present any L-TGF-β on their cell surface ( Fig. S7 ). In contrast, stable transfection of these lines with a HA-GARP construct with a blastacidin resistance cassette followed by selection and sorting resulted in high surface expression of TGF-β1/GARP or TGF-β1 (R249A)/GARP, as measured by anti-HA (clone 5E11D8, GenScript, Piscataway, NJ) or anti LAP (R&D Systems, AF426). To confirm lack of releasable of TGF-β from the L-TGF-β R249A/GARP cell surface complex, L-TGF-β (R249A)/GARP expressing or WT L-TGF-β/GARP TMLC cells were surface biotinylated using EZ-link sulfo-NHS biotin (Thermo Fisher Scientific) and immunoprecipitated using anti-HA, resolved by 4–12% gradient SDS-PAGE, under non-reducing conditions, immunoblotted, probed with streptavidin-HRP and detected by chemiluminescence, essentially as described ( Mu, et al, 2002 ). Immunoprecipitations confirmed association of GARP with WT L-TGF-β, L-TGF-β (R249A), and absence of cleavage of LAP from mature TGF-β in the L-TGF-β (R249A)/GARP TMLC cells ( Fig. S7 ). A TMLC assay to measure integrin-mediated cell-intrinsic TGF-β signaling The αvβ8 ectodomain was coated along with the controls αvβ3 (R&D Systems), BSA (Sigma-Aldrich) or anti-LAP (R&D AF426, 1 μg/ml) onto ELISA plates in PBS (1mM Ca 2+ and 1mM Mg 2+ ) 1 hour at RT. Wells were subsequently washed in PBS and blocked in PBS with 1% BSA WT L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP expressing TMLC cells were plated at a density of 1×10 5 cells/ml in basal media. After 16 hrs, media was removed and applied to wells containing TMLC reporter cells to measure diffusible mature TGF-β, which were incubated overnight prior to lysis and determination of luciferase activity (Promega) as reported ( Mu, et al, 2002 ). To measure cell-intrinsic TGF-β1 activation, the attached L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP or parental TMLC cells were lysed and assayed for luciferase activity. To facilitate comparison between different TMLC lines expressing WT L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP (or parental TMLC cells) normalized luciferase activity was expressed as activated TGF-β in pg/ml. Normalization was performed by interpolating luciferase activity against standard curves generated using each TMLC line with varying doses of recombinant human TGF-β1 (R&D Systems).

