RESUMO
Integrin αvß8 binds with exquisite specificity to latent transforming growth factor-ß (L-TGF-ß). This binding is essential for activating L-TGF-ß presented by a variety of cell types. Inhibiting αvß8-mediated TGF-ß activation blocks immunosuppressive regulatory T cell differentiation, which is a potential therapeutic strategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvß8 ectodomain and its intact natural ligand, L-TGF-ß, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvß8 binding specificity and TGF-ß activation. Our studies reveal a mechanism of TGF-ß activation where mature TGF-ß signals within the confines of L-TGF-ß and the release and diffusion of TGF-ß are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvß8-mediated L-TGF-ß activation.
Assuntos
Microscopia Crioeletrônica/métodos , Integrinas/química , Integrinas/metabolismo , Proteínas de Ligação a TGF-beta Latente/química , Proteínas de Ligação a TGF-beta Latente/metabolismo , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta1/metabolismo , Animais , Anticorpos/imunologia , Sítios de Ligação , Brônquios/citologia , Células CHO , Cricetulus , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Integrinas/imunologia , Ativação Linfocitária , Masculino , Vison , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Linfócitos T Reguladores/imunologiaRESUMO
DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity.
Assuntos
Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Nucleossomos/enzimologia , Proteínas de Plantas/metabolismo , Animais , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/ultraestrutura , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Microscopia Eletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/ultraestrutura , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Cryo-EM samples prepared using traditional methods often suffer from too few particles, poor particle distribution, strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.
Assuntos
Microscopia Crioeletrônica/métodos , Grafite/química , Imagem Individual de Molécula/métodos , Água/química , Aminas/química , Polietilenoglicóis/químicaRESUMO
The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.
Assuntos
Antivirais/farmacologia , Citosina Desaminase/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/imunologia , Desaminases APOBEC , Antivirais/química , Proteínas Culina/química , Proteínas Culina/metabolismo , Citidina Desaminase , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Ubiquitina/metabolismo , Ubiquitinação , Montagem de Vírus , Produtos do Gene vif do Vírus da Imunodeficiência Humana/químicaRESUMO
Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. The NO-signaling pathways mediate diverse physiological processes, including vasodilation, neurotransmission, and myocardial functions. sGC is a heterodimer assembled from two homologous subunits, each comprised of four domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length sGC. We used single-particle electron microscopy to obtain the structure of the complete sGC heterodimer and determine its higher-order domain architecture. Overall, the protein is formed of two rigid modules: the catalytic dimer and the clustered Per/Art/Sim and heme-NO/O2-binding domains, connected by a parallel coiled coil at two hinge points. The quaternary assembly demonstrates a very high degree of flexibility. We captured hundreds of individual conformational snapshots of free sGC, NO-bound sGC, and guanosine-5'-[(α,ß)-methylene]triphosphate-bound sGC. The molecular architecture and pronounced flexibility observed provides a significant step forward in understanding the mechanism of NO signaling.
Assuntos
Guanilato Ciclase/química , Guanilato Ciclase/ultraestrutura , Modelos Moleculares , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/ultraestrutura , Animais , Clonagem Molecular , Ativadores de Enzimas/metabolismo , Guanilato Ciclase/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica de Transmissão , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Guanilil Ciclase SolúvelRESUMO
NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoforms--inducible, endothelial, and neuronal NOS--that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.
Assuntos
Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo I/química , Animais , Calmodulina/química , Calmodulina/metabolismo , Cristalização , Dimerização , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Imageamento Tridimensional , Mamíferos , Camundongos , Microscopia Eletrônica de Transmissão , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredutases/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RatosRESUMO
A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7 Å resolution reconstruction of Nudaurelia capensis ω virus and a 4.2 Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome.
Assuntos
Microscopia Crioeletrônica/métodos , Proteínas/química , Cristalografia por Raios X/métodos , Thermoplasma/química , Vírus/químicaRESUMO
Integrin α5ß1 is crucial for cell attachment and migration in development and tissue regeneration, and α5ß1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5ß1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5ß1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.
