Balancing the Nanoscale Organization in Multivalent Materials for Functional Inhibition of the Programmed Death-1 Immune CheckpointClick to copy article linkArticle link copied!
- Kaltrina PalojaKaltrina PalojaProgrammable Biomaterials Laboratory, Institute of Materials, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne 1015, SwitzerlandMore by Kaltrina Paloja
- Jorieke WeidenJorieke WeidenProgrammable Biomaterials Laboratory, Institute of Materials, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne 1015, SwitzerlandMore by Jorieke Weiden
- Joschka HellmeierJoschka HellmeierMax Planck Institute of Biochemistry, Planegg 82152, GermanyMore by Joschka Hellmeier
- Alexandra S. EklundAlexandra S. EklundMax Planck Institute of Biochemistry, Planegg 82152, GermanyMore by Alexandra S. Eklund
- Susanne C. M. ReinhardtSusanne C. M. ReinhardtMax Planck Institute of Biochemistry, Planegg 82152, GermanyFaculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich 80539, GermanyMore by Susanne C. M. Reinhardt
- Ian A. ParishIan A. ParishPeter MacCallum Cancer Centre, Melbourne, VIC 3000, AustraliaSir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC 3128, AustraliaMore by Ian A. Parish
- Ralf JungmannRalf JungmannMax Planck Institute of Biochemistry, Planegg 82152, GermanyFaculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich 80539, GermanyMore by Ralf Jungmann
- Maartje M. C. Bastings*Maartje M. C. Bastings*Email: [email protected]Programmable Biomaterials Laboratory, Institute of Materials, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne 1015, SwitzerlandInterfaculty Bioengineering Institute, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne 1015, SwitzerlandMore by Maartje M. C. Bastings
Abstract
Dendritic cells (DCs) regulate immune priming by expressing programmed death ligand 1 (PD-L1) and PD-L2, which interact with the inhibitory receptor PD-1 on activated T cells. PD-1 signaling regulates T cell effector functions and limits autoimmunity. Tumor cells can hijack this pathway by overexpressing PD-L1 to suppress antitumor T cell responses. Blocking this inhibitory pathway has been beneficial for the treatment of various cancer types, although only a subset of patients responds. A deepened understanding of the spatial organization and molecular interplay between PD-1 and its ligands may inform the design of more efficacious nanotherapeutics. We visualized the natural molecular PD-L1 organization on DCs by DNA-PAINT microscopy and created a template to engineer DNA-based nanoclusters presenting PD-1 at defined valencies, distances, and patterns. These multivalent nanomaterials were examined for their cellular binding and blocking ability. Our data show that PD-1 nano-organization has profound effects on ligand interaction and that the valency of PD-1 molecules modulates the effectiveness in restoring T cell function. This work highlights the power of spatially controlled functional materials to unravel the importance of multivalent patterns in the PD-1 pathway and presents alternative design strategies for immune-engineering.
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Results and Discussion
PD-L1 Receptors Organize into Nanoclusters on the DC Surface
Figure 1
Figure 1. Super-resolution DNA-PAINT imaging of PD-L1 protein on the DC surface. (a) Schematic outline of the parameters that were studied. Change in PD-L1 molecular arrangement between nonactivated DCs and cells stimulated with CpG and IFNγ for 24 h was investigated using DNA-PAINT imaging. Spatial characteristics, including the distribution of PD-L1 (whether random or clustered), inter-PD-L1 spacing, and clustering order, were determined from super-resolved images. (b) DNA-PAINT images of PD-L1 on DCs before (top) and after (bottom) stimulation. Zoom-in images are presented in the middle and on the right. Each spot represents individual PD-L1 molecules. Scale bars = 5 μm, 500 nm, and 100 nm from left to right. (c,d) NND distribution (c) before and (d) after stimulation. Experimental data are highlighted in orange and CSR simulations in blue. (e) Quantitative analysis of the oligomerization order for clusters defined in nonactivated (white) and stimulated (gray) DCs. Error bars indicate SD and statistical analysis was performed using two-tailed Mann–Whitney test (***p < 0.001). Data were derived from two independent experiments and ≥12 cells.
Preparation of Nanocontrolled PD-1 Displays on DNA Origami Disks
Figure 2
Figure 2. Production of PD-1-DNA conjugates and functionalization on DNA origami disks. (a) Schematic representation of the DNA disk library used to investigate the influence of valency and pattern of PD-1 on the interaction with target ligands. Each black dot represents the position of PD-1 on the DNA disk. (b) Schematic overview of the experimental workflow used to conjugate PD-1 to ssDNA via (1) sortase-mediated reaction and (2) copper-free click chemistry, and (3) hybridize PD-1 to DNA origami disks. Z = poly histidine tag. (c) Schematic representation of the DNA-PAINT experimental setup used for the characterization of 6LH, 6SH, and 6Lin PD-1 DNA disks. PD-1-DNA conjugates were prepared with a ssDNA handle extended with a DNA-PAINT docking sequence (R1). Each PD-1 was visualized by transient hybridization of the imager strand (Cy3B-labeled R1 imager strand) and the docking strand. (d) DNA-PAINT sum images of 6LH, 6SH, and 6Lin PD-1 DNA disks (top). Cross-sectional histogram analysis was performed for the highlighted areas (white boxes) to measure inter-PD-1 distances (bottom). Scale bars: 15 nm.
Binding Behavior of PD-1 DNA Disks on PD-L1 Surfaces
Figure 3
Figure 3. Analysis of PD-1 DNA disk binding behavior on PD-L1 surfaces by SPR. (a) Representative binding curves of DNA disks presenting 0, 1, 3, 6 (in LH, SH, and Lin patterns), or 12 PD-1. Bare DNA disk shows no binding to PD-L1, excluding nonspecific interactions with the ligand. Curves represent increasing particle concentrations (10 nM and 2-fold dilution). (b) Quantification of affinity constants obtained from fitted curves. Error bars indicate SEM (n = 2 independent experiments).
sample | KD [M] | koff [s–1] | KD fold increase | relative KD fold increasea |
---|---|---|---|---|
Monomeric PD-1 | 7.9 ± 0.2 × 10–7 | NDb | – | – |
3 PD-1 Disk | 1.2 ± 0.4 × 10–8 | 1.2 ± 0.1 × 10–3 | 70 | 20 |
6LH PD-1 Disk | 1.4 ± 0.4 × 10–10 | 1.4 ± 0.2 × 10–4 | 5600 | 900 |
6SH PD-1 Disk | 1.6 ± 0.4 × 10–10 | 1.5 ± 0.3 × 10–4 | 4900 | 800 |
6Lin PD-1 Disk | 2.9 ± 0.2 × 10–10 | 1.9 ± 0.9 × 10–4 | 2700 | 500 |
12 PD-1 Disk | 5.6 ± 1.3 × 10–11 | 5.1 ± 1.0 × 10–5 | 14100 | 1200 |
Relative to the PD-1 number on the disk.
Not determined.
Binding Behavior of PD-1 DNA Disks on the DC Surface
Figure 4
Figure 4. Effect of valency and pattern of PD-1 DNA disks on binding to DCs. (a) Representative confocal microscopy images of the surface staining of DCs using (top) αPD-L1 and αPD-L2 antibodies and (bottom) 12 PD-1 DNA disk. Nucleus stained with DAPI in gray, cytoplasm (GFP reporter in CD11c+) in cyan, and PD-L1/PD-L2 in pink. Scale bars: 10 μm. (b) Representative flow cytometric images of DCs stained with 12 PD-1 DNA disk acquired on Amnis ImageStream system. Cytoplasm (GFP reporter in CD11c+) in cyan and PD-L1/PD-L2 in pink. Scale bar: 5 μm. (c) Binding of PD-1 DNA disks with different valencies and patterns to DCs, presented as the geometric mean fluorescence intensity (gMFI) of Cy5 integrated in the DNA disks. The data were normalized by subtracting the gMFI of the 0 DNA disks for each experiment. Error bars indicate SEM (n ≥ 8 independent experiments). Raw data of each group (Figure S10f) were compared to each other using a Kruskal–Wallis test, followed by Dunn’s post-test (*p < 0.05, **p < 0.01, ***p < 0.001).
A High Valency of PD-1 on DNA Disks Restores T Cell Function
Figure 5
Figure 5. Influence of PD-1 valency and pattern on immune checkpoint blockade. (a) Schematic overview of the experimental setup. Primary CD8α+ OT-I T cells were stimulated with αCD3/αCD28 antibodies to induce PD-1 expression. After resting in IL-7, T cells were cocultured with stimulated WT DCs, IL-10 DCs or cancer cells (B16) in the presence of PD-1 DNA disks or control blocking αPD-L1/αPD-L2 antibodies. Cytokine production in the supernatants after 24 h was measured as T cell activation readout. (b) Representative IFNγ production after 24 h of blockade with PD-1 DNA disks. Error bars indicate SEM (n = 2 replicates in one experiment). Replicates can be found in Figure S15. Statistical analysis was performed independently for each cell type. PD-1 DNA disk groups are compared to 0 DNA disk groups with ordinary one-way ANOVA, followed by Dunnett’s test (*p < 0.05, **p < 0.01, ***p < 0.001).
Conclusion
Materials and Methods
Materials
DNA-PAINT Imaging of PD-L1 on DCs
Buffers
Enzymatic Antibody-DNA Conjugation
Sample Preparation for PD-L1 DNA-PAINT Imaging
Microscope Setup
Image Analysis
Data Analysis: PD-L1 Organization
Protein Production and Purification
Mouse PD-1
Sortase A5
PD-1-DNA Site-Specific Conjugation and Purification
DNA Disk Preparation and Purification
PD-1 DNA Disk Preparation and Characterization
DNA-PAINT Characterization of 6LIN, 6SH, and 6Lin PD-1 DNA Disk Constructs
Sample Preparation
Buffers
Microscopy Slide Preparation
Microscope Setup
Image Analysis
Surface Plasmon Resonance Measurements
Cell Culture
Confocal Microscopy Imaging
Slide Preparation
Microscope Setup
Coating of DNA Disks with K10-PEG1K
Antibody-DNA Disk Preparation and Purification
DC Staining Experiments by Flow Cytometry
Mice
T Cell Isolation
Cell Preparation for Immune Checkpoint Blockade Assay
DCs and B16
OT-I T Cells
In Vitro Immune Checkpoint Blockade Assay
Statistical Analysis
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.3c06552.
PD-L1 density on DCs measured by DNA-PAINT; DNA origami designs and characterization by AGE; PD-1-DNA conjugate characterization by PAGE; AGE analysis of PD-1 DNA disks; DNA-PAINT overview images of hexavalent PD-1 DNA disk pattern library and controls; SPR binding sensorgrams of PD-1 and PD-1-DNA conjugate; control DC staining experiments for PD-1 DNA disks and antibody DNA disks; characterization of immune markers on WT DC, IL-10 DC, and B16 by flow cytometry; additional data on cytokine production in immune checkpoint blockade assay; PD-1 and SrtA purification data; K10-PEG1K DNA disk coating characterization by AGE; sequences for DNA origami disk constructs used in this work; imaging parameters used in DNA-PAINT experiments (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
We thank Prof. Bruno E. Correia for scientific discussion, the Protein Production and Structure Core Facility (PTPSP, EPFL, Switzerland) for performing the PD-1 expression in HEK293 cells, the Flow Cytometry Facility (University of Lausanne, Switzerland) for their help with ImageStream flow cytometry, the Flow Cytometry Core Facility (EPFL, Lausanne) and the BioImaging & Optics Core Facility (EPFL, Lausanne) for providing access to their instruments, and the Center of Phenogenomics (CPG, EPFL, Switzerland) for housing the animals. We furthermore thank Christine Lavanchy for technical assistance, and Prof. Hans Acha-Orbea and Dr. Marianna Koga for providing the mutuDCs.
References
This article references 63 other publications.
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- 8Page, D. B.; Postow, M. A.; Callahan, M. K.; Allison, J. P.; Wolchok, J. D. Immune Modulation in Cancer with Antibodies. Annual Review of Medicine 2014, 65 (1), 185– 202, DOI: 10.1146/annurev-med-092012-112807Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXktFeltbo%253D&md5=3f6332a9af46e4f57fc5e6716678b66bImmune modulation in cancer with antibodiesPage, David B.; Postow, Michael A.; Callahan, Margaret K.; Allison, James P.; Wolchok, Jedd D.Annual Review of Medicine (2014), 65 (), 185-202CODEN: ARMCAH; ISSN:0066-4219. (Annual Reviews)A review. Ipilimumab is the prototypical immunomodulatory antibody, approved by the FDA in 2011 for advanced melanoma on the basis of survival benefit. Since that time, we have made significant strides in optimizing this therapy: we have characterized the spectrum of immune-related adverse events and learned how to mitigate them with treatment algorithms, discovered potential biomarkers of activity, and identified the potential synergy between checkpoint modulation and other therapeutic modalities. Recent phase I trials have established the efficacy and safety of next-generation checkpoint agents, including PD-1 and PD-L1 inhibitors, across multiple tumor types. Much work lies ahead in developing these next-generation checkpoint agents, testing them in combination, and detg. how to integrate them into the treatment paradigms of various tumor types.
- 9Acúrcio, R. C.; Pozzi, S.; Carreira, B.; Pojo, M.; Gómez-Cebrián, N.; Casimiro, S.; Fernandes, A.; Barateiro, A.; Farricha, V.; Brito, J.; Leandro, A. P.; Salvador, J. A. R.; Graça, L.; Puchades-Carrasco, L.; Costa, L.; Satchi-Fainaro, R.; Guedes, R. C.; Florindo, H. F. Therapeutic Targeting of PD-1/PD-L1 Blockade by Novel Small-Molecule Inhibitors Recruits Cytotoxic T Cells into Solid Tumor Microenvironment. J. Immunother Cancer 2022, 10 (7), e004695 DOI: 10.1136/jitc-2022-004695Google ScholarThere is no corresponding record for this reference.
- 10Yang, J.; Hu, L. Immunomodulators Targeting the PD-1/PD-L1 Protein-Protein Interaction: From Antibodies to Small Molecules. Medicinal Research Reviews 2019, 39 (1), 265– 301, DOI: 10.1002/med.21530Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisVylsLnI&md5=3b37b2a848554f6b2af82fc802a03168Immunomodulators targeting the PD-1/PD-L1 protein-protein interaction: From antibodies to small moleculesYang, Jeffrey; Hu, LongqinMedicinal Research Reviews (2019), 39 (1), 265-301CODEN: MRREDD; ISSN:0198-6325. (John Wiley & Sons, Inc.)A review. Cancer immunotherapy has made great strides in the recent decade, esp. in the area of immune checkpoint blockade. The outstanding efficacy, prolonged durability of effect, and rapid assimilation of anti-PD-1 and anti-PD-L1 monoclonal antibodies in clin. practice have been nothing short of a medical breakthrough in the treatment of numerous malignancies. The major advantages of these therapeutic antibodies over their small mol. counterparts have been their high binding affinity and target specificity. However, antibodies do have their flaws including immune-related toxicities, inadequate pharmacokinetics and tumor penetration, and high cost burden to manufacturers and consumers. These limitations hinder broader clin. applications of the antibodies and have heightened interests in developing the alternative small mol. platform that includes peptidomimetics and peptides to target the PD-1/PD-L1 immune checkpoint system. The progress on these small mol. alternatives has been relatively slow compared to that of the antibodies. Fortunately, recent structural studies of the interactions among PD-1, PD-L1, and their resp. antibodies have revealed key hotspots on PD-1 and PD-L1 that may facilitate drug discovery efforts for small mol. immunotherapeutics. This review is intended to discuss key concepts in immuno-oncol., describe the successes and shortcomings of PD-1/PD-L1 antibody-based therapies, and to highlight the recent development of small mol. inhibitors of the PD-1/PD-L1 protein-protein interaction.
- 11Guo, L.; Wei, R.; Lin, Y.; Kwok, H. F. Clinical and Recent Patents Applications of PD-1/PD-L1 Targeting Immunotherapy in Cancer Treatment─Current Progress, Strategy, and Future Perspective. Front. Immunol. 2020, DOI: 10.3389/fimmu.2020.01508Google ScholarThere is no corresponding record for this reference.
- 12Day, D.; Hansen, A. R. Immune-Related Adverse Events Associated with Immune Checkpoint Inhibitors. BioDrugs 2016, 30 (6), 571– 584, DOI: 10.1007/s40259-016-0204-3Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvV2gtL3F&md5=a776b5026dcbd50a50708664b7132f92Immune-Related Adverse Events Associated with Immune Checkpoint InhibitorsDay, Daphne; Hansen, Aaron R.BioDrugs (2016), 30 (6), 571-584CODEN: BIDRF4; ISSN:1173-8804. (Springer International Publishing AG)Immune checkpoint inhibitors (ICIs), including antibodies targeting cytotoxic T-lymphocyte-assocd. antigen 4 (CTLA-4) and programmed cell death protein-1 (PD-1), have shown durable treatment responses in multiple tumor types by enhancing antitumor immunity. However, removal of self-tolerance can induce autoimmunity and produce a unique immune-driven toxicity profile, termed immune-related adverse events (irAEs). As ICIs gain approval for a growing no. of indications, it is imperative clinicians increase their knowledge of and ability to manage irAEs. This review examines the etiol., presentation, kinetics, and treatment of irAEs and aims to provide practical guidance for clinicians.
- 13Xu-Monette, Z. Y.; Zhang, M.; Li, J.; Young, K. H. PD-1/PD-L1 Blockade: Have We Found the Key to Unleash the Antitumor Immune Response?. Front. Immunol. 2017, DOI: 10.3389/fimmu.2017.01597Google ScholarThere is no corresponding record for this reference.