Integrin DNA constructs

Wild-type and mutant recombinant human integrin αvβ8 and αvβ6 truncated at the junction of the ectodomains and transmembrane domains in pcDNA1neo have been described ( Nishimura et al., 1994 ; Weinreb et al., 2004 ). β8 Δ SDL was prepared as described ( Takasaka et al., 2018 ). To create mutant β8 subunits, SOE was performed using PCR with WT β8 as a template to create mutant constructs all in pcDNA6 V5HisA (Invitrogen) with a stop codon inserted before the V5/His tag. The following mutagenic primers (all in 5 ‘to 3’ orientation) were used: β8 I208R5’-CAGAAGATCTCTGGAAACAGAGATACACC-3’; 5’-GAAGTTTGGTCGACATAATGC-3’ β8 Y172N: 5’-CAATGCAGTGACAACAATTTAGACTGC-3’, 5’-GCAGTCTAAATTGTTGTCACTGCATTG-3’; β8 Y172M: 5’-GATTCATAATCAATGCAGTGACATGAATTTAGACTGCATGCC-3’, 5’-GGCATGCAGTCTAAATTCATGTCACTGCATTGATTATGAATC-3’; β8 Y172A: 5’-GATTCATAATCAATGCAGTGACGCCAATTTAGACTGCATGCC-3’, 5’-GGCATGCAGTCTAAATTGGCGTCACTGCATTGATTATGAATC-3’; β6 I183N: 5’-CCCTTGCAGTAGTAATCCATACTTCTG-3’, 5’-CAGAAGTATGGATTACTACTGCAAGGG-3’. TGF-β DNA constructs Porcine L-TGF-β1 with a C4S mutation, to improve secretion and prevent association with L-TGF-β binding proteins, and an N-terminal cleavable (HRV 3C) 7x Histidine-streptavidin binding protein tag to facilitate purification ( Shi et al., 2011 ), were joined by SOE PCR from pcDNA-GS-TGF-β1 (gift from Dr. Sun, National Institutes of Health, Bethesda, MD ( Zou and Sun, 2004 )) and subcloned into pcDNA6 (Invitrogen). Porcine L-TGF-β1 C4S pcDNA6 was modified using the following mutagenic primers, RGE (a mutation that disrupts the integrin binding recognition sequence on L-TGF-β and therefore minimizes the number of L-TGF-βs that bind two integrins simultaneously): 5’-CCGCCGGGGTGAACTGGCCAC-3’; 5’-GTGGCCAGTTCACCCCGGCGG-3’; R249A: 5’-ACCTGCACAGCTCCCGGCACCGCGCAGCCCTGG-3’. Wild type human TGFβ1 was derived from human TGFβ1_pLX307 ( Rosenbluh et al., 2016 ) by removing the C-terminal V5 tag using PCR with the following primers; 5’-ATGGCCACCCCGCTGG-3’, 5’-CTCTACTAGTCTCGAGTTATCAGCTGCACTTGCAGGAGCGCAC-3’. The WT human TGF-β1 IRES GFP cassette was then subcloned into a version of pcDNA6 (Invitrogen) with a puromycin resistance cassette which was amplified from TGFβ1_pLX307 using 5’-ATCGTTTCAGACCCACCTCCC-3’ and 5’-CTCTGCTTAGCGAATTCGTTAACTGGCACCGGG-3’. A R249A mutant of this construct was produced using SOE PCR employing the following mutagenic primers; 5’-CACCGCGCAGCCCTGGACACCAAC-3’, 5’-CCAGGGCTGCGCGGTGCCGGGAG-3’. GARP DNA constructs N-terminal HA tagged human GARP pcDNA3 ( Cuende et al., 2015 ) was provided by Sophie Lucas (de Duve Institute, UCLouvain, Brussels, Belgium) and the entire HA-GARP reading frame transferred into pcDNA6 (Invitrogen). All constructs were verified by sequencing.

Secreted protein expression and purification

Integrin constructs were expressed using stably expressing CHOLec 3.2.8.1 cells grown in spinner cultures in CHO SFMII (Thermo Fisher) with antibiotics, for structural studies, or transiently transfected in HEK293 cells using 293 SFMII (Thermo Fisher) for biochemical studies. Integrin purification was carried out by affinity chromatography using a Protein G-clone 8B8 column followed by size exclusion chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare) in 20mM Tris-HCL pH 7.5, 150 mM NaCl, 1mM CaCl 2 and 1mM MgCl 2 . To produce L-TGF-β for structural studies, 293 cells were transiently transfected with equal amounts of porcine L-TGF-β1 C4S R249A RGD and C4S R249A RGE plasmids, to favor formation of L-TGF-β1 with a single intact RGD binding site to favor 1:1 binding stoichiometry to reduce sample heterogeneity, and purified as described ( Shi et al., 2011 ). Briefly, supernatants were collected from spinner cultures, clarified by centrifugation, filtered though a PES (polyethersulfone) membrane, 0.2 mm pore size (Millipore), concentrated, and purified using Ni-NTA agarose (Qiagen), washed with three column volumes of 0.6 M NaCl, 0.01 M Tris (pH 8.0) and eluted with 0.25 M imidazole in TBS. The was adjusted to pH 7.4 then applied to Strep-tactin agarose (IBA) (1 ml per 1 L of culture supernatant) and washed with TBS (pH 7.4). Tag was cleaved with recombinant His-tagged HRV-3C protease (Novagen, 100 U mg–1, 1 mg ml–1), diluted 20-fold in TBS (pH 7.4) with 10% glycerol, applied to the column, and incubated at 4 °C for 16 h. The flow-through was washed with two column volumes of TBS (pH 7.4), containing untagged proTGF-β1, then concentrated using centrifugal concentrators (Millipore) to about 1 mg ml–1 in 10 mM Tris (pH 7.5), 75 mM NaCl. The homogeneity and purity of all protein preparations were verified by SDS-PAGE stained with Coomassie blue and protein concentrations were measured by bicinchoninic acid assay (Pierce).