RESUMO
Single-particle electron cryomicroscopy is undergoing a technical revolution due to the recent developments of direct detectors. These new recording devices detect electrons directly (i.e. without conversion into light) and feature significantly improved detective quantum efficiencies and readout rates as compared to photographic films or CCDs. We evaluated here the potential of one such detector (Gatan K2 Summit) to enable the achievement of near-atomic resolution reconstructions of biological specimens when coupled to a widely used, mid-range transmission electron microscope (FEI TF20 Twin). Compensating for beam-induced motion and stage drift provided a 4.4Å resolution map of Sulfolobus turreted icosahedral virus (STIV), which we used as a test particle in this study. Several motion correction and dose fractionation procedures were explored and we describe their influence on the resolution of the final reconstruction. We also compared the quality of this data to that collected with a FEI Titan Krios microscope equipped with a Falcon I direct detector, which provides a benchmark for data collected using a high-end electron microscope.
Assuntos
Microscopia Crioeletrônica/instrumentação , Microscopia Eletrônica de Transmissão/instrumentação , Rudiviridae/ultraestrutura , Modelos Moleculares , Rudiviridae/química , Sulfolobus/virologia , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/ultraestrutura , Vírion/química , Vírion/ultraestruturaRESUMO
Heterodimeric integrin proteins transmit signals through conformational changes upon ligand binding between their alpha (α) and beta (ß) subunits. Early in chordate evolution, some α subunits acquired an "inserted" (I) domain, which expanded their ligand binding capacity but simultaneously obstructed the ancestral ligand-binding pocket. While this would seemingly impede conventional ligand-mediated integrin activation, it was proposed that the I domain itself could serve both as a ligand replacement and an activation trigger. Here, we provide compelling evidence in support of this longstanding hypothesis using high-resolution cryo-electron microscopy structures of two distinct integrin complexes: the ligand-free and E-cadherin-bound states of the αEß7 integrin with the I domain, as well as the α4ß7 integrin lacking the I domain in both a ligand-free state and bound to MadCAM-1. We trace the evolutionary origin of the I domain to an ancestral collagen-collagen interaction domain. Our analyses illuminate how the I domain intrinsically mimics an extrinsic ligand, enabling integrins to undergo the canonical allosteric cascade of conformational activation and dramatically expanding the range of cellular communication mechanisms in vertebrates.
RESUMO
The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
RESUMO
The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
Assuntos
Integrinas , Fibrose Pulmonar , Animais , Camundongos , Membrana Celular , Microscopia Crioeletrônica , Modelos Animais de DoençasRESUMO
The single-coat protein (CP) of bacteriophage Qß self-assembles into T = 3 icosahedral virus-like particles (VLPs), of interest for a wide range of applications. These VLPs are very stable, but identification of the specific molecular determinants of this stability is lacking. To investigate these determinants along with manipulations that confer more capabilities to our VLP material, we manipulated the CP primary structure to test the importance of various putative stabilizing interactions. Optimization of a procedure to incorporate fused CP subunits allowed for good control over the average number of covalent dimers in each VLP. We confirmed that the disulfide linkages are the most important stabilizing elements for the capsid and that acidic conditions significantly enhance the resistance of VLPs to thermal degradation. Interdimer interactions were found to be less important for VLP assembly than intradimer interactions. Finally, a single point mutation in the CP resulted in a population of smaller VLPs in three distinct structural forms.
Assuntos
Allolevivirus/genética , Substituição de Aminoácidos , Proteínas do Capsídeo/genética , Motivos de Aminoácidos , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/química , Escherichia coli , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Sequências Repetidas Invertidas , Luz , Modelos Moleculares , Mutação Puntual , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Desdobramento de Proteína , RNA Viral/química , RNA Viral/genética , Espalhamento de RadiaçãoRESUMO
The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.
RESUMO
The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.