- 14Wang, Z.; Wu, X. Study and Analysis of Antitumor Resistance Mechanism of PD1/PD-L1 Immune Checkpoint Blocker. Cancer Medicine 2020, 9 (21), 8086– 8121, DOI: 10.1002/cam4.3410Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlWhtLzM&md5=8c4ea127feb88c43818dd33ddd677cb4Study and analysis of antitumor resistance mechanism of PD1/PD-L1 immune checkpoint blockerWang, Zhengyi; Wu, XiaoyingCancer Medicine (2020), 9 (21), 8086-8121CODEN: CMAEDL; ISSN:2045-7634. (John Wiley & Sons Ltd.)Immunocheckpoint proteins of tumor infiltrating lymphocytes play an important role in tumor prognosis in the course of tumor clinicopathol. PD-1 (Programed cell death protein 1) is an important immunosuppressive mol. By binding to PD-L1 (programed cell death-ligand 1), it blocks TCR and its costimulus signal transduction, inhibits the activation and proliferation of T cells, depletes the function of effector T cells, and enables tumor cells to achieve immune escape. In recent years, immunocheckpoint blocking therapy targeting the PD-1/PD-L1 axis has achieved good results in a variety of malignant tumors, pushing tumor immunotherapy to a new milestone, such as anti-PD-1 monoclonal antibody Nivolumab, Pembrolizumab, and anti-PD-L1 monoclonal antibody Atezolizumab, which are considered as potential antitumor drugs. It was found in clin. use that some patients obtained long-term efficacy, but most of them developed drug resistance recurrence in the later stage. The high incidence of drug resistance (including primary and acquired drug resistance) still cannot be ignored, which limited its clin. application and became a new problem in this field. Due to tumor heterogeneity, current limited research shows that PD-1 or PD-L1 monoclonal antibody drug resistance may be related to the following factors: mutation of tumor antigen and antigen presentation process, multiple immune checkpoint interactions, immune microenvironment changes dynamically, activation of oncogenic pathways, gene mutation and epigenetic changes of key proteins in tumors, tumor competitive metab., and accumulation of metabolites, etc, mechanisms of resistance are complex. Therefore, it is the most urgent task to further elucidate the mechanism of immune checkpoint inhibitor resistance, discover multitumor universal biomarkers, and develop new target agents to improve the response rate of immunotherapy in patients. In this study, the mechanism of anti-PD-1/PD-L1 drug resistance in tumors, the potential biomarkers for predicting PD-1 acquired resistance, and the recent development of combination therapy were reviewed one by one. It is believed that, based on the complex mechanism of drug resistance, it is of no clin. significance to simply search for and regulate drug resistance targets, and it may even produce drug resistance again soon. It is speculated that according to the possible tumor characteristics, three types of treatment methods should be combined to change the tumor microenvironment ecol. and eliminate various heterogeneous tumor subsets, so as to reduce tumor drug resistance and improve long-term clin. efficacy.
- 15Sun, J.-Y.; Zhang, D.; Wu, S.; Xu, M.; Zhou, X.; Lu, X.-J.; Ji, J. Resistance to PD-1/PD-L1 Blockade Cancer Immunotherapy: Mechanisms, Predictive Factors, and Future Perspectives. Biomark Res. 2020, 8, 35, DOI: 10.1186/s40364-020-00212-5Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38bitlSmsw%253D%253D&md5=4812ff408d7aa9ce2e9503ddc8658cd8Resistance to PD-1/PD-L1 blockade cancer immunotherapy: mechanisms, predictive factors, and future perspectivesSun Jin-Yu; Zhang Dengke; Wu Songquan; Xu Min; Ji Jiansong; Zhang Dengke; Wu Songquan; Xu Min; Ji Jiansong; Zhou Xiao; Lu Xiao-Jie; Ji JiansongBiomarker research (2020), 8 (), 35 ISSN:2050-7771.PD-1/PD-L1 blockade therapy is a promising cancer treatment strategy, which has revolutionized the treatment landscape of malignancies. Over the last decade, PD-1/PD-L1 blockade therapy has been trialed in a broad range of malignancies and achieved clinical success. Despite the potentially cure-like survival benefit, only a minority of patients are estimated to experience a positive response to PD-1/PD-L1 blockade therapy, and the primary or acquired resistance might eventually lead to cancer progression in patients with clinical responses. Accordingly, the resistance to PD-1/PD-L1 blockade remains a significant challenge hindering its further application. To overcome the limitation in therapy resistance, substantial effort has been made to improve or develop novel anti-PD-1/PD-L1 based immunotherapy strategies with better clinical response and reduced immune-mediated toxicity. In this review, we provide an overview on the resistance to PD-1/PD-L1 blockade and briefly introduce the mechanisms underlying therapy resistance. Moreover, we summarize potential predictive factors for the resistance to PD-1/PD-L1 blockade. Furthermore, we give an insight into the possible solutions to improve efficacy and clinical response. In the following research, combined efforts of basic researchers and clinicians are required to address the limitation of therapy resistance.
- 16Dustin, M. L.; Tseng, S.-Y.; Varma, R.; Campi, G. T Cell–Dendritic Cell Immunological Synapses. Current Opinion in Immunology 2006, 18 (4), 512– 516, DOI: 10.1016/j.coi.2006.05.017Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmsFSks70%253D&md5=24a4098e5f4538882c3b25c078253cf9T cell-dendritic cell immunological synapsesDustin, Michael L.; Tseng, Su-Yi; Varma, Rajat; Campi, GabrieleCurrent Opinion in Immunology (2006), 18 (4), 512-516CODEN: COPIEL; ISSN:0952-7915. (Elsevier Ltd.)A review. Dendritic cells (DCs) are myeloid lineage cells that are imprinted by their environment and that mature in response to microbial products. A crucial role of the DC is to impart this context-specific information to T cells as well as to present self and foreign MHC-peptide complexes through formation of an immunol. synapse. The structure of the T cell-DC immunol. synapse departs from the canonical structure formed with B cells or with supported planar bilayers in that it has multiple foci of T-cell receptor interactions rather than a central focus. Recent studies on model systems provide insight into the mechanisms and biol. consequences of the unique T cell-DC synaptic patterns.
- 17Dustin, M. L.; Chakraborty, A. K.; Shaw, A. S. Understanding the Structure and Function of the Immunological Synapse. Cold Spring Harb Perspect Biol. 2010, 2 (10), a002311 DOI: 10.1101/cshperspect.a002311Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWgtr3M&md5=890c57aa9978a99a5aee45b4f886f9a7Understanding the structure and function of the immunological synapseDustin, Michael L.; Chakraborty, Arup K.; Shaw, Andrey S.Cold Spring Harbor Perspectives in Biology (2010), 2 (10), a002311CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)A review. The immunol. synapse has been an area of very active scientific interest over the last decade. Surprisingly, much about the synapse remains unknown or is controversial. Here we review some of these current issues in the field: how the synapse is defined, its potential role in T-cell function, and our current understanding about how the synapse is formed.
- 18Sallusto, F.; Lanzavecchia, A. The Instructive Role of Dendritic Cells on T-Cell Responses. Arthritis Res. 2002, 4 (Suppl 3), S127– S132, DOI: 10.1186/ar567Google ScholarThere is no corresponding record for this reference.
- 19Doh, J.; Irvine, D. J. Immunological Synapse Arrays: Patterned Protein Surfaces That Modulate Immunological Synapse Structure Formation in T Cells. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (15), 5700– 5705, DOI: 10.1073/pnas.0509404103Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XktFaisLg%253D&md5=a55746389e7d38f219fdac35d9ce2b0cImmunological synapse arrays: patterned protein surfaces that modulate immunological synapse structure formation in T cellsDoh, Junsang; Irvine, Darrell J.Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (15), 5700-5705CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)T cells are activated by recognition of foreign peptides displayed on the surface of antigen presenting cells (APCs), an event that triggers assembly of a complex microscale structure at the T cell-APC interface known as the immunol. synapse (IS). It remains unresolved whether the unique phys. structure of the synapse itself impacts the functional response of T cells, independent of the quantity and quality of ligands encountered by the T cell. As a first step toward addressing this question, we created multicomponent protein surfaces presenting lithog. defined patterns of tethered T cell receptor (TCR) ligands (anti-CD3 "activation sites") surrounded by a field of tethered intercellular adhesion mol.-1 (ICAM-1), as a model substrate on which T cells could be seeded to mimic T cell-APC interactions. CD4+ T cells seeded on these surfaces polarized and migrated; on contact with activation sites, T cells assembled an IS with a structure modulated by the phys. pattern of ligand encountered. On surfaces patterned with focal spots of TCR ligand, T cells stably interacted with activation sites, proliferated, and secreted cytokines. In contrast, T cells interacting with activation sites patterned to preclude centralized clustering of TCR ligand failed to form stable contacts with activation sites, exhibited aberrant PKC-θ clustering in a fraction of cells, and had significantly reduced prodn. of IFN-γ. These results suggest that focal clustering of TCR ligand characteristic of the "mature" IS may be required under some conditions for full T cell activation.
- 20Oh, S. A.; Wu, D.-C.; Cheung, J.; Navarro, A.; Xiong, H.; Cubas, R.; Totpal, K.; Chiu, H.; Wu, Y.; Comps-Agrar, L.; Leader, A. M.; Merad, M.; Roose-Germa, M.; Warming, S.; Yan, M.; Kim, J. M.; Rutz, S.; Mellman, I. PD-L1 Expression by Dendritic Cells Is a Key Regulator of T-Cell Immunity in Cancer. Nat. Cancer 2020, 1 (7), 681– 691, DOI: 10.1038/s43018-020-0075-xGoogle Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjsFegsb8%253D&md5=ea68fa5a9028234cb36d9f1e8bc68a1dPD-L1 expression by dendritic cells is a key regulator of T-cell immunity in cancerOh, Soyoung A.; Wu, Dai-Chen; Cheung, Jeanne; Navarro, Armando; Xiong, Huizhong; Cubas, Rafael; Totpal, Klara; Chiu, Henry; Wu, Yan; Comps-Agrar, Laetitia; Leader, Andrew M.; Merad, Miriam; Roose-Germa, Merone; Warming, Soren; Yan, Minhong; Kim, Jeong M.; Rutz, Sascha; Mellman, IraNature Cancer (2020), 1 (7), 681-691CODEN: NCAADQ; ISSN:2662-1347. (Springer International Publishing AG)Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a crit. source of PD-L1, despite being vastly outnumbered by PD-L1+ macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8+ T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1+ tumor cells.
- 21Yokosuka, T.; Takamatsu, M.; Kobayashi-Imanishi, W.; Hashimoto-Tane, A.; Azuma, M.; Saito, T. Programmed Cell Death 1 Forms Negative Costimulatory Microclusters That Directly Inhibit T Cell Receptor Signaling by Recruiting Phosphatase SHP2. J. Exp Med. 2012, 209 (6), 1201– 1217, DOI: 10.1084/jem.20112741Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XoslShsr4%253D&md5=5c1fad62b4e3e69e177be5718ce92d41Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2Yokosuka, Tadashi; Takamatsu, Masako; Kobayashi-Imanishi, Wakana; Hashimoto-Tane, Akiko; Azuma, Miyuki; Saito, TakashiJournal of Experimental Medicine (2012), 209 (6), 1201-1217CODEN: JEMEAV; ISSN:0022-1007. (Rockefeller University Press)Programmed cell death 1 (PD-1) is a neg. costimulatory receptor crit. for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a mol. mechanism of PD-1-mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently assocd. with the phosphatase SHP2 (Src homol. 2 domain-contg. tyrosine phosphatase 2). These neg. costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling mols. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addn. to PD-1 clustering, PD-1-TCR colocalization within microclusters is required for efficient PD-1-mediated suppression. This inhibitory mechanism also functions in PD-1hi T cells generated in vivo and can be overridden by a neutralizing anti-PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.
- 22Pentcheva-Hoang, T.; Chen, L.; Pardoll, D. M.; Allison, J. P. Programmed Death-1 Concentration at the Immunological Synapse Is Determined by Ligand Affinity and Availability. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (45), 17765– 17770, DOI: 10.1073/pnas.0708767104Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht12ltr3L&md5=02b184fa0f86ae1b680e84df45271c42Programmed death-1 concentration at the immunological synapse is determined by ligand affinity and availabilityPentcheva-Hoang, Tsvetelina; Chen, Lieping; Pardoll, Drew M.; Allison, James P.Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (45), 17765-17770CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Despite the importance of programmed death-1 (PD-1) for T cell inhibition, little is known about its intracellular trafficking or requirements for localization to the immunol. synapse. Here, we show that in activated T cells, PD-1 is present at the plasma membrane, near the Golgi and in the trans-Golgi network. Unlike CD28 and CTLA-4, PD-1 accumulation at the synapse is extensive only when T cells interact with dendritic cells (DCs) expressing high B7-DC levels. However, B7-H1 is also critically important, esp. when the DCs have little B7-DC. Despite this preference, B7-H1-/- DCs elicit greater cytokine secretion than B7-DC-/- DCs during T cell restimulation, possibly because they also express less B7-DC. PD-1 and CD28 have similar kinetics of synaptic accumulation, suggesting that the process involves T cell receptor-triggered cytoskeletal reorganization followed by ligand binding.
- 23Fang, T.; Alvelid, J.; Spratt, J.; Ambrosetti, E.; Testa, I.; Teixeira, A. I. Spatial Regulation of T-Cell Signaling by Programmed Death-Ligand 1 on Wireframe DNA Origami Flat Sheets. ACS Nano 2021, 15 (2), 3441– 3452, DOI: 10.1021/acsnano.0c10632Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjt1Khsrw%253D&md5=43b13eb11ccbfae436ccae1612284a58Spatial Regulation of T-Cell Signaling by Programmed Death-Ligand 1 on Wireframe DNA Origami Flat SheetsFang, Trixy; Alvelid, Jonatan; Spratt, Joel; Ambrosetti, Elena; Testa, Ilaria; Teixeira, Ana I.ACS Nano (2021), 15 (2), 3441-3452CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Programmed Death-1 (PD-1) is a coinhibitory receptor expressed on activated T cells that suppresses T-cell signaling and effector functions. It has been previously shown that binding to its ligand PD-L1 induces a spatial reorganization of PD-1 receptors into microclusters on the cell membrane. However, the roles of the spatial organization of PD-L1 on PD-1 clustering and T-cell signaling have not been elucidated. Here, we used DNA origami flat sheets to display PD-L1 ligands at defined nanoscale distances and investigated their ability to inhibit T-cell activation in vitro. We found that DNA origami flat sheets modified with CD3 and CD28 activating antibodies (FS-α-CD3-CD28) induced robust T-cell activation. Co-treatment with flat sheets presenting PD-L1 ligands sepd. by ∼200 nm (FS-PD-L1-200), but not 13 nm (FS-PD-L1-13) or 40 nm (FS-PD-L1-40), caused an inhibition of T-cell signaling, which increased with increasing molar ratio of FS-PD-L1-200 to FS-α-CD3-CD28. Furthermore, FS-PD-L1-200 induced the formation of smaller PD-1 nanoclusters and caused a larger redn. in IL-2 expression compared to FS-PD-L1-13. Together, these findings suggest that the spatial organization of PD-L1 dets. its ability to regulate T-cell signaling and may guide the development of future nano medicine-based immunomodulatory therapies.
- 24Ruglioni, M.; Civita, S.; Salvadori, T.; Cristiani, S.; Carnicelli, V.; Barachini, S.; Petrini, I.; Diaspro, A.; Bianchini, P.; Storti, B.; Bizzarri, R.; Fogli, S.; Danesi, R. Nanoscale Engagement of Immune Checkpoint Inhibitor PD-L1 in Membrane Lipid Rafts. bioRxiv , August 11, 2022. DOI: 10.1101/2022.08.09.503318 .Google ScholarThere is no corresponding record for this reference.
- 25Zhou, L.; Chai, F.; He, Y.; Zhou, Z.; Guo, S.; Li, P.; Sun, Q.; Zu, X.; Liu, X.; Huang, Q.; Zhong, Y.; Zhou, A.; Wang, X.; Ren, H. Homodimerized Cytoplasmic Domain of PD-L1 Regulates Its Complex Glycosylation in Living Cells. Commun. Biol. 2022, 5 (1), 1– 12, DOI: 10.1038/s42003-022-03845-4Google ScholarThere is no corresponding record for this reference.
- 26Rothemund, P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440 (7082), 297– 302, DOI: 10.1038/nature04586Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XitlKgu7g%253D&md5=583caefdda9b1deb5d3f2ef78d9e6ecbFolding DNA to create nanoscale shapes and patternsRothemund, Paul W. K.Nature (London, United Kingdom) (2006), 440 (7082), 297-302CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)'Bottom-up fabrication', which exploits the intrinsic properties of atoms and mols. to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA mols. provides an attractive route towards this goal. Here the author describe a simple method for folding long, single-stranded DNA mols. into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diam. and approx. desired shapes such as squares, disks and five-pointed stars with a spatial resoln. of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton mol. complex).
- 27Seeman, N. C.; Sleiman, H. F. DNA Nanotechnology. Nat. Rev. Mater. 2018, 3 (1), 17068 DOI: 10.1038/natrevmats.2017.68Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslOntLbM&md5=a8683cd5a65e013464f37bb6383853b4DNA nanotechnologySeeman, Nadrian C.; Sleiman, Hanadi F.Nature Reviews Materials (2018), 3 (1), 17068CODEN: NRMADL; ISSN:2058-8437. (Nature Research)DNA is the mol. that stores and transmits genetic information in biol. systems. The field of DNA nanotechnol. takes this mol. out of its biol. context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnol., and has been revolutionary in our ability to control mol. self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomol. structure detn., drug delivery and synthetic biol. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.
- 28Zhan, P.; Peil, A.; Jiang, Q.; Wang, D.; Mousavi, S.; Xiong, Q.; Shen, Q.; Shang, Y.; Ding, B.; Lin, C.; Ke, Y.; Liu, N. Recent Advances in DNA Origami-Engineered Nanomaterials and Applications. Chem. Rev. 2023, 123 (7), 3976– 4050, DOI: 10.1021/acs.chemrev.3c00028Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXmt1Gltrw%253D&md5=42555f8f95f32e7fc6240b77d51fa063Recent Advances in DNA Origami-Engineered Nanomaterials and ApplicationsZhan, Pengfei; Peil, Andreas; Jiang, Qiao; Wang, Dongfang; Mousavi, Shikufa; Xiong, Qiancheng; Shen, Qi; Shang, Yingxu; Ding, Baoquan; Lin, Chenxiang; Ke, Yonggang; Liu, NaChemical Reviews (Washington, DC, United States) (2023), 123 (7), 3976-4050CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. DNA nanotechnol. is a unique field, where physics, chem., biol., mathematics, engineering, and materials science can elegantly converge. Since the original proposal of Nadrian Seeman, significant advances have been achieved in the past four decades. During this glory time, the DNA origami technique developed by Paul Rothemund further pushed the field forward with a vigorous momentum, fostering a plethora of concepts, models, methodologies, and applications that were not thought of before. This review focuses on the recent progress in DNA origami-engineered nanomaterials in the past five years, outlining the exciting achievements as well as the unexplored research avenues. We believe that the spirit and assets that Seeman left for scientists will continue to bring interdisciplinary innovations and useful applications to this field in the next decade.