Cryo-EM sample preparation

To prepare integrin-Fab or integrin-L-TGF-β complexes, 100 mg of recombinant αvβ8 was incubated in a 2-fold molar excess of each Fab or L-TGF-β, incubated at room temperature for 30 min, subjected to size exclusion chromatography and concentrated to 6 to 9 mg/ml. For cryo-EM grid preparation, 2.5 μL of purified αvβ8 complex were deposited onto Quantifoil grids. For the αvβ8/L-TGF-β-R249A at 0.25 mg/ml, a 400 mesh 1.2/1/3 holey carbon gold grid was used. For the αvβ8/L-TGF-β/RGD-RGE complex at 0.075 mg/mL, a 400 mesh 2/2 holey carbon copper grid that had been covered with a thin layer of graphene oxide was used. For the αvβ8/C6D4/11d12v2 and the αvβ8/C6-RGD3/11d12v2 complexes, both at 6.8 mg/mL, 300 mesh 1.2/1.3 holey carbon gold grids were used. Except for the graphene-coated grid, grids were glow-discharged for 60s at 15 mA prior to sample application and freezing. The αvβ8/L-TGF-β C4S R249A RGD and αvβ8/L-TGF-β C4S R249A RGD/RGE complexes were frozen using a FEI Vitrobot Mark IV using a 4 second blot time. The αvβ8/C6D4/11d12v2 and αvβ8/C6-RGD3/11d12v2 complexes were frozen using a FEI Vitrobot Mark III using a 3–4 second blotting time. All grids were frozen with 100% humidity at 20°C and plunge-frozen in liquid ethane cooled by liquid nitrogen.

Cryo-EM data acquisition

Four datasets were acquired on a FEI Titan Krios transmission electron microscope operated in nano-probe mode at 300 kV equipped with a Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV and a Gatan K2 Summit direct detector. Automated data collection was carried out using the SerialEM software ( Mastronarde, 2005 ). Movies were recorded in super-resolution mode with a super-resolution pixel size of 0.673 Å/px and a nominal magnification of 105kx at a dose rate of ~8 e − /px/s. For the αvβ8/L-TGF-β-R249A complex and the αvβ8/LTGF-β/RGD-RGE, each 16 second movie contained 80 frames of 200 ms each, which corresponds to a total dose of ~70 e − /Å 2 . For the αvβ8/C6D4/11d12v2 complex and the αvβ8/C6RGD3/11d12v2 complex, each 12 second movie contained 60 frames of 200 ms, which corresponds to a total dose of ~50 e − /Å 2 . Each dataset was collected in a single session with a nominal defocus range of 1.0 – 2.5 μm under focus. Total micrographs collected for each dataset are as follows: αvβ8/L-TGF-β-R249A complex: 1684 micrographs; αvβ8/L-TGF-β-RGDRGE complex: 2682 micrographs; αvβ8/C6D4/11d12v2 complex: 1644 micrographs; αvβ8/C6RGD3/11d12v2 complex: 4033 micrographs.

Imaging Processing

Dose fractionated super-resolution image stacks were motion corrected and binned 2 × 2 by Fourier cropping using MotionCor2 ( Zheng et al., 2017 ). Motion corrected sums without dose-weighting were used for contrast transfer function (CTF) determination using GCTF ( Zhang, 2016 ) or CTFFIND4 ( Rohou and Grigorieff, 2015 ). Particles were picked using the reference-free method using Gautomatch ( http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch ) and boxed out using Relion 3.0 ( Zivanov et al., 2018 ) with a box size of 300 pixels and binned to 64 pixels. After 2D alignment and classification was carried out using cryoSPARC ( Punjani et al., 2017 ), selected particles were re-extracted in Relion 3.0 and binned to 128 pixels to generate ab initio initial models using cryoSPARC. 3D classification schemes are outlined for the αvβ8/LTGFB-R249A complex and the αvβ8/L-TGF-β-RGD-RGE complex in Fig. S5 and for the αvβ8/C6RGD3/11d12v2 complex in Fig. S4 . No 3D classification was used for the αvβ8/C6D4/11d12v2 complex dataset. For all final maps, non-uniform refinement, local resolution refinement, local resolution estimation, sharpening, and local filtering was carried out using cryoSPARC to yield maps with a final pixel size of 1.345Å/px. The number of particles contributing to the final maps are as follows: αvβ8/L-TGF-β (conformation iv): 43,600 particles; αvβ8/C6D4/11d12v: 84,266 particles; αvβ8/C6-RGD3/11d12v2: 221,159 particles. Images were rendered using UCSF Chimera ( Pettersen et al., 2004 ) and PyMol ( DeLano, 2002 ).