RESUMO
In cryogenic electron microscopy (cryo-EM) of radiation-sensitive biological samples, both the signal-to-noise ratio (SNR) and the contrast of images are critically important in the image-processing pipeline. Classic methods improve low-frequency image contrast experimentally, by imaging with high defocus, or computationally, by applying various types of low-pass filter. These contrast improvements typically come at the expense of the high-frequency SNR, which is suppressed by high-defocus imaging and removed by low-pass filtration. Recently, convolutional neural networks (CNNs) trained to denoise cryo-EM images have produced impressive gains in image contrast, but it is not clear how these algorithms affect the information content of the image. Here, a denoising CNN for cryo-EM images was implemented and a quantitative evaluation of SNR enhancement, induced bias and the effects of denoising on image processing and three-dimensional reconstructions was performed. The study suggests that besides improving the visual contrast of cryo-EM images, the enhanced SNR of denoised images may be used in other parts of the image-processing pipeline, such as classification and 3D alignment. These results lay the groundwork for the use of denoising CNNs in the cryo-EM image-processing pipeline beyond particle picking.
RESUMO
Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Separase/metabolismo , Motivos de Aminoácidos , Anáfase , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Metáfase , Securina/genética , Securina/metabolismo , Separase/química , Especificidade por Substrato , CoesinasRESUMO
Transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed cation channel associated with a variety of cardiovascular disorders. TRPM4 is activated by increased intracellular calcium in a voltage-dependent manner but, unlike many other TRP channels, is permeable to monovalent cations only. Here we present two structures of full-length human TRPM4 embedded in lipid nanodiscs at ~3-angstrom resolution, as determined by single-particle cryo-electron microscopy. These structures, with and without calcium bound, reveal a general architecture for this major subfamily of TRP channels and a well-defined calcium-binding site within the intracellular side of the S1-S4 domain. The structures correspond to two distinct closed states. Calcium binding induces conformational changes that likely prime the channel for voltage-dependent opening.
Assuntos
Canais de Cátion TRPM/química , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Microscopia Crioeletrônica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos , Modelos Moleculares , Nanoestruturas , Conformação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/ultraestruturaRESUMO
Integrins are conformationally flexible cell surface receptors that survey the extracellular environment for their cognate ligands. Interactions with ligands are thought to be linked to global structural rearrangements involving transitions between bent, extended-closed and extended-open forms. Thus far, structural details are lacking for integrins in the extended conformations due to extensive flexibility between the headpiece and legs in this conformation. Here we present single-particle electron cryomicroscopy structures of human αvß8 integrin in the extended-closed conformation, which has been considered to be a low-affinity intermediate. Our structures show the headpiece rotating about a flexible αv knee, suggesting a ligand surveillance mechanism for integrins in their extended-closed form. Our model predicts that the extended conformation is mainly stabilized by an interface formed between flexible loops in the upper and lower domains of the αv leg. Confirming these findings with the αvß3 integrin suggests that our model of stabilizing the extended-closed conformation is generalizable to other integrins.
Assuntos
Microscopia Crioeletrônica/métodos , Integrinas/metabolismo , Sequência de Aminoácidos , Humanos , Integrinas/química , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
TGF-ß is a promising immunotherapeutic target. It is expressed ubiquitously in a latent form that must be activated to function. Determination of where and how latent TGF-ß (L-TGF-ß) is activated in the tumor microenvironment could facilitate cell- and mechanism-specific approaches to immunotherapeutically target TGF-ß. Binding of L-TGF-ß to integrin αvß8 results in activation of TGF-ß. We engineered and used αvß8 antibodies optimized for blocking or detection, which - respectively - inhibit tumor growth in syngeneic tumor models or sensitively and specifically detect ß8 in human tumors. Inhibition of αvß8 potentiates cytotoxic T cell responses and recruitment of immune cells to tumor centers - effects that are independent of PD-1/PD-L1. ß8 is expressed on the cell surface at high levels by tumor cells, not immune cells, while the reverse is true of L-TGF-ß, suggesting that tumor cell αvß8 serves as a platform for activating cell-surface L-TGF-ß presented by immune cells. Transcriptome analysis of tumor-associated lymphoid cells reveals macrophages as a key cell type responsive to ß8 inhibition with major increases in chemokine and tumor-eliminating genes. High ß8 expression in tumor cells is seen in 20%-80% of various cancers, which rarely coincides with high PD-L1 expression. These data suggest tumor cell αvß8 is a PD-1/PD-L1-independent immunotherapeutic target.