- 29Comberlato, A.; Koga, M. M.; Nüssing, S.; Parish, I. A.; Bastings, M. M. C. Spatially Controlled Activation of Toll-like Receptor 9 with DNA-Based Nanomaterials. Nano Lett. 2022, 22 (6), 2506– 2513, DOI: 10.1021/acs.nanolett.2c00275Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XmsFGitL8%253D&md5=b034d8a7e87d43f14b89a6c3e0e6d8beSpatially Controlled Activation of Toll-like Receptor 9 with DNA-Based NanomaterialsComberlato, Alice; Koga, Marianna M.; Nussing, Simone; Parish, Ian A.; Bastings, Maartje M. C.Nano Letters (2022), 22 (6), 2506-2513CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)First evidence of geometrical patterns and defined distances of biomols. as fundamental parameters to regulate receptor binding and cell signaling have emerged recently. Here, we demonstrate the importance of controlled nanospacing of immunostimulatory agents for the activation of immune cells by exploiting DNA-based nanomaterials and pre-existing crystallog. data. We created DNA origami nanoparticles that present CpG-motifs in rationally designed spatial patterns to activate Toll-like Receptor 9 in RAW 264.7 macrophages. We demonstrated that stronger immune activation is achieved when active mols. are positioned at the distance of 7 nm, matching the active dimer structure of the receptor. Moreover, we show how the introduction of linkers between particle and ligand can influence the spatial tolerance of binding. These findings are fundamental for a fine-tuned manipulation of the immune system, considering the importance of spatially controlled presentation of therapeutics to increase efficacy and specificity of immune-modulating nanomaterials where multivalent binding is involved.
- 30Kurisinkal, E. E.; Caroprese, V.; Koga, M. M.; Morzy, D.; Bastings, M. M. C. Selective Integrin A5β1 Targeting through Spatially Constrained Multivalent DNA-Based Nanoparticles. Molecules 2022, 27 (15), 4968, DOI: 10.3390/molecules27154968Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitFGms77J&md5=7f7b3b2e2176adba59b0642ceb39bd3aSelective Integrin α5β1 Targeting through Spatially Constrained Multivalent DNA-Based NanoparticlesKurisinkal, Eva E.; Caroprese, Vincenzo; Koga, Marianna M.; Morzy, Diana; Bastings, Maartje M. C.Molecules (2022), 27 (15), 4968CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor d. across healthy and diseased cells. Engaging receptors above a threshold concn. would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α5β1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding obsd. across HUVEC, CHO and HeLa in resting vs. activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ∼7 nm and ∼24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was obsd., indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technol. imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.
- 31Hellmeier, J.; Platzer, R.; Eklund, A. S.; Schlichthaerle, T.; Karner, A.; Motsch, V.; Schneider, M. C.; Kurz, E.; Bamieh, V.; Brameshuber, M.; Preiner, J.; Jungmann, R.; Stockinger, H.; Schütz, G. J.; Huppa, J. B.; Sevcsik, E. DNA Origami Demonstrate the Unique Stimulatory Power of Single PMHCs as T Cell Antigens. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (4), e2016857118 DOI: 10.1073/pnas.2016857118Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFait7Y%253D&md5=20d8b5c4b8380028ad0e7a3690330a12DNA origami demonstrate the unique stimulatory power of single pMHCs as T cell antigensHellmeier, Joschka; Platzer, Rene; Eklund, Alexandra S.; Schlichthaerle, Thomas; Karner, Andreas; Motsch, Viktoria; Schneider, Magdalena C.; Kurz, Elke; Bamieh, Victor; Brameshuber, Mario; Preiner, Johannes; Jungmann, Ralf; Stockinger, Hannes; Schuetz, Gerhard J.; Huppa, Johannes B.; Sevcsik, EvaProceedings of the National Academy of Sciences of the United States of America (2021), 118 (4), e2016857118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a crit. factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the min. signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC mols.
- 32Shaw, A.; Lundin, V.; Petrova, E.; Fördős, F.; Benson, E.; Al-Amin, A.; Herland, A.; Blokzijl, A.; Högberg, B.; Teixeira, A. I. Spatial Control of Membrane Receptor Function Using Ligand Nanocalipers. Nat. Methods 2014, 11 (8), 841– 846, DOI: 10.1038/nmeth.3025Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFSlsLzO&md5=75998e2c18b132b9b86cc224c4b24ccdSpatial control of membrane receptor function using ligand nanocalipersShaw, Alan; Lundin, Vanessa; Petrova, Ekaterina; Foerdos, Ferenc; Benson, Erik; Al-Amin, Abdullah; Herland, Anna; Blokzijl, Andries; Hoegberg, Bjoern; Teixeira, Ana I.Nature Methods (2014), 11 (8), 841-846CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor-mediated signaling.
- 33Veneziano, R.; Moyer, T. J.; Stone, M. B.; Wamhoff, E.-C.; Read, B. J.; Mukherjee, S.; Shepherd, T. R.; Das, J.; Schief, W. R.; Irvine, D. J.; Bathe, M. Role of Nanoscale Antigen Organization on B-Cell Activation Probed Using DNA Origami. Nat. Nanotechnol. 2020, 15 (8), 716– 723, DOI: 10.1038/s41565-020-0719-0Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1Ontr3M&md5=94f9c3ffdf3b617e1320510ce5fcf86cRole of nanoscale antigen organization on B-cell activation probed using DNA origamiVeneziano, Remi; Moyer, Tyson J.; Stone, Matthew B.; Wamhoff, Eike-Christian; Read, Benjamin J.; Mukherjee, Sayak; Shepherd, Tyson R.; Das, Jayajit; Schief, William R.; Irvine, Darrell J.; Bathe, MarkNature Nanotechnology (2020), 15 (8), 716-723CODEN: NNAABX; ISSN:1748-3387. (Nature Research)Abstr.: Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However, the effects of antigen copy no., spacing and affinity, as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here, we display the clin. vaccine immunogen eOD-GT8, an engineered outer domain of the HIV-1 glycoprotein-120, on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to ∼25-30 nm monotonically increases B-cell receptor activation. Moreover, scaffold rigidity is essential for robust B-cell triggering. These results reveal mol. vaccine design principles that may be used to drive functional B-cell responses.
- 34Berger, R. M. L.; Weck, J. M.; Kempe, S. M.; Hill, O.; Liedl, T.; Rädler, J. O.; Monzel, C.; Heuer-Jungemann, A. Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells. Small 2021, 17 (26), 2101678 DOI: 10.1002/smll.202101678Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVKmtr3J&md5=b2b54e5a9858d6e345b6aab81cd0356dNanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in CellsBerger, Ricarda M. L.; Weck, Johann M.; Kempe, Simon M.; Hill, Oliver; Liedl, Tim; Radler, Joachim O.; Monzel, Cornelia; Heuer-Jungemann, AmelieSmall (2021), 17 (26), 2101678CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their mol. organization and activation are hitherto lacking. Among the well-known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami-based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time-to-death kinetics for hexagonal FasL arrangements with 10 nm inter-mol. spacing. Compared to naturally occurring sol. FasL, this trigger is faster and 100x more efficient. Nanoagents with different spacing, lower FasL no. or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over mol. no. and geometry. They define mol. benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses.
- 35Bila, H.; Kurisinkal, E. E.; Bastings, M. M. C. Engineering a Stable Future for DNA-Origami as a Biomaterial. Biomaterials Science 2019, 7, 532, DOI: 10.1039/C8BM01249KGoogle Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlClsLvF&md5=26d82aa33cd092a354c1e200503a8c53Engineering a stable future for DNA-origami as a biomaterialBila, Hale; Kurisinkal, Eva E.; Bastings, Maartje M. C.Biomaterials Science (2019), 7 (2), 532-541CODEN: BSICCH; ISSN:2047-4849. (Royal Society of Chemistry)DNA as a biomaterial has evoked great interest as a potential platform for therapeutics and diagnostics and as hydrogel scaffolds due to the relative ease of programming its robust and uniform shape, site-specific functionality and controlled responsive behavior. However, for a stable self-assembled product, a relatively high cation concn. is required to prevent denaturation. Physiol. and cell-culture conditions do not match these concns. and present addnl. nucleases that cause a serious threat to the integrity of DNA-based materials. For the translation of this promising technol. towards bioengineering challenges, stability needs to be guaranteed. Over the past years, various methods have been developed addressing the stability-related weaknesses of DNA-origami. This mini-review explains the common stability issues and compares the stabilization strategies recently developed. We present a detailed overview of each method in order to ease the selection process on which method to use for future users of DNA-origami as a biomaterial.
- 36Koga, M. M.; Comberlato, A.; Rodríguez-Franco, H. J.; Bastings, M. M. C. Strategic Insights into Engineering Parameters Affecting Cell Type-Specific Uptake of DNA-Based Nanomaterials. Biomacromolecules 2022, 23 (6), 2586– 2594, DOI: 10.1021/acs.biomac.2c00282Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xhtl2nu7nI&md5=c022ca022821738c0bf0307a9be52b20Strategic Insights into Engineering Parameters Affecting Cell Type-Specific Uptake of DNA-Based NanomaterialsKoga, Marianna M.; Comberlato, Alice; Rodriguez-Franco, Hugo J.; Bastings, Maartje M. C.Biomacromolecules (2022), 23 (6), 2586-2594CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)DNA-based nanomaterials are gaining popularity as uniform and programmable bioengineering tools as a result of recent solns. to their weak stability under biol. conditions. The DNA nanotechnol. platform uniquely allows decoupling of engineering parameters to comprehensively study the effect of each upon cellular encounter. We here present a systematic anal. of the effect of surface parameters of DNA-based nanoparticles on uptake in three different cell models: tumor cells, macrophages, and dendritic cells. The influence of surface charge, stabilizing coating, fluorophore types, functionalization technique, and particle concn. employed is found to cause significant differences in material uptake among these cell types. We therefore provide new insights into the large variance in cell type-specific uptake, highlighting the necessity of proper engineering and careful assay development when DNA-based materials are used as tools in bioengineering and as future nanotherapeutic agents.
- 37Rodríguez-Franco, H. J.; Weiden, J.; Bastings, M. M. C. Stabilizing Polymer Coatings Alter the Protein Corona of DNA Origami and Can Be Engineered to Bias the Cellular Uptake. ACS Polym. Au 2023, 3, 344 DOI: 10.1021/acspolymersau.3c00009Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhtFGmsb3P&md5=70096fef16a47ea6cf30569c2d6892c6Stabilizing Polymer Coatings Alters the Protein Corona of DNA Origami and Can Be Engineered to Bias the Cellular UptakeRodriguez-Franco, Hugo J.; Weiden, Jorieke; Bastings, Maartje M. C.ACS Polymers Au (2023), 3 (4), 344-353CODEN: APACCD; ISSN:2694-2453. (American Chemical Society)With DNA-based nanomaterials being designed for applications in cellular environments, the need arises to accurately understand their surface interactions toward biol. targets. As for any material exposed to protein-rich cell culture conditions, a protein corona will establish around DNA nanoparticles, potentially altering the a-priori designed particle function. Here, we first set out to identify the protein corona around DNA origami nanomaterials, taking into account the application of stabilizing block co-polymer coatings (oligolysine-1kPEG or oligolysine-5kPEG) widely used to ensure particle integrity. By implementing a label-free methodol., the distinct polymer coating conditions show unique protein profiles, predominantly defined by differences in the mol. wt. and isoelec. point of the adsorbed proteins. Interestingly, none of the applied coatings reduced the diversity of the proteins detected within the specific coronae. We then biased the protein corona through pre-incubation with selected proteins and show significant changes in the cell uptake. Our study contributes to a deeper understanding of the complex interplay between DNA nanomaterials, proteins, and cells at the bio-interface.
- 38Peng, Q.; Qiu, X.; Zhang, Z.; Zhang, S.; Zhang, Y.; Liang, Y.; Guo, J.; Peng, H.; Chen, M.; Fu, Y.-X.; Tang, H. PD-L1 on Dendritic Cells Attenuates T Cell Activation and Regulates Response to Immune Checkpoint Blockade. Nat. Commun. 2020, 11 (1), 4835, DOI: 10.1038/s41467-020-18570-xGoogle Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFOhsLvJ&md5=34a8cf2f133b76392b86fc9ceb781b69PD-L1 on dendritic cells attenuates T cell activation and regulates response to immune checkpoint blockadePeng, Qi; Qiu, Xiangyan; Zhang, Zihan; Zhang, Silin; Zhang, Yuanyuan; Liang, Yong; Guo, Jingya; Peng, Hua; Chen, Mingyi; Fu, Yang-Xin; Tang, HaidongNature Communications (2020), 11 (1), 4835CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Immune checkpoint blockade therapies have shown clin. promise in a variety of cancers, but how tumor-infiltrating T cells are activated remains unclear. In this study, we explore the functions of PD-L1 on dendritic cells (DCs), which highly express PD-L1. We observe that PD-L1 on DC plays a crit. role in limiting T cell responses. Type 1 conventional DCs are essential for PD-L1 blockade and they upregulate PD-L1 upon antigen uptake. Upregulation of PD-L1 on DC is mediated by type II interferon. While DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, subsequent PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Blocking PD-L1 in established tumors promotes re-activation of tumor-infiltrating T cells for tumor control. Our study identifies a crit. and dynamic role of PD-L1 on DC, which needs to be harnessed for better invigoration of antitumor immune responses.
- 39Jungmann, R.; Steinhauer, C.; Scheible, M.; Kuzyk, A.; Tinnefeld, P.; Simmel, F. C. Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami. Nano Lett. 2010, 10 (11), 4756– 4761, DOI: 10.1021/nl103427wGoogle Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWiu7rK&md5=e93210e6c0d8347498b003e120ca8248Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA OrigamiJungmann, Ralf; Steinhauer, Christian; Scheible, Max; Kuzyk, Anton; Tinnefeld, Philip; Simmel, Friedrich C.Nano Letters (2010), 10 (11), 4756-4761CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)DNA origami is a powerful method for the programmable assembly of nanoscale mol. structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resoln. becomes increasingly important. We present a single-mol. assay for the study of binding and unbinding kinetics on DNA origami. The authors find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, the authors exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topog.) imaging with <30 nm resoln. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.
- 40Schnitzbauer, J.; Strauss, M. T.; Schlichthaerle, T.; Schueder, F.; Jungmann, R. Super-Resolution Microscopy with DNA-PAINT. Nat. Protoc. 2017, 12 (6), 1198– 1228, DOI: 10.1038/nprot.2017.024Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnvF2rsLo%253D&md5=83998c799df527fa89eb74000aee4889Super-resolution microscopy with DNA-PAINTSchnitzbauer, Joerg; Strauss, Maximilian T.; Schlichthaerle, Thomas; Schueder, Florian; Jungmann, RalfNature Protocols (2017), 12 (6), 1198-1228CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Super-resoln. techniques have begun to transform biol. and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topog. (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resoln. microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resoln. microscopy. Using the programmability and specificity of DNA mols. as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resoln. techniques, making them compatible with virtually any single-mol.-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise mol. counting and ultra-high, mol.-scale (sub-5-nm) spatial resoln., reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample prepn., multiplexed data acquisition, data simulation, super-resoln. image reconstruction and post-processing such as drift correction, mol. counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.
- 41Eklund, A. S.; Comberlato, A.; Parish, I. A.; Jungmann, R.; Bastings, M. M. C. Quantification of Strand Accessibility in Biostable DNA Origami with Single-Staple Resolution. ACS Nano 2021, 15 (11), 17668– 17677, DOI: 10.1021/acsnano.1c05540Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFOht7jM&md5=f3ce356964f59ca2b5bdbc23bb762370Quantification of Strand Accessibility in Biostable DNA Origami with Single-Staple ResolutionEklund, Alexandra S.; Comberlato, Alice; Parish, Ian A.; Jungmann, Ralf; Bastings, Maartje M. C.ACS Nano (2021), 15 (11), 17668-17677CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)DNA-based nanostructures are actively gaining interest as tools for biomedical and therapeutic applications following the recent development of protective coating strategies prolonging structural integrity in physiol. conditions. For tailored biol. action, these nanostructures are often functionalized with targeting or imaging labels using DNA base pairing. Only if these labels are accessible on the structure's surface will they be able to interact with their intended biol. target. However, the accessibility of functional sites for different geometries and environments, specifically after the application of a protective coating, is currently not known. Here, we assay this accessibility on the level of single handle strands with two- and three-dimensional resoln. using DNA-PAINT and show that the hybridization kinetics of top and bottom sides on the same nanostructure linked to a surface remain unaltered. We furthermore demonstrate that the functionality of the structures remains available after an oligolysine-PEG coating is applied, enabling bioassays where functionality and stability are imperative.
- 42Vanamee, É. S.; Faustman, D. L. Structural Principles of Tumor Necrosis Factor Superfamily Signaling. Science Signaling 2018, 11 (511), eaao4910 DOI: 10.1126/scisignal.aao4910Google ScholarThere is no corresponding record for this reference.