Model Building and Refinement

The atomic model of the αv headpiece from the crystal structure of αvβ6 (PDB: 4UM8) with glycans removed was fitted to the cryo-EM map as a rigid body. An atomic model of the β8 headpiece was generated by rigid body fitting of a homology model based on the same crystal structure (4UM8) using Modeller ( Webb and Sali, 2014 ) and adjusted around the SyMBS cation area using the crystal structure of αvβ3 (PDB: 3IJE), then fitted into the cryo-EM density map as a rigid body. Atomic models of Fabs were generated with RosettaAntibody using multiple-template grafting and H3 loop modelization ( Lyskov et al., 2013 ) based on the primary sequence of their V h /V k . An atomic model of the arm domain of L-TGF-β1 was generated from the crystal structure of L-TGF-β1 bound to αvβ6 (PDB: 5FFO). The models for Fab C6D4 and Fab C6-RGD3 or L-TGF-β1 were then fitted as a rigid body to the map. Prototypical CHOLec3.2.8.1 glycans were added back to the model at the solvent exposed N-glycosylation consensus sites using GLYCAM ( Singh et al., 2016 ). The sugar base of glycans were trimmed to fit into the corresponding densities and further refined in Rosetta using a dedicated protocol that uses physically realistic geometries based on prior knowledge of saccharide chemical properties ( Frenz et al., 2019 ). After rigid body fitting, all models were manually adjusted to fit the cryo-EM density maps in COOT ( Emsley et al., 2010 ), followed by real space refinement using Phenix ( Adams et al., 2010 ), and Rosetta ( Wang et al., 2016 ). All modeling was aided by using EM maps that were focused on specific regions, as well as sharpened and unsharpened maps. All maps used for modeling have been deposited.

Antibody binding assays

ELISA plates were coated with recombinant αv-integrins (1 mg/ml coating concentration, all from R&D systems) blocked with 5% BSA in PBS for 1 hour, and antibodies allowed to bind for 2 hours at RT and detected with anti-mouse-HRP.

Cell adhesion assays

ELISA plates were coated with integrins (2 mg/ml coating concentration) and blocked with PBS with 5% BSA for 1 hour and then CHO-GARP/L-TGF-β, or CHO mock (5 ×10 5 ) transfectants in the presence of various concentrations of C6D4, C6-RGD3 or 3G9 were centrifuged onto integrin coated wells (10 × g) for 5 min allowed to adhere for 30 min at RT after which the plates were inverted and centrifuged (10 × g) for 5 min and then immediated fixed and stained (1% formaldehyde, 20% MeOH, 0.5% crystal violet) and after extensive washing, dye was solubilized in PBS with 1% T-X100 for 1 hr at RT and attached cells estimated by absorbance (A 595 ).

L-TGF-β1 binding assays

ELISA plates were coated with recombinant porcine L-TGF-β1 (0.5 μg/ml coating concentration), blocked with 5% BSA in PBS for 1 hour, and integrins at various concentrations were allowed to bind for 2 hours at RT, and detected with 8B8 antibody and anti-mouse-HRP. Cell staining, flow cytometry and cell sorting Cell staining for L-TGF-β1 was confirmed by flow cytometry using anti-LAP (R&D biotinylated anti-LAP BAF246 and streptavidin APC) and GARP cell surface expression was confirmed by anti-HA staining (clone 5E11D8 (Genscript, Piscataway, NJ)). EGFP expression was also used as a surrogate marker for L-TGF-β1 expression. High-expressing pools of expressing cells were established by sorting (BD FACSAria, BD Biosciences, US).