- 43Kwon, P. S.; Ren, S.; Kwon, S.-J.; Kizer, M. E.; Kuo, L.; Xie, M.; Zhu, D.; Zhou, F.; Zhang, F.; Kim, D.; Fraser, K.; Kramer, L. D.; Seeman, N. C.; Dordick, J. S.; Linhardt, R. J.; Chao, J.; Wang, X. Designer DNA Architecture Offers Precise and Multivalent Spatial Pattern-Recognition for Viral Sensing and Inhibition. Nat. Chem. 2020, 12 (1), 26– 35, DOI: 10.1038/s41557-019-0369-8Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1OitbjK&md5=29cdaa47c143af624754127ea377eaf9Designer DNA architecture offers precise and multivalent spatial pattern-recognition for viral sensing and inhibitionKwon, Paul S.; Ren, Shaokang; Kwon, Seok-Joon; Kizer, Megan E.; Kuo, Lili; Xie, Mo; Zhu, Dan; Zhou, Feng; Zhang, Fuming; Kim, Domyoung; Fraser, Keith; Kramer, Laura D.; Seeman, Nadrian C.; Dordick, Jonathan S.; Linhardt, Robert J.; Chao, Jie; Wang, XingNature Chemistry (2020), 12 (1), 26-35CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)DNA, when folded into nanostructures with a specific shape, is capable of spacing and arranging binding sites into a complex geometric pattern with nanometer precision. Here the authors demonstrate a designer DNA nanostructure that can act as a template to display multiple binding motifs with precise spatial pattern-recognition properties, and that this approach can confer exceptional sensing and potent viral inhibitory capabilities. A star-shaped DNA architecture, carrying five mol. beacon-like motifs, was constructed to display ten dengue envelope protein domain III (ED3)-targeting aptamers into a two-dimensional pattern precisely matching the spatial arrangement of ED3 clusters on the dengue (DENV) viral surface. The resulting multivalent interactions provide high DENV-binding avidity. This structure is a potent viral inhibitor and it can act as a sensor by including a fluorescent output to report binding. The authors' mol.-platform design strategy could be adapted to detect and combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures.
- 44Castro, M.; van Santen, H. M.; Férez, M.; Alarcón, B.; Lythe, G.; Molina-París, C. Receptor Pre-Clustering and T Cell Responses: Insights into Molecular Mechanisms. Front. Immunol. 2014, DOI: 10.3389/fimmu.2014.00132Google ScholarThere is no corresponding record for this reference.
- 45Parker, K.; Trampert, P.; Tinnemann, V.; Peckys, D.; Dahmen, T.; de Jonge, N. Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy. Biophys. J. 2018, 115 (3), 503– 513, DOI: 10.1016/j.bpj.2018.06.016Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1eis77L&md5=f20860ed1db0cc9c5a511a224bf08baeLinear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron MicroscopyParker, Kelly; Trampert, Patrick; Tinnemann, Verena; Peckys, Diana; Dahmen, Tim; de Jonge, NielsBiophysical Journal (2018), 115 (3), 503-513CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The spatial distribution of the human epidermal growth factor 2 (HER2) receptor in the plasma membrane of SKBR3 and HCC1954 breast cancer cells was studied. The receptor was labeled with quantum dot nanoparticles, and fixed whole cells were imaged in their native liq. state with environmental SEM using scanning transmission electron microscopy detection. The locations of individual HER2 positions were detd. in a total plasma membrane area of 991 μm2 for several SKBR3 cells and 1062 μm2 for HCC1954 cells. Some of the HER2 receptors were arranged in a linear chain with interlabel distances of 40 ± 7 and 32 ± 10 nm in SKBR3 and HCC1954 cells, resp. The finding was tested against randomly occurring linear chains of six or more positions, from which it was concluded that the exptl. finding is significant and did not arise from random label distributions. Because the measured interlabel distance in the HER2 chains is similar to the 36-nm helix-repetition distance of actin filaments, it is proposed that a linking mechanism between HER2 and actin filaments leads to linearly aligned oligomers.
- 46Bila, H.; Paloja, K.; Caroprese, V.; Kononenko, A.; Bastings, M. M. C. Multivalent Pattern Recognition through Control of Nano-Spacing in Low-Valency Super-Selective Materials. J. Am. Chem. Soc. 2022, 144 (47), 21576– 21586, DOI: 10.1021/jacs.2c08529Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XivVOksbnM&md5=b978e943b726b821bb876b2a28b5a419Multivalent Pattern Recognition through Control of Nano-Spacing in Low-Valency Super-Selective MaterialsBila, Hale; Paloja, Kaltrina; Caroprese, Vincenzo; Kononenko, Artem; Bastings, Maartje M. C.Journal of the American Chemical Society (2022), 144 (47), 21576-21586CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Super-selective multivalent ligand-receptor interactions display a signature step-like onset in binding when meeting a characteristic d. of target receptors. Materials engineered for super-selective binding generally display a high no. of flexible ligands to enhance the systems' avidity. In many biol. processes, however, ligands are present in moderate copy nos. and arranged in spatio-temporal patterns. In this low-valency regime, the rigidity of the ligand-presenting architecture plays a crit. role in the selectivity of the multivalent complex through decrease of the entropic penalty of binding. Exploiting the precision in spatial design inherent to the DNA nanotechnol., we engineered a library of rigid architectures to explore how valency, affinity, and nano-spacing control the presence of super-selectivity in multivalent binding. A micromolar monovalent affinity was required for super-selective binding to be obsd. within low-valency systems, and the transition point for stable interactions was measured at hexavalent ligand presentation, setting the limits of the low-valency regime. Super-selective binding was obsd. for all hexavalent architectures, and, more strikingly, the ligand pattern detd. the selectivity onset. Hereby, we demonstrate for the first time that nano-control of geometric patterns can be used to discriminate between receptor densities in a super-selective manner. Materials that were indistinguishable in their mol. compn. and ligand valency bound with various efficacies on surfaces with const. receptor densities. We define this new phenomenon in super-selective binding as multivalent pattern recognition.
- 47Reinhardt, S. C. M.; Masullo, L. A.; Baudrexel, I.; Steen, P. R.; Kowalewski, R.; Eklund, A. S.; Strauss, S.; Unterauer, E. M.; Schlichthaerle, T.; Strauss, M. T.; Klein, C.; Jungmann, R. Ångström-Resolution Fluorescence Microscopy. Nature 2023, 617 (7962), 711– 716, DOI: 10.1038/s41586-023-05925-9Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhtVyisr%252FF&md5=9b60751fedd2939b888ccf218e922e8cAngstrom-resolution fluorescence microscopyReinhardt, Susanne C. M.; Masullo, Luciano A.; Baudrexel, Isabelle; Steen, Philipp R.; Kowalewski, Rafal; Eklund, Alexandra S.; Strauss, Sebastian; Unterauer, Eduard M.; Schlichthaerle, Thomas; Strauss, Maximilian T.; Klein, Christian; Jungmann, RalfNature (London, United Kingdom) (2023), 617 (7962), 711-716CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Fluorescence microscopy, with its mol. specificity, is one of the major characterization methods used in the life sciences to understand complex biol. systems. Super-resoln. approaches1-6 can achieve resoln. in cells in the range of 15 to 20 nm, but interactions between individual biomols. occur at length scales below 10 nm and characterization of intramol. structure requires Angstrom resoln. State-of-the-art super-resoln. implementations7-14 have demonstrated spatial resolns. down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolns. do not directly translate to expts. in cells, and Angstrom resoln. has not been demonstrated to date. Here we introdue a DNA-barcoding method, resoln. enhancement by sequential imaging (RESI), that improves the resoln. of fluorescence microscopy down to the Angstrom scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolns. of >15 nm, we demonstrate that single-protein resoln. can be achieved for biomols. in whole intact cells. Furthermore, we exptl. resolve the DNA backbone distance of single bases in DNA origami with Ångstrom resoln. We use our method in a proof-of-principle demonstration to map the mol. arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the mol. mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramol. imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resoln. microscopy and structural biol. studies and thus delivers information key to understanding complex biol. systems.
- 48Magnez, R.; Thiroux, B.; Taront, S.; Segaoula, Z.; Quesnel, B.; Thuru, X. PD-1/PD-L1 Binding Studies Using Microscale Thermophoresis. Sci. Rep 2017, 7 (1), 17623 DOI: 10.1038/s41598-017-17963-1Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MzitFemug%253D%253D&md5=d5d0ace31a28ebc7f260cced3fce2f03PD-1/PD-L1 binding studies using microscale thermophoresisMagnez Romain; Thiroux Bryan; Taront Solenne; Segaoula Zacharie; Quesnel Bruno; Thuru Xavier; Magnez Romain; Thiroux Bryan; Taront Solenne; Segaoula Zacharie; Quesnel Bruno; Thuru Xavier; Quesnel BrunoScientific reports (2017), 7 (1), 17623 ISSN:.The characterization of protein interactions has become essential in many fields of life science, especially drug discovery. Microscale thermophoresis (MST) is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. In addition, one of the major advantages of this technique is that no tedious purification step is necessary to access the protein of interest. Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 couple, which is involved in tumour escape processes, without purification of the target protein from cell lysates. The method requires the overexpression of fluorescent proteins in CHO-K1 cells and describes the optimal conditions for determining the dissociation constant. The protocol has a variety of potential applications in studying the interactions of these proteins with small molecules and demonstrates that MST is a valuable method for studying the PD-1/PD-L1 pathway.
- 49Lázár-Molnár, E.; Scandiuzzi, L.; Basu, I.; Quinn, T.; Sylvestre, E.; Palmieri, E.; Ramagopal, U. A.; Nathenson, S. G.; Guha, C.; Almo, S. C. Structure-Guided Development of a High-Affinity Human Programmed Cell Death-1: Implications for Tumor Immunotherapy. EBioMedicine 2017, 17, 30– 44, DOI: 10.1016/j.ebiom.2017.02.004Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1czgt1ehtw%253D%253D&md5=4bb2f1751befebc4a0a73b51241619dcStructure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapyLazar-Molnar Eszter; Sylvestre Eliezer; Palmieri Edith; Scandiuzzi Lisa; Basu Indranil; Quinn Thomas; Ramagopal Udupi A; Nathenson Stanley G; Guha Chandan; Almo Steven CEBioMedicine (2017), 17 (), 30-44 ISSN:.Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.
- 50Butte, M. J.; Keir, M. E.; Phamduy, T. B.; Sharpe, A. H.; Freeman, G. J. Programmed Death-1 Ligand 1 Interacts Specifically with the B7–1 Costimulatory Molecule to Inhibit T Cell Responses. Immunity 2007, 27 (1), 111– 122, DOI: 10.1016/j.immuni.2007.05.016Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXosFeqtrk%253D&md5=8fabef0e1244a1d356378867d92a248eProgrammed Death-1 Ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responsesButte, Manish J.; Keir, Mary E.; Phamduy, Theresa B.; Sharpe, Arlene H.; Freeman, Gordon J.Immunity (2007), 27 (1), 111-122CODEN: IUNIEH; ISSN:1074-7613. (Cell Press)Pathways in the B7:CD28 family of costimulatory mols. regulate T cell activation and tolerance. B7-dependent responses in Cd28-/- Ctla4-/- T cells together with reports of stimulatory and inhibitory functions for Programmed Death-1 Ligand 1 or 2 mols. (PD-L1 or PD-L2) have suggested addnl. receptors for these B7 family members. The authors show that B7-1 and PD-L1 interacted with affinity intermediate to that of B7-1:CD28 and B7-1:CTLA-4. The PD-L1:B7-1 interface overlapped with the B7-1:CTLA-4 and PD-L1:PD-1 (Programmed Death-1) interfaces. T cell activation and cytokine prodn. were inhibited by the interaction of B7-1 with PD-L1. The responses of PD-1-deficient vs. PD-1,B7-1 double-deficient T cells to PD-L1 and of CD28,CTLA-4 double-deficient vs. CD28,CTLA-4,PD-L1 triple-deficient T cells to B7-1 demonstrated that PD-L1 and B7-1 interact specifically to inhibit T cell activation. The authors' findings point to a substantial bidirectional inhibitory interaction between B7-1 and PD-L1 and add an addnl. dimension to immunoregulatory functions of the B7:CD28 family.
- 51Ponnuswamy, N.; Bastings, M. M. C.; Nathwani, B.; Ryu, J. H.; Chou, L. Y. T.; Vinther, M.; Li, W. A.; Anastassacos, F. M.; Mooney, D. J.; Shih, W. M. Oligolysine-Based Coating Protects DNA Nanostructures from Low-Salt Denaturation and Nuclease Degradation. Nat. Commun. 2017, 8 (1), 15654 DOI: 10.1038/ncomms15654Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXovFCls7Y%253D&md5=253bfcf6f3cb7cd9632092bad7624b0eOligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradationPonnuswamy, Nandhini; Bastings, Maartje M. C.; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y. T.; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M.; Mooney, David J.; Shih, William M.Nature Communications (2017), 8 (), 15654CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviors. However, their utility in biol. fluids can be compromised through denaturation induced by physiol. salt concns. and degrdn. mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.
- 52Ueno, H.; Klechevsky, E.; Morita, R.; Aspord, C.; Cao, T.; Matsui, T.; Di Pucchio, T.; Connolly, J.; Fay, J. W.; Pascual, V.; Palucka, A. K.; Banchereau, J. Dendritic Cell Subsets in Health and Disease. Immunological Reviews 2007, 219 (1), 118– 142, DOI: 10.1111/j.1600-065X.2007.00551.xGoogle Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1Cnt7zI&md5=0231ed1978d62f49a2aa5ba959d92d30Dendritic cell subsets in health and diseaseUeno, Hideki; Klechevsky, Eynav; Morita, Rimpei; Aspord, Caroline; Cao, Tinghua; Matsui, Toshimichi; Di Pucchio, Tiziana; Connolly, John; Fay, Joseph W.; Pascual, Virginia; Palucka, A. Karolina; Banchereau, JacquesImmunological Reviews (2007), 219 (), 118-142CODEN: IMRED2; ISSN:0105-2896. (Blackwell Publishing Ltd.)A review. The dendritic cell (DC) system of antigen-presenting cells controls immunity and tolerance. DCs initiate and regulate immune responses in a manner that depends on signals they receive from microbes and their cellular environment. They allow the immune system to make qual. distinct responses against different microbial infections. DCs are composed of subsets that express different microbial receptors and express different surface mols. and cytokines. Our studies lead us to propose that interstitial (dermal) DCs preferentially activate humoral immunity, whereas Langerhans cells preferentially induce cellular immunity. Alterations of the DC system result in diseases such as autoimmunity, allergy, and cancer. Conversely, DCs can be exploited for vaccination, and novel vaccines that directly target DCs in vivo are being designed.
- 53Domogalla, M. P.; Rostan, P. V.; Raker, V. K.; Steinbrink, K. Tolerance through Education: How Tolerogenic Dendritic Cells Shape Immunity. Front. Immunol. 2017, DOI: 10.3389/fimmu.2017.01764Google ScholarThere is no corresponding record for this reference.
- 54Iberg, C. A.; Hawiger, D. Natural and Induced Tolerogenic Dendritic Cells. J. Immunol. 2020, 204 (4), 733– 744, DOI: 10.4049/jimmunol.1901121Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXks1SgsL0%253D&md5=eaf2afc58e975a8879c3da4b741f7398Natural and induced tolerogenic dendritic cellsIberg, Courtney A.; Hawiger, DanielJournal of Immunology (2020), 204 (4), 733-744CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)A review. Dendritic cells (DCs) are highly susceptible to extrinsic signals that modify the functions of these crucial APCs. Maturation of DCs induced by diverse proinflammatory conditions promotes immune responses, but certain signals also induce tolerogenic functions in DCs. These "induced tolerogenic DCs" help to moderate immune responses such as those to commensals present at specific anatomical locations. However, also under steady-state conditions, some DCs are characterized by inherent tolerogenic properties. The immunomodulatory mechanisms constitutively present in such "natural tolerogenic DCs" help to promote tolerance to peripheral Ags. By extending tolerance initially established in the thymus, these functions of DCs help to regulate autoimmune and other immune responses. In this review we will discuss the mechanisms and functions of natural and induced tolerogenic DCs and offer further insight into how their possible manipulations may ultimately lead to more precise treatments for various immune-mediated conditions and diseases.
- 55Koga, M. M.; Engel, A.; Pigni, M.; Lavanchy, C.; Stevanin, M.; Laversenne, V.; Schneider, B. L.; Acha-Orbea, H. IL10- and IL35-Secreting MutuDC Lines Act in Cooperation to Inhibit Memory T Cell Activation Through LAG-3 Expression. Front. Immunol. 2021, DOI: 10.3389/fimmu.2021.607315Google ScholarThere is no corresponding record for this reference.
- 56Zhao, Y.; Lee, C. K.; Lin, C.-H.; Gassen, R. B.; Xu, X.; Huang, Z.; Xiao, C.; Bonorino, C.; Lu, L.-F.; Bui, J. D.; Hui, E. PD-L1:CD80 Cis-Heterodimer Triggers the Co-Stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways. Immunity 2019, 51 (6), 1059– 1073, DOI: 10.1016/j.immuni.2019.11.003Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFylsL%252FE&md5=bc7d76d40f9923ffaaafb12934663e7aPD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 PathwaysZhao, Yunlong; Lee, Calvin K.; Lin, Chia-Hao; Gassen, Rodrigo B.; Xu, Xiaozheng; Huang, Zhe; Xiao, Changchun; Bonorino, Cristina; Lu, Li-Fan; Bui, Jack D.; Hui, EnfuImmunity (2019), 51 (6), 1059-1073.e9CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)Combined immunotherapy targeting the immune checkpoint receptors cytotoxic T-lymphocyte-assocd. protein 4 (CTLA-4) and programmed cell death 1 (PD-1), or CTLA-4 and the PD-1 ligand (PD-L1) exhibits superior anti-tumor responses compared with single-agent therapy. Here, we examd. the mol. basis for this synergy. Using reconstitution assays with fluorescence readouts, we found that PD-L1 and the CTLA-4 ligand CD80 heterodimerize in cis but not trans. Quant. biochem. and cell biol. assays revealed that PD-L1:CD80 cis-heterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the T cell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.
- 57Sansom, D. M.; Walker, L. S. K. Dimers Aren’t Forever: CD80 Breaks up with PD-L1. Immunity 2019, 51 (6), 972– 974, DOI: 10.1016/j.immuni.2019.11.011Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtVGlsbzL&md5=429f5e91cd86fcd2cb44adb66b54a75dDimers Aren't Forever: CD80 Breaks up with PD-L1Sansom, David M.; Walker, Lucy S. K.Immunity (2019), 51 (6), 972-974CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)A review. Targeting the CTLA-4 and PD-1 "checkpoints" is an effective treatment for a no. of cancers. In this issue of Immunity, Hui et al. reveal that interaction between a CTLA-4 ligand, CD80, and its counterpart in the PD-1 pathway, PD-L1, affects both PD-1 and CTLA-4 function, raising new questions about the biol. effects of using checkpoint inhibitors alone and in combination.
- 58Martinez-Veracoechea, F. J.; Frenkel, D. Designing Super Selectivity in Multivalent Nano-Particle Binding. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (27), 10963– 10968, DOI: 10.1073/pnas.1105351108Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptV2htrY%253D&md5=79356231cc82dd3344d53f2a85e5daadDesigning super selectivity in multivalent nano-particle bindingMartinez-Veracoechea, Francisco J.; Frenkel, DaanProceedings of the National Academy of Sciences of the United States of America (2011), 108 (27), 10963-10968, S10963/1-S10963/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A key challenge in nano-science is to design ligand-coated nano-particles that can bind selectively to surfaces that display the cognate receptors above a threshold (surface) concn. Nano-particles that bind monovalently to a target surface do not discriminate sharply between surfaces with high and low receptor coverage. In contrast, "multivalent" nano-particles that can bind to a larger no. of ligands simultaneously, display regimes of "super selectivity" where the fraction of bound particles varies sharply with the receptor concn. We present numerical simulations that show that multivalent nano-particles can be designed such that they approach the 'on-off" binding behavior ideal for receptor-concn. selective targeting. We propose a simple anal. model that accounts for the super selective behavior of multivalent nano-particles. The model shows that the super selectivity is due to the fact that the no. of distinct ligand-receptor binding arrangements increases in a highly nonlinear way with receptor coverage. Somewhat counterintuitively, our study shows that selectivity can be improved by making the individual ligand-receptor bonds weaker. We propose a simple rule of thumb to predict the conditions under which super selectivity can be achieved. We validate our model predictions against the Monte Carlo simulations.
- 59Curk, T.; Dobnikar, J.; Frenkel, D. Optimal Multivalent Targeting of Membranes with Many Distinct Receptors. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (28), 7210– 7215, DOI: 10.1073/pnas.1704226114Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhsrnI&md5=588b429ecc50b542e70dda1ef2e56826Optimal multivalent targeting of membranes with many distinct receptorsCurk, Tine; Dobnikar, Jure; Frenkel, DaanProceedings of the National Academy of Sciences of the United States of America (2017), 114 (28), 7210-7215CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Cells can often be recognized by the concns. of receptors expressed on their surface. For better (targeted drug treatment) or worse (targeted infection by pathogens), it is clearly important to be able to target cells selectively. A good targeting strategy would result in strong binding to cells with the desired receptor profile and barely binding to other cells. Using a simple model, the authors formulate optimal design rules for multivalent particles that allow them to distinguish target cells based on their receptor profile. The authors found the following: (1) It is not a good idea to aim for very strong binding between the individual ligands on the guest (delivery vehicle) and the receptors on the host (cell). Rather, one should exploit multivalency: High sensitivity to the receptor d. on the host can be achieved by coating the guest with many ligands that bind only weakly to the receptors on the cell surface. (2) The concn. profile of the ligands on the guest should closely match the compn. of the cognate membrane receptors on the target surface. And (3) irresp. of all details, the effective strength of the ligand-receptor interaction should be of the order of the thermal energy kBT, where T is the abs. temp. and kB is Boltzmann's const. The authors present simulations that support the theor. predictions. The authors speculate that, using the above design rules, it should be possible to achieve targeted drug delivery with a greatly reduced incidence of side effects.
- 60Dubacheva, G. V.; Curk, T.; Richter, R. P. Determinants of Superselectivity─Practical Concepts for Application in Biology and Medicine. Acc. Chem. Res. 2023, 56 (7), 729– 739, DOI: 10.1021/acs.accounts.2c00672Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXkvFyktr4%253D&md5=82e46f198147e0ce1021a8fd4223c987Determinants of Superselectivity-Practical Concepts for Application in Biology and MedicineDubacheva, Galina V.; Curk, Tine; Richter, Ralf P.Accounts of Chemical Research (2023), 56 (7), 729-739CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)Multivalent interactions are common in biol. systems and are also widely deployed for targeting applications in biomedicine. A unique feature of multivalent binding is "superselectivity". Superselectivity refers to the sharp discrimination of surfaces (e.g., on cells or cell compartments) by their comparative surface densities of a given receptor. This feature is different from the conventional "type" selectivity, which discriminates surfaces by their distinct receptor types. In a broader definition, a probe is superselective if it converts a gradual change in any one interaction parameter into a sharp on/off dependency in probe binding. This Account describes our systematic exptl. and theorotical efforts over the past decade to analyze the determinants of superselective binding. It aims to offer chem. biologists, biophysicists, biologists, and biomedical scientists a set of guidelines for the interpretation of multivalent binding data, and design rules for tuning superselective targeting. We first provide a basic introduction that identifies multiple low-affinity interactions and combinatorial entropy as the minimal set of conditions required for superselective recognition. We then introduce the main exptl. and theteoritical tools and analyze how salient features of the multivalent probes (i.e., their concn., size, ligand valency, and scaffold type), of the surface receptors (i.e., their affinity for ligands, surface d., and mobility), and of competitors and cofactors (i.e., their concn. and affinity for the ligands and/or receptors) influence the sharpness and the position of the threshold for superselective recognition. Emerging from this work are a set of relatively simple yet quantative data anal. guidelines and superselectivity design rules that apply to a broad range of probe types and interaction systems. The key finding is the scaling variable xS which faithfully predicts the influence of the surface receptor d., probe ligand valency, receptor-ligand affinity, and competitor/cofactor concns. and affinities on superselective recognition. The scaling variable is a simple yet versatile tool to quant. tune the on/off threshold of superselective probes. We exemplify its application by reviewing and reinterpreting literature data for selected biol. and biomedical interaction systems where superselectivity clearly is important. Our guidelines can be deployed to generate a new mechanistic understanding of multivalent recognition events inside and outside cells and the downstream physiol./pathol. implications. Moreover, the design rules can be harnessed to develop novel superselective probes for anal. purposes in the life sciences and for diagnostic/therapeutic intervention in biomedicine.
- 61Strauss, S.; Jungmann, R. Up to 100-Fold Speed-up and Multiplexing in Optimized DNA-PAINT. Nat. Methods 2020, 17 (8), 789– 791, DOI: 10.1038/s41592-020-0869-xGoogle Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1yjtbfO&md5=14599350694abb5936e9b187d8ce7c15Up to 100-fold speed-up and multiplexing in optimized DNA-PAINTStrauss, Sebastian; Jungmann, RalfNature Methods (2020), 17 (8), 789-791CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)DNA-PAINT's imaging speed has recently been significantly enhanced by optimized sequence design and buffer conditions. However, this implementation has not reached an ultimate speed limit and is only applicable to imaging of single targets. To further improve acquisition speed, we introduce concatenated, periodic DNA sequence motifs, yielding up to 100-fold-faster sampling in comparison to traditional DNA-PAINT. We extend this approach to six orthogonal sequence motifs, now enabling speed-optimized multiplexed imaging.
- 62Fabricius, V.; Lefèbre, J.; Geertsema, H.; Marino, S. F.; Ewers, H. Rapid and Efficient C-Terminal Labeling of Nanobodies for DNA-PAINT. J. Phys. D: Appl. Phys. 2018, 51 (47), 474005 DOI: 10.1088/1361-6463/aae0e2Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlWrtrrO&md5=e4e74321222ca126b2005abad174b4f6Rapid and efficient C-terminal labeling of nanobodies for DNA-PAINTFabricius, Valentin; Lefebre, Jonathan; Geertsema, Hylkje; Marino, Stephen F.; Ewers, HelgeJournal of Physics D: Applied Physics (2018), 51 (47), 474005/1-474005/8CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)Single mol. localization-based approaches to super-resoln. microscopy (SMLM) create images that resolve features smaller than the diffraction limit of light by rendering them from the sequentially measured positions of thousands of individual mols. New SMLM approaches based on the transient binding of very bright dyes via DNA-DNA interaction (DNA-PAINT) allow the resoln. of dyes only a few nanometers apart in vitro. This imaging of cellular structures requires the specific assocn. of dyes to their targets, which results in an addnl. 'linkage error'. This error can be minimized by using extremely small, single-domain antibody-based binders such as nanobodies, but the DNA-oligomers used in DNA-PAINT are of significant size in comparison to nanobodies and may interfere with binding. We have developed an optimized procedure based on enzymic labeling and click-chem. for the coupling of DNA oligomers to the nanobody C-terminus, which is located on the opposite side of the epitope-binding domain. Our approach allows for straightforward labeling, purifn. and DNA-PAINT imaging. We performed high efficiency labeling of two different nanobodies and show dual color multiplexed SMLM to demonstrate the general applicability of our labeling scheme.
- 63Eklund, A. S.; Ganji, M.; Gavins, G.; Seitz, O.; Jungmann, R. Peptide-PAINT Super-Resolution Imaging Using Transient Coiled Coil Interactions. Nano Lett. 2020, 20 (9), 6732– 6737, DOI: 10.1021/acs.nanolett.0c02620Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVKntLrM&md5=541b25b2df246c513591ead462186ea5Peptide-PAINT Super-Resolution Imaging Using Transient Coiled Coil InteractionsEklund, Alexandra S.; Ganji, Mahipal; Gavins, Georgina; Seitz, Oliver; Jungmann, RalfNano Letters (2020), 20 (9), 6732-6737CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Super-resoln. microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale. DNA-PAINT is a relatively easy-to-implement single-mol.-based technique, which uses the programmable and transient interaction of dye-labeled oligonucleotides with their complements for super-resoln. imaging. However, similar to many imaging approaches, it is still hampered by the subpar performance of labeling probes in terms of their large size and limited labeling efficiency. To overcome this, the authors here translate the programmability and transient binding nature of DNA-PAINT to coiled coil interactions of short peptides and introduce Peptide-PAINT. The authors benchmark and optimize its binding kinetics in a single-mol. assay and demonstrate its super-resoln. capability using self-assembled DNA origami structures. Peptide-PAINT outperforms classical DNA-PAINT in terms of imaging speed and efficiency. Finally, the authors prove the suitability of Peptide-PAINT for cellular super-resoln. imaging by visualizing the microtubule and vimentin network in fixed cells.
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Abstract
Figure 1
Figure 1. Super-resolution DNA-PAINT imaging of PD-L1 protein on the DC surface. (a) Schematic outline of the parameters that were studied. Change in PD-L1 molecular arrangement between nonactivated DCs and cells stimulated with CpG and IFNγ for 24 h was investigated using DNA-PAINT imaging. Spatial characteristics, including the distribution of PD-L1 (whether random or clustered), inter-PD-L1 spacing, and clustering order, were determined from super-resolved images. (b) DNA-PAINT images of PD-L1 on DCs before (top) and after (bottom) stimulation. Zoom-in images are presented in the middle and on the right. Each spot represents individual PD-L1 molecules. Scale bars = 5 μm, 500 nm, and 100 nm from left to right. (c,d) NND distribution (c) before and (d) after stimulation. Experimental data are highlighted in orange and CSR simulations in blue. (e) Quantitative analysis of the oligomerization order for clusters defined in nonactivated (white) and stimulated (gray) DCs. Error bars indicate SD and statistical analysis was performed using two-tailed Mann–Whitney test (***p < 0.001). Data were derived from two independent experiments and ≥12 cells.
Figure 2
Figure 2. Production of PD-1-DNA conjugates and functionalization on DNA origami disks. (a) Schematic representation of the DNA disk library used to investigate the influence of valency and pattern of PD-1 on the interaction with target ligands. Each black dot represents the position of PD-1 on the DNA disk. (b) Schematic overview of the experimental workflow used to conjugate PD-1 to ssDNA via (1) sortase-mediated reaction and (2) copper-free click chemistry, and (3) hybridize PD-1 to DNA origami disks. Z = poly histidine tag. (c) Schematic representation of the DNA-PAINT experimental setup used for the characterization of 6LH, 6SH, and 6Lin PD-1 DNA disks. PD-1-DNA conjugates were prepared with a ssDNA handle extended with a DNA-PAINT docking sequence (R1). Each PD-1 was visualized by transient hybridization of the imager strand (Cy3B-labeled R1 imager strand) and the docking strand. (d) DNA-PAINT sum images of 6LH, 6SH, and 6Lin PD-1 DNA disks (top). Cross-sectional histogram analysis was performed for the highlighted areas (white boxes) to measure inter-PD-1 distances (bottom). Scale bars: 15 nm.
Figure 3
Figure 3. Analysis of PD-1 DNA disk binding behavior on PD-L1 surfaces by SPR. (a) Representative binding curves of DNA disks presenting 0, 1, 3, 6 (in LH, SH, and Lin patterns), or 12 PD-1. Bare DNA disk shows no binding to PD-L1, excluding nonspecific interactions with the ligand. Curves represent increasing particle concentrations (10 nM and 2-fold dilution). (b) Quantification of affinity constants obtained from fitted curves. Error bars indicate SEM (n = 2 independent experiments).
Figure 4
Figure 4. Effect of valency and pattern of PD-1 DNA disks on binding to DCs. (a) Representative confocal microscopy images of the surface staining of DCs using (top) αPD-L1 and αPD-L2 antibodies and (bottom) 12 PD-1 DNA disk. Nucleus stained with DAPI in gray, cytoplasm (GFP reporter in CD11c+) in cyan, and PD-L1/PD-L2 in pink. Scale bars: 10 μm. (b) Representative flow cytometric images of DCs stained with 12 PD-1 DNA disk acquired on Amnis ImageStream system. Cytoplasm (GFP reporter in CD11c+) in cyan and PD-L1/PD-L2 in pink. Scale bar: 5 μm. (c) Binding of PD-1 DNA disks with different valencies and patterns to DCs, presented as the geometric mean fluorescence intensity (gMFI) of Cy5 integrated in the DNA disks. The data were normalized by subtracting the gMFI of the 0 DNA disks for each experiment. Error bars indicate SEM (n ≥ 8 independent experiments). Raw data of each group (Figure S10f) were compared to each other using a Kruskal–Wallis test, followed by Dunn’s post-test (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 5
Figure 5. Influence of PD-1 valency and pattern on immune checkpoint blockade. (a) Schematic overview of the experimental setup. Primary CD8α+ OT-I T cells were stimulated with αCD3/αCD28 antibodies to induce PD-1 expression. After resting in IL-7, T cells were cocultured with stimulated WT DCs, IL-10 DCs or cancer cells (B16) in the presence of PD-1 DNA disks or control blocking αPD-L1/αPD-L2 antibodies. Cytokine production in the supernatants after 24 h was measured as T cell activation readout. (b) Representative IFNγ production after 24 h of blockade with PD-1 DNA disks. Error bars indicate SEM (n = 2 replicates in one experiment). Replicates can be found in Figure S15. Statistical analysis was performed independently for each cell type. PD-1 DNA disk groups are compared to 0 DNA disk groups with ordinary one-way ANOVA, followed by Dunnett’s test (*p < 0.05, **p < 0.01, ***p < 0.001).
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- 1Sharpe, A. H.; Pauken, K. E. The Diverse Functions of the PD1 Inhibitory Pathway. Nat. Rev. Immunol 2018, 18 (3), 153– 167, DOI: 10.1038/nri.2017.1081https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1WqsrfP&md5=304a93c872e4beaa8f249390d516527eThe diverse functions of the PD1 inhibitory pathwaySharpe, Arlene H.; Pauken, Kristen E.Nature Reviews Immunology (2018), 18 (3), 153-167CODEN: NRIABX; ISSN:1474-1733. (Nature Research)T cell activation is a highly regulated process involving peptide-MHC engagement of the T cell receptor and pos. costimulatory signals. Upon activation, coinhibitory 'checkpoints', including programmed cell death protein 1 (PD1), become induced to regulate T cells. PD1 has an essential role in balancing protective immunity and immunopathol., homeostasis and tolerance. However, during responses to chronic pathogens and tumors, PD1 expression can limit protective immunity. Recently developed PD1 pathway inhibitors have revolutionized cancer treatment for some patients, but the majority of patients do not show complete responses, and adverse events have been noted. This Review discusses the diverse roles of the PD1 pathway in regulating immune responses and how this knowledge can improve cancer immunotherapy as well as restore and/or maintain tolerance during autoimmunity and transplantation.
- 2Chikuma, S. Basics of PD-1 in Self-Tolerance, Infection, and Cancer Immunity. Int. J. Clin Oncol 2016, 21 (3), 448– 455, DOI: 10.1007/s10147-016-0958-02https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XivVeqs7w%253D&md5=78752623b71d6d39a54dcec0f4807857Basics of PD-1 in self-tolerance, infection, and cancer immunityChikuma, ShunsukeInternational Journal of Clinical Oncology (2016), 21 (3), 448-455CODEN: IJCOF6; ISSN:1341-9625. (Springer Japan)Successful cancer treatment requires understanding host immune response against tumor cells. PD-1 belongs to the CD28 superfamily of receptors that work as "checkpoints" of immune activation. PD-1 maintains immune self-tolerance to prevent autoimmunity and controls T-cell reaction during infection to prevent excessive tissue damage. Tumor cells that arise from normal tissue acquire mutations that can be targeted by lymphocytes. Accumulating lines of evidence suggest that tumor cells evade host immune attack by expressing physiol. PD-1 ligands and stimulating PD-1 on the lymphocytes. Based on this idea, researchers have successfully demonstrated that systemic administration of monoclonal antibodies that inhibit the binding of PD-1 to the ligands reactivated T cells and augmented the anti-cancer immune response. In this review, I summarize the basics of T-cell biol. and its regulation by PD-1 and discuss the current understanding and questions about this multifaceted mol.