Peptide competition assays

For peptide competition assays, ELISA plates were coated with recombinant porcine L-TGF-β1 (0.5 μg/ml coating concentration), blocked with 5% BSA in PBS for 1 hour, and integrins at 0.5 μg/ml were pre-incubated with peptides at various concentrations for 20 minutes, allowed to bind for 2 hours at RT, and detected with 8B8 antibody and anti-mouse-HRP. Sequence alignments Multiple protein sequence alignments for integrins were generated using Clustal Omega ( Madeira et al., 2019 ).

Supplementary Material 1 Fig. S1 Integrins and TGF-β: nomenclature and models of activation, related to Introduction, Fig. 7 (A) Integrin and L-TGF-β domains and subdomains and their membrane or extracellular matrix localization via coupling to adaptor proteins are shown using common nomenclature. (B) In the global-rearrangement or “switchblade” model of integrin activation, the bent conformation moves through an extended-closed conformation to an extended-open conformation. In this model, high-affinity ligand binding and actin cytoskeletal force transduction occur in the extended-open conformation. In the case of αvβ6 it has been proposed that such force releases TGF-β and allows diffusion to its target receptors ( Dong et al., 2017 ). Pink arrows indicate movements in the headpiece. (C) The αvβ8 integrin assumes a single conformation, extended-closed, that accommodates ligand binding and affinity regulation. The essential immune cell functions of αvβ8 have been tied to binding to L-TGF-β presented by type I transmembrane adaptor proteins such as GARP on immune cell surfaces. In this model, we hypothesize that the “latency lasso” of the straitjacket domain of L-TGF-β loosens upon binding to αvβ8 to allow the active domain of TGF-β to interact with its receptors. 2 Fig. S2. Cryo-EM micrographs and statistics for αvβ8 complexes, related to Figs. 1 and 2 Details of data collection and processing data for (A) αvβ8/LTGF-β (subclass iv), (B) αvβ8/C6D4 and (C) αvβ8/C6-RGD3. For each complex the following are shown: First column: a representative motion corrected micrograph of the particles suspended in vitreous ice. Second column: the gold standard FSC (top) and the angular distributions of particles (bottom) used in the final map, as estimated by cryoSPARC. Third column: (A) the unsharpened map displayed at a low thresh, (B,C) the unsharpened map before focused refinement displayed at a low threshold. Fourth column: the map after focused alignment displayed at a high threshold sharpened to a b-factor of (A) −71 (B) −83 or (C) −84. Maps are colored on the same scale, as indicated in row A, based on local resolution estimates. Scale bar = 100 nm. 3 Fig. S3. Comparisons of αvβ8 structures with integrin crystal structures, model quality, and characterization of C6-RGD3, related to Figs. 1 , 2 and 4 (A, B) Superimpositions of ribbon models of αvβ8/L-TGF-β1 with published models from crystal structures of liganded αvβ6 (RGD peptide, PDB: 4UM9 ( Dong et al., 2014 ) (A); L-TGF-β1, PDB: 5FFO ( Dong et al., 2017 ) (B)). From αvβ8/L-TGF-β1 αv-subunit, green; β8-subunit, blue; RGD loop, purple; β6 subunit and L-TGF-β3 RGD peptide, salmon (A); β6 subunit and L-TGF-β1 RGD loop, lime green. (C, D) Close-up of the binding interface of the αvβ8 integrin (light green and blue) and Fab C6D4 (coral, C) or Fab C6-RGD3 (pink, D) with the corresponding sharpened density map (grey volume). (E) Close-up of the binding interface of the β8 integrin subunit SDL2 loop (cyan) and the L-TGF-β1 proximal loop, RGD motif, and ligand-binding helix (purple) superimposed on their respective sharpened density maps (mesh). (F) Close-up of the Fab C6-RGD3 CDRL1 loop (pink) and its sharpened density map (pink mesh). (G, H) Close-up of the integrin β8 SDL1 α1 helix and β6-α7 loop when in complex with L-TGF-β1 (cyan, G) or Fab C6-RGD3 (dark blue, H). (I) Map to model FSC curves for the αvβ8/L-TGF-β1 (purple), αvβ8/C6D4 (orange), αvβ8/C6-RGD3 (magenta) complexes. (J-M) View of the metal ions and MIDAS cation coordination in various liganded integrin structures in ribbon models: αvβ8/L-TGF-β1 complex (J), αvβ3/fibronectin 10 th domain RGD complex (PDB: 4MMX) ( Van Agthoven et al., 2014 ) (K), α iib β3/fibrinogen RGD peptide complex (PDB: 2VDR) ( Springer et al., 2008 )(L), αvβ6/L-TGF-β1 complex (PDB: 5FFO) (M). The coordinating residues are indicated in sticks. (N-S) Superimpositions of the α1-helix, with the β6-α7 loop and MIDAS cation (dotted circle) as ribbon models with superimpositions from unliganded or liganded α iib β3 (PDB: 3T3P ( Zhu et al., 2012 ) or 2VDR( Xiong et al., 2009 ), respectively): (N) liganded (yellow) or unliganded α iib β3, red; (O) liganded α iib β3 (red) or αvβ8/C6-RGD3, green; (P) liganded α iib β3 (yellow) or αvβ8/C6D4, light blue; (Q) liganded α iib β3 (orange) or αvβ8/L-TGF-β1, pink; (R) αvβ8/C6D4 (light blue), αvβ8/C6-RGD3 (green), or αvβ8/L-TGF-β1, pink. Movement of the tip of the SDL1 α1-helix is highlighted by the S116 (β8)/S123 (β3) residues in sticks. The Asp RGD is represented in sticks for liganded structures. (T, U) Superimposition of ribbon models of the αvβ8/C6D4 and α4β7/Act-1 (PDB: 3V4P) ( Yu et al., 2012 ) complexes. Both C6D4 and Act-1 epitopes are located in the SDL2 region of the integrin (front view (T); rotated view, U)). αv-green, β8-blue, C6D4-orange, α4-lime green, β7-light blue, Act-1-magenta. (V) Ribbon model of the αvβ8/C6D4 complex with the CDR L1 loop highlighted in red that was replaced with the L-TGF-β integrin-binding motif and helix to create C6-RGD3 (αv-green, β8-blue, C6D4-gold). (W) Binding assay to immobilized αv-integrins to show specificity of C6D4 for αvβ8, and C6-RGD3 for αvβ6 and αvβ8. Shown is a representative experiment of three (n=3). (X, Z) Inhibition of cell adhesion of CHO cells co-expressing GARP and L-TGF-β1 on their cell surface to immobilized (X) αvβ8 or (Z) αvβ6 in the presence of indicated concentrations of C6D4 or C6-RGD3 or anti-β6 (3G9). TGF-β activation assays of (Y) CHO cells stably transfected with αvβ8 or (AA) human bronchial epithelial cells (HBEC), which naturally express high levels of αvβ6 and low levels of αvβ8 ( Araya et al., 2007 ) in the presence of indicated concentrations of C6D4, C6-RGD3 or 3G9 ( Weinreb et al., 2004 ). Activation is shown relative to a pan-TGF-β neutralizing antibody (1D11). n=3, error bars show s.e.m. 4 Fig. S4. Processing schematic for the αvβ8/C6-RGD3 complex, related to Fig. 2 A schematic flowchart showing the classification scheme of the αvβ8/C6-RGD3 complex. Particle numbers at each step and for each class are indicated. From top to bottom: After 3D classification, the data was separated into three groups: particles with C6-RGD3 bound at the expected angle, particles with C6-RGD3 at a skewed angle, and particles without C6-RGD3. These three groups were processed separately. The map showing C6-RGD3 at the expected angle shows the RGD motif in the alternative, non-helical binding conformation. The map showing C6-RGD3 at the skewed angle shows the RGD motif in a helical conformation. All maps are colored on the same scale, as indicated, based on local resolution estimates. 5 Fig. S5. Processing schematic for the αvβ8/LTGF-β complex, related to Fig. 5 A schematic flowchart showing the classification scheme of the αvβ8/L-TGF-β complex. Due to the preferred orientations adopted by the particles, two types of grids were used and two separate datasets were collected. Upon angular assignment, it was found that although particles on both grids suffered from preferred orientations, the orientations complement each other to form a more complete orientation sampling. Particle numbers at each step and for each class are indicated. 