- 3Ceeraz, S.; Nowak, E. C.; Noelle, R. J. B7 Family Checkpoint Regulators in Immune Regulation and Disease. Trends in Immunology 2013, 34 (11), 556– 563, DOI: 10.1016/j.it.2013.07.0033https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1KltbjJ&md5=395a88bdda0a83ec10fe4845d5fa76c9B7 family checkpoint regulators in immune regulation and diseaseCeeraz, Sabrina; Nowak, Elizabeth C.; Noelle, Randolph J.Trends in Immunology (2013), 34 (11), 556-563CODEN: TIRMAE; ISSN:1471-4906. (Elsevier Ltd.)A review. Fine-tuning the immune response and maintaining tolerance to self-antigens involves a complex network of co-stimulatory and co-inhibitory mols. The recent FDA approval of ipilimumab, a monoclonal antibody blocking cytotoxic T lymphocyte antigen (CTLA)-4, demonstrates the impact of checkpoint regulators in disease. This is reinforced by ongoing clin. trials targeting not only CTLA-4, but also the programmed death (PD)-1 and B7-H4 pathways in various disease states. Recently, two new B7 family inhibitory ligands, V-domain Ig suppressor of T cell activation (VISTA) and B7-H6 were identified. Here, we review recent understanding of B7 family members and their concerted regulation of the immune response to either self or foreign pathogens. We also discuss clin. developments in targeting these pathways in different disease settings, and introduce VISTA as a putative therapeutic target.
- 4Francisco, L. M.; Sage, P. T.; Sharpe, A. H. The PD-1 Pathway in Tolerance and Autoimmunity. Immunol Rev. 2010, 236, 219– 242, DOI: 10.1111/j.1600-065X.2010.00923.x4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXovFaks7s%253D&md5=8323a6c78c3172b79287894c4a00ae8eThe PD-1 pathway in tolerance and autoimmunityFrancisco, Loise M.; Sage, Peter T.; Sharpe, Arlene H.Immunological Reviews (2010), 236 (1), 219-242CODEN: IMRED2; ISSN:1600-065X. (John Wiley & Sons, Inc.)A review. Regulatory T cells (Tregs) and the PD-1: PD-ligand (PD-L) pathway are both crit. to terminating immune responses. Elimination of either can result in the breakdown of tolerance and the development of autoimmunity. The PD-1: PD-L pathway can thwart self-reactive T cells and protect against autoimmunity in many ways. In this review, we highlight how PD-1 and its ligands defend against potentially pathogenic self-reactive effector T cells by simultaneously harnessing two mechanisms of peripheral tolerance: (i) the promotion of Treg development and function and (ii) the direct inhibition of potentially pathogenic self-reactive T cells that have escaped into the periphery. Treg cells induced by the PD-1 pathway may also assist in maintaining immune homeostasis, keeping the threshold for T-cell activation high enough to safeguard against autoimmunity. PD-L1 expression on non-hematopoietic cells as well as hematopoietic cells endows PD-L1 with the capacity to promote Treg development and enhance Treg function in lymphoid organs and tissues that are targets of autoimmune attack. At sites where transforming growth factor-β is present (e.g. sites of immune privilege or inflammation), PD-L1 may promote the de novo generation of Tregs. When considering the consequences of uncontrolled immunity, it would be therapeutically advantageous to manipulate Treg development and sustain Treg function. Thus, this review also discusses how the PD-1 pathway regulates a no. of autoimmune diseases and the therapeutic potential of PD-1: PD-L modulation.
- 5Cha, J.-H.; Chan, L.-C.; Li, C.-W.; Hsu, J. L.; Hung, M.-C. Mechanisms Controlling PD-L1 Expression in Cancer. Mol. Cell 2019, 76 (3), 359– 370, DOI: 10.1016/j.molcel.2019.09.0305https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVCgtbrL&md5=593d644ba9c97932fb6c3326eafd7e92Mechanisms Controlling PD-L1 Expression in CancerCha, Jong-Ho; Chan, Li-Chuan; Li, Chia-Wei; Hsu, Jennifer L.; Hung, Mien-ChieMolecular Cell (2019), 76 (3), 359-370CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)A review. The engagement of programmed cell death protein 1 (PD-1; encoded by the PDCD1 gene) receptor expressed on activated T cells and its ligand, programmed death-ligand 1 (PD-L1; encoded by the CD274 gene), is a major co-inhibitory checkpoint signaling that controls T cell activities. Various types of cancers express high levels of PD-L1 and exploit PD-L1/PD-1 signaling to evade T cell immunity. Blocking the PD-L1/PD-1 pathway has consistently shown remarkable anti-tumor effects in patients with advanced cancers and is recognized as the gold std. for developing new immune checkpoint blockade (ICB) and combination therapies. However, the response rates of anti-PD-L1 have been limited in several solid tumors. Therefore, furthering our understanding of the regulatory mechanisms of PD-L1 can bring substantial benefits to patients with cancer by improving the efficacy of current PD-L1/PD-1 blockade or other ICBs. In this review, we provide current knowledge of PD-L1 regulatory mechanisms at the transcriptional, posttranscriptional, post-translational, and extracellular levels, and discuss the implications of these findings in cancer diagnosis and immunotherapy.
- 6Juneja, V. R.; McGuire, K. A.; Manguso, R. T.; LaFleur, M. W.; Collins, N.; Haining, W. N.; Freeman, G. J.; Sharpe, A. H. PD-L1 on Tumor Cells Is Sufficient for Immune Evasion in Immunogenic Tumors and Inhibits CD8 T Cell Cytotoxicity. J. Exp Med. 2017, 214 (4), 895– 904, DOI: 10.1084/jem.201608016https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtV2ju7nJ&md5=95ceac8e66dd13f327bfc0eb3dab42c1PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicityJuneja, Vikram R.; McGuire, Kathleen A.; Manguso, Robert T.; LaFleur, Martin W.; Collins, Natalie; Haining, W. Nicholas; Freeman, Gordon J.; Sharpe, Arlene H.Journal of Experimental Medicine (2017), 214 (4), 895-904CODEN: JEMEAV; ISSN:1540-9538. (Rockefeller University Press)It is unclear whether PD-L1 on tumor cells is sufficient for tumor immune evasion or simply correlates with an inflamed tumor microenvironment. We used three mouse tumor models sensitive to PD-1 blockade to evaluate the significance of PD-L1 on tumor vs. nontumor cells. PD-L1 on nontumor cells is crit. for inhibiting antitumor immunity in B16 melanoma and a genetically engineered melanoma. In contrast, PD-L1 on MC38 colorectal adenocarcinoma cells is sufficient to suppress antitumor immunity, as deletion of PD-L1 on highly immunogenic MC38 tumor cells allows effective antitumor immunity. MC38-derived PD-L1 potently inhibited CD8+ T cell cytotoxicity. Wild-type MC38 cells outcompeted PD-L1-deleted MC38 cells in vivo, demonstrating tumor PD-L1 confers a selective advantage. Thus, both tumor- and host-derived PD-L1 can play crit. roles in immunosuppression. Differences in tumor immunogenicity appear to underlie their relative importance. Our findings establish reduced cytotoxicity as a key mechanism by which tumor PD-L1 suppresses antitumor immunity and demonstrate that tumor PD-L1 is not just a marker of suppressed antitumor immunity.
- 7Jiang, X.; Wang, J.; Deng, X.; Xiong, F.; Ge, J.; Xiang, B.; Wu, X.; Ma, J.; Zhou, M.; Li, X.; Li, Y.; Li, G.; Xiong, W.; Guo, C.; Zeng, Z. Role of the Tumor Microenvironment in PD-L1/PD-1-Mediated Tumor Immune Escape. Molecular Cancer 2019, 18 (1), 10, DOI: 10.1186/s12943-018-0928-47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cjhslyltg%253D%253D&md5=2bb5915587023a05d999f0c6f7f89e14Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escapeJiang Xianjie; Ge Junshang; Xiang Bo; Ma Jian; Zhou Ming; Li Xiaoling; Li Guiyuan; Xiong Wei; Guo Can; Zeng Zhaoyang; Jiang Xianjie; Wang Jie; Deng Xiangying; Xiong Fang; Ge Junshang; Xiang Bo; Wu Xu; Ma Jian; Zhou Ming; Li Xiaoling; Li Yong; Li Guiyuan; Xiong Wei; Guo Can; Zeng Zhaoyang; Jiang Xianjie; Xiang Bo; Ma Jian; Zhou Ming; Li Xiaoling; Li Guiyuan; Xiong Wei; Guo Can; Zeng Zhaoyang; Wu Xu; Li YongMolecular cancer (2019), 18 (1), 10 ISSN:.Tumor immune escape is an important strategy of tumor survival. There are many mechanisms of tumor immune escape, including immunosuppression, which has become a research hotspot in recent years. The programmed death ligand-1/programmed death-1 (PD-L1/PD-1) signaling pathway is an important component of tumor immunosuppression, which can inhibit the activation of T lymphocytes and enhance the immune tolerance of tumor cells, thereby achieving tumor immune escape. Therefore, targeting the PD-L1/PD-1 pathway is an attractive strategy for cancer treatment; however, the therapeutic effectiveness of PD-L1/PD-1 remains poor. This situation requires gaining a deeper understanding of the complex and varied molecular mechanisms and factors driving the expression and activation of the PD-L1/PD-1 signaling pathway. In this review, we summarize the regulation mechanisms of the PD-L1/PD-1 signaling pathway in the tumor microenvironment and their roles in mediating tumor escape. Overall, the evidence accumulated to date suggests that induction of PD-L1 by inflammatory factors in the tumor microenvironment may be one of the most important factors affecting the therapeutic efficiency of PD-L1/PD-1 blocking.
- 8Page, D. B.; Postow, M. A.; Callahan, M. K.; Allison, J. P.; Wolchok, J. D. Immune Modulation in Cancer with Antibodies. Annual Review of Medicine 2014, 65 (1), 185– 202, DOI: 10.1146/annurev-med-092012-1128078https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXktFeltbo%253D&md5=3f6332a9af46e4f57fc5e6716678b66bImmune modulation in cancer with antibodiesPage, David B.; Postow, Michael A.; Callahan, Margaret K.; Allison, James P.; Wolchok, Jedd D.Annual Review of Medicine (2014), 65 (), 185-202CODEN: ARMCAH; ISSN:0066-4219. (Annual Reviews)A review. Ipilimumab is the prototypical immunomodulatory antibody, approved by the FDA in 2011 for advanced melanoma on the basis of survival benefit. Since that time, we have made significant strides in optimizing this therapy: we have characterized the spectrum of immune-related adverse events and learned how to mitigate them with treatment algorithms, discovered potential biomarkers of activity, and identified the potential synergy between checkpoint modulation and other therapeutic modalities. Recent phase I trials have established the efficacy and safety of next-generation checkpoint agents, including PD-1 and PD-L1 inhibitors, across multiple tumor types. Much work lies ahead in developing these next-generation checkpoint agents, testing them in combination, and detg. how to integrate them into the treatment paradigms of various tumor types.
- 9Acúrcio, R. C.; Pozzi, S.; Carreira, B.; Pojo, M.; Gómez-Cebrián, N.; Casimiro, S.; Fernandes, A.; Barateiro, A.; Farricha, V.; Brito, J.; Leandro, A. P.; Salvador, J. A. R.; Graça, L.; Puchades-Carrasco, L.; Costa, L.; Satchi-Fainaro, R.; Guedes, R. C.; Florindo, H. F. Therapeutic Targeting of PD-1/PD-L1 Blockade by Novel Small-Molecule Inhibitors Recruits Cytotoxic T Cells into Solid Tumor Microenvironment. J. Immunother Cancer 2022, 10 (7), e004695 DOI: 10.1136/jitc-2022-004695There is no corresponding record for this reference.
- 10Yang, J.; Hu, L. Immunomodulators Targeting the PD-1/PD-L1 Protein-Protein Interaction: From Antibodies to Small Molecules. Medicinal Research Reviews 2019, 39 (1), 265– 301, DOI: 10.1002/med.2153010https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisVylsLnI&md5=3b37b2a848554f6b2af82fc802a03168Immunomodulators targeting the PD-1/PD-L1 protein-protein interaction: From antibodies to small moleculesYang, Jeffrey; Hu, LongqinMedicinal Research Reviews (2019), 39 (1), 265-301CODEN: MRREDD; ISSN:0198-6325. (John Wiley & Sons, Inc.)A review. Cancer immunotherapy has made great strides in the recent decade, esp. in the area of immune checkpoint blockade. The outstanding efficacy, prolonged durability of effect, and rapid assimilation of anti-PD-1 and anti-PD-L1 monoclonal antibodies in clin. practice have been nothing short of a medical breakthrough in the treatment of numerous malignancies. The major advantages of these therapeutic antibodies over their small mol. counterparts have been their high binding affinity and target specificity. However, antibodies do have their flaws including immune-related toxicities, inadequate pharmacokinetics and tumor penetration, and high cost burden to manufacturers and consumers. These limitations hinder broader clin. applications of the antibodies and have heightened interests in developing the alternative small mol. platform that includes peptidomimetics and peptides to target the PD-1/PD-L1 immune checkpoint system. The progress on these small mol. alternatives has been relatively slow compared to that of the antibodies. Fortunately, recent structural studies of the interactions among PD-1, PD-L1, and their resp. antibodies have revealed key hotspots on PD-1 and PD-L1 that may facilitate drug discovery efforts for small mol. immunotherapeutics. This review is intended to discuss key concepts in immuno-oncol., describe the successes and shortcomings of PD-1/PD-L1 antibody-based therapies, and to highlight the recent development of small mol. inhibitors of the PD-1/PD-L1 protein-protein interaction.
- 11Guo, L.; Wei, R.; Lin, Y.; Kwok, H. F. Clinical and Recent Patents Applications of PD-1/PD-L1 Targeting Immunotherapy in Cancer Treatment─Current Progress, Strategy, and Future Perspective. Front. Immunol. 2020, DOI: 10.3389/fimmu.2020.01508There is no corresponding record for this reference.
- 12Day, D.; Hansen, A. R. Immune-Related Adverse Events Associated with Immune Checkpoint Inhibitors. BioDrugs 2016, 30 (6), 571– 584, DOI: 10.1007/s40259-016-0204-312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvV2gtL3F&md5=a776b5026dcbd50a50708664b7132f92Immune-Related Adverse Events Associated with Immune Checkpoint InhibitorsDay, Daphne; Hansen, Aaron R.BioDrugs (2016), 30 (6), 571-584CODEN: BIDRF4; ISSN:1173-8804. (Springer International Publishing AG)Immune checkpoint inhibitors (ICIs), including antibodies targeting cytotoxic T-lymphocyte-assocd. antigen 4 (CTLA-4) and programmed cell death protein-1 (PD-1), have shown durable treatment responses in multiple tumor types by enhancing antitumor immunity. However, removal of self-tolerance can induce autoimmunity and produce a unique immune-driven toxicity profile, termed immune-related adverse events (irAEs). As ICIs gain approval for a growing no. of indications, it is imperative clinicians increase their knowledge of and ability to manage irAEs. This review examines the etiol., presentation, kinetics, and treatment of irAEs and aims to provide practical guidance for clinicians.
- 13Xu-Monette, Z. Y.; Zhang, M.; Li, J.; Young, K. H. PD-1/PD-L1 Blockade: Have We Found the Key to Unleash the Antitumor Immune Response?. Front. Immunol. 2017, DOI: 10.3389/fimmu.2017.01597There is no corresponding record for this reference.
- 14Wang, Z.; Wu, X. Study and Analysis of Antitumor Resistance Mechanism of PD1/PD-L1 Immune Checkpoint Blocker. Cancer Medicine 2020, 9 (21), 8086– 8121, DOI: 10.1002/cam4.341014https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlWhtLzM&md5=8c4ea127feb88c43818dd33ddd677cb4Study and analysis of antitumor resistance mechanism of PD1/PD-L1 immune checkpoint blockerWang, Zhengyi; Wu, XiaoyingCancer Medicine (2020), 9 (21), 8086-8121CODEN: CMAEDL; ISSN:2045-7634. (John Wiley & Sons Ltd.)Immunocheckpoint proteins of tumor infiltrating lymphocytes play an important role in tumor prognosis in the course of tumor clinicopathol. PD-1 (Programed cell death protein 1) is an important immunosuppressive mol. By binding to PD-L1 (programed cell death-ligand 1), it blocks TCR and its costimulus signal transduction, inhibits the activation and proliferation of T cells, depletes the function of effector T cells, and enables tumor cells to achieve immune escape. In recent years, immunocheckpoint blocking therapy targeting the PD-1/PD-L1 axis has achieved good results in a variety of malignant tumors, pushing tumor immunotherapy to a new milestone, such as anti-PD-1 monoclonal antibody Nivolumab, Pembrolizumab, and anti-PD-L1 monoclonal antibody Atezolizumab, which are considered as potential antitumor drugs. It was found in clin. use that some patients obtained long-term efficacy, but most of them developed drug resistance recurrence in the later stage. The high incidence of drug resistance (including primary and acquired drug resistance) still cannot be ignored, which limited its clin. application and became a new problem in this field. Due to tumor heterogeneity, current limited research shows that PD-1 or PD-L1 monoclonal antibody drug resistance may be related to the following factors: mutation of tumor antigen and antigen presentation process, multiple immune checkpoint interactions, immune microenvironment changes dynamically, activation of oncogenic pathways, gene mutation and epigenetic changes of key proteins in tumors, tumor competitive metab., and accumulation of metabolites, etc, mechanisms of resistance are complex. Therefore, it is the most urgent task to further elucidate the mechanism of immune checkpoint inhibitor resistance, discover multitumor universal biomarkers, and develop new target agents to improve the response rate of immunotherapy in patients. In this study, the mechanism of anti-PD-1/PD-L1 drug resistance in tumors, the potential biomarkers for predicting PD-1 acquired resistance, and the recent development of combination therapy were reviewed one by one. It is believed that, based on the complex mechanism of drug resistance, it is of no clin. significance to simply search for and regulate drug resistance targets, and it may even produce drug resistance again soon. It is speculated that according to the possible tumor characteristics, three types of treatment methods should be combined to change the tumor microenvironment ecol. and eliminate various heterogeneous tumor subsets, so as to reduce tumor drug resistance and improve long-term clin. efficacy.
- 15Sun, J.-Y.; Zhang, D.; Wu, S.; Xu, M.; Zhou, X.; Lu, X.-J.; Ji, J. Resistance to PD-1/PD-L1 Blockade Cancer Immunotherapy: Mechanisms, Predictive Factors, and Future Perspectives. Biomark Res. 2020, 8, 35, DOI: 10.1186/s40364-020-00212-515https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38bitlSmsw%253D%253D&md5=4812ff408d7aa9ce2e9503ddc8658cd8Resistance to PD-1/PD-L1 blockade cancer immunotherapy: mechanisms, predictive factors, and future perspectivesSun Jin-Yu; Zhang Dengke; Wu Songquan; Xu Min; Ji Jiansong; Zhang Dengke; Wu Songquan; Xu Min; Ji Jiansong; Zhou Xiao; Lu Xiao-Jie; Ji JiansongBiomarker research (2020), 8 (), 35 ISSN:2050-7771.PD-1/PD-L1 blockade therapy is a promising cancer treatment strategy, which has revolutionized the treatment landscape of malignancies. Over the last decade, PD-1/PD-L1 blockade therapy has been trialed in a broad range of malignancies and achieved clinical success. Despite the potentially cure-like survival benefit, only a minority of patients are estimated to experience a positive response to PD-1/PD-L1 blockade therapy, and the primary or acquired resistance might eventually lead to cancer progression in patients with clinical responses. Accordingly, the resistance to PD-1/PD-L1 blockade remains a significant challenge hindering its further application. To overcome the limitation in therapy resistance, substantial effort has been made to improve or develop novel anti-PD-1/PD-L1 based immunotherapy strategies with better clinical response and reduced immune-mediated toxicity. In this review, we provide an overview on the resistance to PD-1/PD-L1 blockade and briefly introduce the mechanisms underlying therapy resistance. Moreover, we summarize potential predictive factors for the resistance to PD-1/PD-L1 blockade. Furthermore, we give an insight into the possible solutions to improve efficacy and clinical response. In the following research, combined efforts of basic researchers and clinicians are required to address the limitation of therapy resistance.
- 16Dustin, M. L.; Tseng, S.-Y.; Varma, R.; Campi, G. T Cell–Dendritic Cell Immunological Synapses. Current Opinion in Immunology 2006, 18 (4), 512– 516, DOI: 10.1016/j.coi.2006.05.01716https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmsFSks70%253D&md5=24a4098e5f4538882c3b25c078253cf9T cell-dendritic cell immunological synapsesDustin, Michael L.; Tseng, Su-Yi; Varma, Rajat; Campi, GabrieleCurrent Opinion in Immunology (2006), 18 (4), 512-516CODEN: COPIEL; ISSN:0952-7915. (Elsevier Ltd.)A review. Dendritic cells (DCs) are myeloid lineage cells that are imprinted by their environment and that mature in response to microbial products. A crucial role of the DC is to impart this context-specific information to T cells as well as to present self and foreign MHC-peptide complexes through formation of an immunol. synapse. The structure of the T cell-DC immunol. synapse departs from the canonical structure formed with B cells or with supported planar bilayers in that it has multiple foci of T-cell receptor interactions rather than a central focus. Recent studies on model systems provide insight into the mechanisms and biol. consequences of the unique T cell-DC synaptic patterns.
- 17Dustin, M. L.; Chakraborty, A. K.; Shaw, A. S. Understanding the Structure and Function of the Immunological Synapse. Cold Spring Harb Perspect Biol. 2010, 2 (10), a002311 DOI: 10.1101/cshperspect.a00231117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWgtr3M&md5=890c57aa9978a99a5aee45b4f886f9a7Understanding the structure and function of the immunological synapseDustin, Michael L.; Chakraborty, Arup K.; Shaw, Andrey S.Cold Spring Harbor Perspectives in Biology (2010), 2 (10), a002311CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)A review. The immunol. synapse has been an area of very active scientific interest over the last decade. Surprisingly, much about the synapse remains unknown or is controversial. Here we review some of these current issues in the field: how the synapse is defined, its potential role in T-cell function, and our current understanding about how the synapse is formed.
- 18Sallusto, F.; Lanzavecchia, A. The Instructive Role of Dendritic Cells on T-Cell Responses. Arthritis Res. 2002, 4 (Suppl 3), S127– S132, DOI: 10.1186/ar567There is no corresponding record for this reference.
- 19Doh, J.; Irvine, D. J. Immunological Synapse Arrays: Patterned Protein Surfaces That Modulate Immunological Synapse Structure Formation in T Cells. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (15), 5700– 5705, DOI: 10.1073/pnas.050940410319https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XktFaisLg%253D&md5=a55746389e7d38f219fdac35d9ce2b0cImmunological synapse arrays: patterned protein surfaces that modulate immunological synapse structure formation in T cellsDoh, Junsang; Irvine, Darrell J.Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (15), 5700-5705CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)T cells are activated by recognition of foreign peptides displayed on the surface of antigen presenting cells (APCs), an event that triggers assembly of a complex microscale structure at the T cell-APC interface known as the immunol. synapse (IS). It remains unresolved whether the unique phys. structure of the synapse itself impacts the functional response of T cells, independent of the quantity and quality of ligands encountered by the T cell. As a first step toward addressing this question, we created multicomponent protein surfaces presenting lithog. defined patterns of tethered T cell receptor (TCR) ligands (anti-CD3 "activation sites") surrounded by a field of tethered intercellular adhesion mol.-1 (ICAM-1), as a model substrate on which T cells could be seeded to mimic T cell-APC interactions. CD4+ T cells seeded on these surfaces polarized and migrated; on contact with activation sites, T cells assembled an IS with a structure modulated by the phys. pattern of ligand encountered. On surfaces patterned with focal spots of TCR ligand, T cells stably interacted with activation sites, proliferated, and secreted cytokines. In contrast, T cells interacting with activation sites patterned to preclude centralized clustering of TCR ligand failed to form stable contacts with activation sites, exhibited aberrant PKC-θ clustering in a fraction of cells, and had significantly reduced prodn. of IFN-γ. These results suggest that focal clustering of TCR ligand characteristic of the "mature" IS may be required under some conditions for full T cell activation.
- 20Oh, S. A.; Wu, D.-C.; Cheung, J.; Navarro, A.; Xiong, H.; Cubas, R.; Totpal, K.; Chiu, H.; Wu, Y.; Comps-Agrar, L.; Leader, A. M.; Merad, M.; Roose-Germa, M.; Warming, S.; Yan, M.; Kim, J. M.; Rutz, S.; Mellman, I. PD-L1 Expression by Dendritic Cells Is a Key Regulator of T-Cell Immunity in Cancer. Nat. Cancer 2020, 1 (7), 681– 691, DOI: 10.1038/s43018-020-0075-x20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjsFegsb8%253D&md5=ea68fa5a9028234cb36d9f1e8bc68a1dPD-L1 expression by dendritic cells is a key regulator of T-cell immunity in cancerOh, Soyoung A.; Wu, Dai-Chen; Cheung, Jeanne; Navarro, Armando; Xiong, Huizhong; Cubas, Rafael; Totpal, Klara; Chiu, Henry; Wu, Yan; Comps-Agrar, Laetitia; Leader, Andrew M.; Merad, Miriam; Roose-Germa, Merone; Warming, Soren; Yan, Minhong; Kim, Jeong M.; Rutz, Sascha; Mellman, IraNature Cancer (2020), 1 (7), 681-691CODEN: NCAADQ; ISSN:2662-1347. (Springer International Publishing AG)Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a crit. source of PD-L1, despite being vastly outnumbered by PD-L1+ macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8+ T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1+ tumor cells.
- 21Yokosuka, T.; Takamatsu, M.; Kobayashi-Imanishi, W.; Hashimoto-Tane, A.; Azuma, M.; Saito, T. Programmed Cell Death 1 Forms Negative Costimulatory Microclusters That Directly Inhibit T Cell Receptor Signaling by Recruiting Phosphatase SHP2. J. Exp Med. 2012, 209 (6), 1201– 1217, DOI: 10.1084/jem.2011274121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XoslShsr4%253D&md5=5c1fad62b4e3e69e177be5718ce92d41Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2Yokosuka, Tadashi; Takamatsu, Masako; Kobayashi-Imanishi, Wakana; Hashimoto-Tane, Akiko; Azuma, Miyuki; Saito, TakashiJournal of Experimental Medicine (2012), 209 (6), 1201-1217CODEN: JEMEAV; ISSN:0022-1007. (Rockefeller University Press)Programmed cell death 1 (PD-1) is a neg. costimulatory receptor crit. for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a mol. mechanism of PD-1-mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently assocd. with the phosphatase SHP2 (Src homol. 2 domain-contg. tyrosine phosphatase 2). These neg. costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling mols. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addn. to PD-1 clustering, PD-1-TCR colocalization within microclusters is required for efficient PD-1-mediated suppression. This inhibitory mechanism also functions in PD-1hi T cells generated in vivo and can be overridden by a neutralizing anti-PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.
- 22Pentcheva-Hoang, T.; Chen, L.; Pardoll, D. M.; Allison, J. P. Programmed Death-1 Concentration at the Immunological Synapse Is Determined by Ligand Affinity and Availability. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (45), 17765– 17770, DOI: 10.1073/pnas.070876710422https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht12ltr3L&md5=02b184fa0f86ae1b680e84df45271c42Programmed death-1 concentration at the immunological synapse is determined by ligand affinity and availabilityPentcheva-Hoang, Tsvetelina; Chen, Lieping; Pardoll, Drew M.; Allison, James P.Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (45), 17765-17770CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Despite the importance of programmed death-1 (PD-1) for T cell inhibition, little is known about its intracellular trafficking or requirements for localization to the immunol. synapse. Here, we show that in activated T cells, PD-1 is present at the plasma membrane, near the Golgi and in the trans-Golgi network. Unlike CD28 and CTLA-4, PD-1 accumulation at the synapse is extensive only when T cells interact with dendritic cells (DCs) expressing high B7-DC levels. However, B7-H1 is also critically important, esp. when the DCs have little B7-DC. Despite this preference, B7-H1-/- DCs elicit greater cytokine secretion than B7-DC-/- DCs during T cell restimulation, possibly because they also express less B7-DC. PD-1 and CD28 have similar kinetics of synaptic accumulation, suggesting that the process involves T cell receptor-triggered cytoskeletal reorganization followed by ligand binding.
- 23Fang, T.; Alvelid, J.; Spratt, J.; Ambrosetti, E.; Testa, I.; Teixeira, A. I. Spatial Regulation of T-Cell Signaling by Programmed Death-Ligand 1 on Wireframe DNA Origami Flat Sheets. ACS Nano 2021, 15 (2), 3441– 3452, DOI: 10.1021/acsnano.0c1063223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjt1Khsrw%253D&md5=43b13eb11ccbfae436ccae1612284a58Spatial Regulation of T-Cell Signaling by Programmed Death-Ligand 1 on Wireframe DNA Origami Flat SheetsFang, Trixy; Alvelid, Jonatan; Spratt, Joel; Ambrosetti, Elena; Testa, Ilaria; Teixeira, Ana I.ACS Nano (2021), 15 (2), 3441-3452CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Programmed Death-1 (PD-1) is a coinhibitory receptor expressed on activated T cells that suppresses T-cell signaling and effector functions. It has been previously shown that binding to its ligand PD-L1 induces a spatial reorganization of PD-1 receptors into microclusters on the cell membrane. However, the roles of the spatial organization of PD-L1 on PD-1 clustering and T-cell signaling have not been elucidated. Here, we used DNA origami flat sheets to display PD-L1 ligands at defined nanoscale distances and investigated their ability to inhibit T-cell activation in vitro. We found that DNA origami flat sheets modified with CD3 and CD28 activating antibodies (FS-α-CD3-CD28) induced robust T-cell activation. Co-treatment with flat sheets presenting PD-L1 ligands sepd. by ∼200 nm (FS-PD-L1-200), but not 13 nm (FS-PD-L1-13) or 40 nm (FS-PD-L1-40), caused an inhibition of T-cell signaling, which increased with increasing molar ratio of FS-PD-L1-200 to FS-α-CD3-CD28. Furthermore, FS-PD-L1-200 induced the formation of smaller PD-1 nanoclusters and caused a larger redn. in IL-2 expression compared to FS-PD-L1-13. Together, these findings suggest that the spatial organization of PD-L1 dets. its ability to regulate T-cell signaling and may guide the development of future nano medicine-based immunomodulatory therapies.
- 24Ruglioni, M.; Civita, S.; Salvadori, T.; Cristiani, S.; Carnicelli, V.; Barachini, S.; Petrini, I.; Diaspro, A.; Bianchini, P.; Storti, B.; Bizzarri, R.; Fogli, S.; Danesi, R. Nanoscale Engagement of Immune Checkpoint Inhibitor PD-L1 in Membrane Lipid Rafts. bioRxiv , August 11, 2022. DOI: 10.1101/2022.08.09.503318 .There is no corresponding record for this reference.
- 25Zhou, L.; Chai, F.; He, Y.; Zhou, Z.; Guo, S.; Li, P.; Sun, Q.; Zu, X.; Liu, X.; Huang, Q.; Zhong, Y.; Zhou, A.; Wang, X.; Ren, H. Homodimerized Cytoplasmic Domain of PD-L1 Regulates Its Complex Glycosylation in Living Cells. Commun. Biol. 2022, 5 (1), 1– 12, DOI: 10.1038/s42003-022-03845-4There is no corresponding record for this reference.
- 26Rothemund, P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440 (7082), 297– 302, DOI: 10.1038/nature0458626https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XitlKgu7g%253D&md5=583caefdda9b1deb5d3f2ef78d9e6ecbFolding DNA to create nanoscale shapes and patternsRothemund, Paul W. K.Nature (London, United Kingdom) (2006), 440 (7082), 297-302CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)'Bottom-up fabrication', which exploits the intrinsic properties of atoms and mols. to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA mols. provides an attractive route towards this goal. Here the author describe a simple method for folding long, single-stranded DNA mols. into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diam. and approx. desired shapes such as squares, disks and five-pointed stars with a spatial resoln. of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton mol. complex).
- 27Seeman, N. C.; Sleiman, H. F. DNA Nanotechnology. Nat. Rev. Mater. 2018, 3 (1), 17068 DOI: 10.1038/natrevmats.2017.6827https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslOntLbM&md5=a8683cd5a65e013464f37bb6383853b4DNA nanotechnologySeeman, Nadrian C.; Sleiman, Hanadi F.Nature Reviews Materials (2018), 3 (1), 17068CODEN: NRMADL; ISSN:2058-8437. (Nature Research)DNA is the mol. that stores and transmits genetic information in biol. systems. The field of DNA nanotechnol. takes this mol. out of its biol. context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnol., and has been revolutionary in our ability to control mol. self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomol. structure detn., drug delivery and synthetic biol. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.
- 28Zhan, P.; Peil, A.; Jiang, Q.; Wang, D.; Mousavi, S.; Xiong, Q.; Shen, Q.; Shang, Y.; Ding, B.; Lin, C.; Ke, Y.; Liu, N. Recent Advances in DNA Origami-Engineered Nanomaterials and Applications. Chem. Rev. 2023, 123 (7), 3976– 4050, DOI: 10.1021/acs.chemrev.3c0002828https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXmt1Gltrw%253D&md5=42555f8f95f32e7fc6240b77d51fa063Recent Advances in DNA Origami-Engineered Nanomaterials and ApplicationsZhan, Pengfei; Peil, Andreas; Jiang, Qiao; Wang, Dongfang; Mousavi, Shikufa; Xiong, Qiancheng; Shen, Qi; Shang, Yingxu; Ding, Baoquan; Lin, Chenxiang; Ke, Yonggang; Liu, NaChemical Reviews (Washington, DC, United States) (2023), 123 (7), 3976-4050CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. DNA nanotechnol. is a unique field, where physics, chem., biol., mathematics, engineering, and materials science can elegantly converge. Since the original proposal of Nadrian Seeman, significant advances have been achieved in the past four decades. During this glory time, the DNA origami technique developed by Paul Rothemund further pushed the field forward with a vigorous momentum, fostering a plethora of concepts, models, methodologies, and applications that were not thought of before. This review focuses on the recent progress in DNA origami-engineered nanomaterials in the past five years, outlining the exciting achievements as well as the unexplored research avenues. We believe that the spirit and assets that Seeman left for scientists will continue to bring interdisciplinary innovations and useful applications to this field in the next decade.
- 29Comberlato, A.; Koga, M. M.; Nüssing, S.; Parish, I. A.; Bastings, M. M. C. Spatially Controlled Activation of Toll-like Receptor 9 with DNA-Based Nanomaterials. Nano Lett. 2022, 22 (6), 2506– 2513, DOI: 10.1021/acs.nanolett.2c0027529https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XmsFGitL8%253D&md5=b034d8a7e87d43f14b89a6c3e0e6d8beSpatially Controlled Activation of Toll-like Receptor 9 with DNA-Based NanomaterialsComberlato, Alice; Koga, Marianna M.; Nussing, Simone; Parish, Ian A.; Bastings, Maartje M. C.Nano Letters (2022), 22 (6), 2506-2513CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)First evidence of geometrical patterns and defined distances of biomols. as fundamental parameters to regulate receptor binding and cell signaling have emerged recently. Here, we demonstrate the importance of controlled nanospacing of immunostimulatory agents for the activation of immune cells by exploiting DNA-based nanomaterials and pre-existing crystallog. data. We created DNA origami nanoparticles that present CpG-motifs in rationally designed spatial patterns to activate Toll-like Receptor 9 in RAW 264.7 macrophages. We demonstrated that stronger immune activation is achieved when active mols. are positioned at the distance of 7 nm, matching the active dimer structure of the receptor. Moreover, we show how the introduction of linkers between particle and ligand can influence the spatial tolerance of binding. These findings are fundamental for a fine-tuned manipulation of the immune system, considering the importance of spatially controlled presentation of therapeutics to increase efficacy and specificity of immune-modulating nanomaterials where multivalent binding is involved.