3D reconstructions of seven out of sixteen subclasses showed significant density for L-TGF-β, and reached high resolution after refinement. All maps are colored on the same scale, as indicated, based on local resolution estimates. 6 Fig. S6. β8 and β6 SDL mutations, and structure-guided mutagenesis of the β8 SDL2/3 binding pocket, related to Fig. 6 (A, B) CHOlec 3.2.8.1 cells were stably transfected with various β8, β6 WT or SDL mutants, as indicated, sorted several times to establish highly expressing pools, and tested for expression using C6D4 (A) compared to clone 68 (B), a βI domain antibody that binds to the tail of the β8 α1-helix ( Minagawa et al., 2014 ). Note that the mutant I208R does not bind C6D4 as well as other mutants, as this mutation would be expected to clash with the position of C6D4 CDR L1 . (C, D) Binding of L-TGF-β1 to truncated secreted forms of C) WT and a Y172A αvβ8 mutant, or D) αvβ6 WT and a I183N αvβ6 mutant. n=3, error bars show s.e.m. (E-G) Ribbon models of the αvβ8 head domain of the CDR L1 loops of (E) C6D4, (F) C6-RGD3 and (G) L-TGF-β (gold sticks), with boxed ligand pocket (upper panels) shown in close up views in middle and lower panels. Color code: αv-subunit (green), β8-SDL1 (light blue), -SDL2 (cyan) and -SDL3 (purple) loops. The positioning of the C6D4 CDR L1 ; C6-RGD3 CDR L1 ; or L-TGF-β1 integrin-binding loops are shown positioned in the binding cleft of β8 wild-type (middle panels); and the I208R mutant (lower panels). Middle panel color code: L-TGF-β integrin-binding loop R215 (gold), the β8 SDL2 Y172 (red) and SDL1 S116 (red). Lower panel color code: the SDL3 I208R mutation is indicated in red spheres demonstrating clash with the C6D4 CDRL1; C6-RGD3 CDR L1 but not the canonical position of the L-TGF-β integrin-binding loop. The SyMBS (grey) and MIDAS (green) cations are shown. 7 Fig. S7. Expression and characterization of WT L-TGF-β, L-TGF-β (R249A), WT L-TGF-β/GARP and L-TGF-β (R249A)/GARP TMLC reporter cells, related to Fig. 7 (A-D) WT L-TGF-β/GARP and L-TGF-β (R249A)/GARP are equally expressed on the surface of TGF-β reporter cells (TMLC). TMLC cells were first stably transfected with either (A) WT L-TGF-β IRES GFP or, (B) L-TGF-β (R249A) IRES GFP and sorted for equal expression of GFP. Histograms show GFP expression on the x-axis and surface localization of L-TGF-β, as assessed by anti-LAP-APC staining, on the y-axis. Stably transfected and sorted cells show no surface staining for L-TGF-β, despite showing ~40–50% GFP bright cells. In contrast, when (C) WT L-TGF-β IRES GFP or (D) L-TGF-β (R249A) IRES GFP expressing TMLC were subsequently stably transfected with a N-terminal HA tagged GARP construct, bright surface expression of L-TGF-β was detected in ~40% of cells. (C) WT L-TGF-β IRES GFP/HA GARP or, (D) L-TGF-β (R249A) IRES GFP/HA GARP TMLC were sorted for equal surface expression of L-TGF-β, as assessed by anti-LAP-APC staining. (E) Cartoon representation of the experiment. Individual wells of 96-well ELISA plates were coated with either αvβ8, αvβ3 (which binds with low affinity to L-TGF-β), polyclonal anti-LAP (which binds to LAP, but does not activate or inhibit activation of L-TGF-β), or BSA as a non-specific binding control. TMLC cells either non-transfected or transfected as in A-D with WT L-TGF-β or L-TGF-β (R249A) with or without GARP were applied to coated wells. (F) Parental TMLC cells, WT L-TGF-β IRES GFP, L-TGF-β (R249A) IRES GFP, WT L-TGF-β IRES GFP/HA GARP or, L-TGF-β (R249A) IRES GFP/HA GARP expressing TMLC cells were added to wells coated as indicated in the legend with each substrate and assayed for luciferase activity after 16–20 hr. Each condition was repeated in triplicate with three independent experiments. *p < 0.05, ** p