- 30Kurisinkal, E. E.; Caroprese, V.; Koga, M. M.; Morzy, D.; Bastings, M. M. C. Selective Integrin A5β1 Targeting through Spatially Constrained Multivalent DNA-Based Nanoparticles. Molecules 2022, 27 (15), 4968, DOI: 10.3390/molecules2715496830https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitFGms77J&md5=7f7b3b2e2176adba59b0642ceb39bd3aSelective Integrin α5β1 Targeting through Spatially Constrained Multivalent DNA-Based NanoparticlesKurisinkal, Eva E.; Caroprese, Vincenzo; Koga, Marianna M.; Morzy, Diana; Bastings, Maartje M. C.Molecules (2022), 27 (15), 4968CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor d. across healthy and diseased cells. Engaging receptors above a threshold concn. would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α5β1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding obsd. across HUVEC, CHO and HeLa in resting vs. activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ∼7 nm and ∼24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was obsd., indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technol. imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.
- 31Hellmeier, J.; Platzer, R.; Eklund, A. S.; Schlichthaerle, T.; Karner, A.; Motsch, V.; Schneider, M. C.; Kurz, E.; Bamieh, V.; Brameshuber, M.; Preiner, J.; Jungmann, R.; Stockinger, H.; Schütz, G. J.; Huppa, J. B.; Sevcsik, E. DNA Origami Demonstrate the Unique Stimulatory Power of Single PMHCs as T Cell Antigens. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (4), e2016857118 DOI: 10.1073/pnas.201685711831https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFait7Y%253D&md5=20d8b5c4b8380028ad0e7a3690330a12DNA origami demonstrate the unique stimulatory power of single pMHCs as T cell antigensHellmeier, Joschka; Platzer, Rene; Eklund, Alexandra S.; Schlichthaerle, Thomas; Karner, Andreas; Motsch, Viktoria; Schneider, Magdalena C.; Kurz, Elke; Bamieh, Victor; Brameshuber, Mario; Preiner, Johannes; Jungmann, Ralf; Stockinger, Hannes; Schuetz, Gerhard J.; Huppa, Johannes B.; Sevcsik, EvaProceedings of the National Academy of Sciences of the United States of America (2021), 118 (4), e2016857118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a crit. factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the min. signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC mols.
- 32Shaw, A.; Lundin, V.; Petrova, E.; Fördős, F.; Benson, E.; Al-Amin, A.; Herland, A.; Blokzijl, A.; Högberg, B.; Teixeira, A. I. Spatial Control of Membrane Receptor Function Using Ligand Nanocalipers. Nat. Methods 2014, 11 (8), 841– 846, DOI: 10.1038/nmeth.302532https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFSlsLzO&md5=75998e2c18b132b9b86cc224c4b24ccdSpatial control of membrane receptor function using ligand nanocalipersShaw, Alan; Lundin, Vanessa; Petrova, Ekaterina; Foerdos, Ferenc; Benson, Erik; Al-Amin, Abdullah; Herland, Anna; Blokzijl, Andries; Hoegberg, Bjoern; Teixeira, Ana I.Nature Methods (2014), 11 (8), 841-846CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor-mediated signaling.
- 33Veneziano, R.; Moyer, T. J.; Stone, M. B.; Wamhoff, E.-C.; Read, B. J.; Mukherjee, S.; Shepherd, T. R.; Das, J.; Schief, W. R.; Irvine, D. J.; Bathe, M. Role of Nanoscale Antigen Organization on B-Cell Activation Probed Using DNA Origami. Nat. Nanotechnol. 2020, 15 (8), 716– 723, DOI: 10.1038/s41565-020-0719-033https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1Ontr3M&md5=94f9c3ffdf3b617e1320510ce5fcf86cRole of nanoscale antigen organization on B-cell activation probed using DNA origamiVeneziano, Remi; Moyer, Tyson J.; Stone, Matthew B.; Wamhoff, Eike-Christian; Read, Benjamin J.; Mukherjee, Sayak; Shepherd, Tyson R.; Das, Jayajit; Schief, William R.; Irvine, Darrell J.; Bathe, MarkNature Nanotechnology (2020), 15 (8), 716-723CODEN: NNAABX; ISSN:1748-3387. (Nature Research)Abstr.: Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However, the effects of antigen copy no., spacing and affinity, as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here, we display the clin. vaccine immunogen eOD-GT8, an engineered outer domain of the HIV-1 glycoprotein-120, on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to ∼25-30 nm monotonically increases B-cell receptor activation. Moreover, scaffold rigidity is essential for robust B-cell triggering. These results reveal mol. vaccine design principles that may be used to drive functional B-cell responses.
- 34Berger, R. M. L.; Weck, J. M.; Kempe, S. M.; Hill, O.; Liedl, T.; Rädler, J. O.; Monzel, C.; Heuer-Jungemann, A. Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells. Small 2021, 17 (26), 2101678 DOI: 10.1002/smll.20210167834https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVKmtr3J&md5=b2b54e5a9858d6e345b6aab81cd0356dNanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in CellsBerger, Ricarda M. L.; Weck, Johann M.; Kempe, Simon M.; Hill, Oliver; Liedl, Tim; Radler, Joachim O.; Monzel, Cornelia; Heuer-Jungemann, AmelieSmall (2021), 17 (26), 2101678CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their mol. organization and activation are hitherto lacking. Among the well-known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami-based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time-to-death kinetics for hexagonal FasL arrangements with 10 nm inter-mol. spacing. Compared to naturally occurring sol. FasL, this trigger is faster and 100x more efficient. Nanoagents with different spacing, lower FasL no. or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over mol. no. and geometry. They define mol. benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses.
- 35Bila, H.; Kurisinkal, E. E.; Bastings, M. M. C. Engineering a Stable Future for DNA-Origami as a Biomaterial. Biomaterials Science 2019, 7, 532, DOI: 10.1039/C8BM01249K35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlClsLvF&md5=26d82aa33cd092a354c1e200503a8c53Engineering a stable future for DNA-origami as a biomaterialBila, Hale; Kurisinkal, Eva E.; Bastings, Maartje M. C.Biomaterials Science (2019), 7 (2), 532-541CODEN: BSICCH; ISSN:2047-4849. (Royal Society of Chemistry)DNA as a biomaterial has evoked great interest as a potential platform for therapeutics and diagnostics and as hydrogel scaffolds due to the relative ease of programming its robust and uniform shape, site-specific functionality and controlled responsive behavior. However, for a stable self-assembled product, a relatively high cation concn. is required to prevent denaturation. Physiol. and cell-culture conditions do not match these concns. and present addnl. nucleases that cause a serious threat to the integrity of DNA-based materials. For the translation of this promising technol. towards bioengineering challenges, stability needs to be guaranteed. Over the past years, various methods have been developed addressing the stability-related weaknesses of DNA-origami. This mini-review explains the common stability issues and compares the stabilization strategies recently developed. We present a detailed overview of each method in order to ease the selection process on which method to use for future users of DNA-origami as a biomaterial.
- 36Koga, M. M.; Comberlato, A.; Rodríguez-Franco, H. J.; Bastings, M. M. C. Strategic Insights into Engineering Parameters Affecting Cell Type-Specific Uptake of DNA-Based Nanomaterials. Biomacromolecules 2022, 23 (6), 2586– 2594, DOI: 10.1021/acs.biomac.2c0028236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xhtl2nu7nI&md5=c022ca022821738c0bf0307a9be52b20Strategic Insights into Engineering Parameters Affecting Cell Type-Specific Uptake of DNA-Based NanomaterialsKoga, Marianna M.; Comberlato, Alice; Rodriguez-Franco, Hugo J.; Bastings, Maartje M. C.Biomacromolecules (2022), 23 (6), 2586-2594CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)DNA-based nanomaterials are gaining popularity as uniform and programmable bioengineering tools as a result of recent solns. to their weak stability under biol. conditions. The DNA nanotechnol. platform uniquely allows decoupling of engineering parameters to comprehensively study the effect of each upon cellular encounter. We here present a systematic anal. of the effect of surface parameters of DNA-based nanoparticles on uptake in three different cell models: tumor cells, macrophages, and dendritic cells. The influence of surface charge, stabilizing coating, fluorophore types, functionalization technique, and particle concn. employed is found to cause significant differences in material uptake among these cell types. We therefore provide new insights into the large variance in cell type-specific uptake, highlighting the necessity of proper engineering and careful assay development when DNA-based materials are used as tools in bioengineering and as future nanotherapeutic agents.
- 37Rodríguez-Franco, H. J.; Weiden, J.; Bastings, M. M. C. Stabilizing Polymer Coatings Alter the Protein Corona of DNA Origami and Can Be Engineered to Bias the Cellular Uptake. ACS Polym. Au 2023, 3, 344 DOI: 10.1021/acspolymersau.3c0000937https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhtFGmsb3P&md5=70096fef16a47ea6cf30569c2d6892c6Stabilizing Polymer Coatings Alters the Protein Corona of DNA Origami and Can Be Engineered to Bias the Cellular UptakeRodriguez-Franco, Hugo J.; Weiden, Jorieke; Bastings, Maartje M. C.ACS Polymers Au (2023), 3 (4), 344-353CODEN: APACCD; ISSN:2694-2453. (American Chemical Society)With DNA-based nanomaterials being designed for applications in cellular environments, the need arises to accurately understand their surface interactions toward biol. targets. As for any material exposed to protein-rich cell culture conditions, a protein corona will establish around DNA nanoparticles, potentially altering the a-priori designed particle function. Here, we first set out to identify the protein corona around DNA origami nanomaterials, taking into account the application of stabilizing block co-polymer coatings (oligolysine-1kPEG or oligolysine-5kPEG) widely used to ensure particle integrity. By implementing a label-free methodol., the distinct polymer coating conditions show unique protein profiles, predominantly defined by differences in the mol. wt. and isoelec. point of the adsorbed proteins. Interestingly, none of the applied coatings reduced the diversity of the proteins detected within the specific coronae. We then biased the protein corona through pre-incubation with selected proteins and show significant changes in the cell uptake. Our study contributes to a deeper understanding of the complex interplay between DNA nanomaterials, proteins, and cells at the bio-interface.
- 38Peng, Q.; Qiu, X.; Zhang, Z.; Zhang, S.; Zhang, Y.; Liang, Y.; Guo, J.; Peng, H.; Chen, M.; Fu, Y.-X.; Tang, H. PD-L1 on Dendritic Cells Attenuates T Cell Activation and Regulates Response to Immune Checkpoint Blockade. Nat. Commun. 2020, 11 (1), 4835, DOI: 10.1038/s41467-020-18570-x38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFOhsLvJ&md5=34a8cf2f133b76392b86fc9ceb781b69PD-L1 on dendritic cells attenuates T cell activation and regulates response to immune checkpoint blockadePeng, Qi; Qiu, Xiangyan; Zhang, Zihan; Zhang, Silin; Zhang, Yuanyuan; Liang, Yong; Guo, Jingya; Peng, Hua; Chen, Mingyi; Fu, Yang-Xin; Tang, HaidongNature Communications (2020), 11 (1), 4835CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Immune checkpoint blockade therapies have shown clin. promise in a variety of cancers, but how tumor-infiltrating T cells are activated remains unclear. In this study, we explore the functions of PD-L1 on dendritic cells (DCs), which highly express PD-L1. We observe that PD-L1 on DC plays a crit. role in limiting T cell responses. Type 1 conventional DCs are essential for PD-L1 blockade and they upregulate PD-L1 upon antigen uptake. Upregulation of PD-L1 on DC is mediated by type II interferon. While DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, subsequent PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Blocking PD-L1 in established tumors promotes re-activation of tumor-infiltrating T cells for tumor control. Our study identifies a crit. and dynamic role of PD-L1 on DC, which needs to be harnessed for better invigoration of antitumor immune responses.
- 39Jungmann, R.; Steinhauer, C.; Scheible, M.; Kuzyk, A.; Tinnefeld, P.; Simmel, F. C. Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami. Nano Lett. 2010, 10 (11), 4756– 4761, DOI: 10.1021/nl103427w39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWiu7rK&md5=e93210e6c0d8347498b003e120ca8248Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA OrigamiJungmann, Ralf; Steinhauer, Christian; Scheible, Max; Kuzyk, Anton; Tinnefeld, Philip; Simmel, Friedrich C.Nano Letters (2010), 10 (11), 4756-4761CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)DNA origami is a powerful method for the programmable assembly of nanoscale mol. structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resoln. becomes increasingly important. We present a single-mol. assay for the study of binding and unbinding kinetics on DNA origami. The authors find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, the authors exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topog.) imaging with <30 nm resoln. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.
- 40Schnitzbauer, J.; Strauss, M. T.; Schlichthaerle, T.; Schueder, F.; Jungmann, R. Super-Resolution Microscopy with DNA-PAINT. Nat. Protoc. 2017, 12 (6), 1198– 1228, DOI: 10.1038/nprot.2017.02440https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnvF2rsLo%253D&md5=83998c799df527fa89eb74000aee4889Super-resolution microscopy with DNA-PAINTSchnitzbauer, Joerg; Strauss, Maximilian T.; Schlichthaerle, Thomas; Schueder, Florian; Jungmann, RalfNature Protocols (2017), 12 (6), 1198-1228CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Super-resoln. techniques have begun to transform biol. and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topog. (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resoln. microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resoln. microscopy. Using the programmability and specificity of DNA mols. as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resoln. techniques, making them compatible with virtually any single-mol.-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise mol. counting and ultra-high, mol.-scale (sub-5-nm) spatial resoln., reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample prepn., multiplexed data acquisition, data simulation, super-resoln. image reconstruction and post-processing such as drift correction, mol. counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.
- 41Eklund, A. S.; Comberlato, A.; Parish, I. A.; Jungmann, R.; Bastings, M. M. C. Quantification of Strand Accessibility in Biostable DNA Origami with Single-Staple Resolution. ACS Nano 2021, 15 (11), 17668– 17677, DOI: 10.1021/acsnano.1c0554041https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFOht7jM&md5=f3ce356964f59ca2b5bdbc23bb762370Quantification of Strand Accessibility in Biostable DNA Origami with Single-Staple ResolutionEklund, Alexandra S.; Comberlato, Alice; Parish, Ian A.; Jungmann, Ralf; Bastings, Maartje M. C.ACS Nano (2021), 15 (11), 17668-17677CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)DNA-based nanostructures are actively gaining interest as tools for biomedical and therapeutic applications following the recent development of protective coating strategies prolonging structural integrity in physiol. conditions. For tailored biol. action, these nanostructures are often functionalized with targeting or imaging labels using DNA base pairing. Only if these labels are accessible on the structure's surface will they be able to interact with their intended biol. target. However, the accessibility of functional sites for different geometries and environments, specifically after the application of a protective coating, is currently not known. Here, we assay this accessibility on the level of single handle strands with two- and three-dimensional resoln. using DNA-PAINT and show that the hybridization kinetics of top and bottom sides on the same nanostructure linked to a surface remain unaltered. We furthermore demonstrate that the functionality of the structures remains available after an oligolysine-PEG coating is applied, enabling bioassays where functionality and stability are imperative.
- 42Vanamee, É. S.; Faustman, D. L. Structural Principles of Tumor Necrosis Factor Superfamily Signaling. Science Signaling 2018, 11 (511), eaao4910 DOI: 10.1126/scisignal.aao4910There is no corresponding record for this reference.
- 43Kwon, P. S.; Ren, S.; Kwon, S.-J.; Kizer, M. E.; Kuo, L.; Xie, M.; Zhu, D.; Zhou, F.; Zhang, F.; Kim, D.; Fraser, K.; Kramer, L. D.; Seeman, N. C.; Dordick, J. S.; Linhardt, R. J.; Chao, J.; Wang, X. Designer DNA Architecture Offers Precise and Multivalent Spatial Pattern-Recognition for Viral Sensing and Inhibition. Nat. Chem. 2020, 12 (1), 26– 35, DOI: 10.1038/s41557-019-0369-843https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1OitbjK&md5=29cdaa47c143af624754127ea377eaf9Designer DNA architecture offers precise and multivalent spatial pattern-recognition for viral sensing and inhibitionKwon, Paul S.; Ren, Shaokang; Kwon, Seok-Joon; Kizer, Megan E.; Kuo, Lili; Xie, Mo; Zhu, Dan; Zhou, Feng; Zhang, Fuming; Kim, Domyoung; Fraser, Keith; Kramer, Laura D.; Seeman, Nadrian C.; Dordick, Jonathan S.; Linhardt, Robert J.; Chao, Jie; Wang, XingNature Chemistry (2020), 12 (1), 26-35CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)DNA, when folded into nanostructures with a specific shape, is capable of spacing and arranging binding sites into a complex geometric pattern with nanometer precision. Here the authors demonstrate a designer DNA nanostructure that can act as a template to display multiple binding motifs with precise spatial pattern-recognition properties, and that this approach can confer exceptional sensing and potent viral inhibitory capabilities. A star-shaped DNA architecture, carrying five mol. beacon-like motifs, was constructed to display ten dengue envelope protein domain III (ED3)-targeting aptamers into a two-dimensional pattern precisely matching the spatial arrangement of ED3 clusters on the dengue (DENV) viral surface. The resulting multivalent interactions provide high DENV-binding avidity. This structure is a potent viral inhibitor and it can act as a sensor by including a fluorescent output to report binding. The authors' mol.-platform design strategy could be adapted to detect and combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures.
- 44Castro, M.; van Santen, H. M.; Férez, M.; Alarcón, B.; Lythe, G.; Molina-París, C. Receptor Pre-Clustering and T Cell Responses: Insights into Molecular Mechanisms. Front. Immunol. 2014, DOI: 10.3389/fimmu.2014.00132There is no corresponding record for this reference.
- 45Parker, K.; Trampert, P.; Tinnemann, V.; Peckys, D.; Dahmen, T.; de Jonge, N. Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy. Biophys. J. 2018, 115 (3), 503– 513, DOI: 10.1016/j.bpj.2018.06.01645https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1eis77L&md5=f20860ed1db0cc9c5a511a224bf08baeLinear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron MicroscopyParker, Kelly; Trampert, Patrick; Tinnemann, Verena; Peckys, Diana; Dahmen, Tim; de Jonge, NielsBiophysical Journal (2018), 115 (3), 503-513CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The spatial distribution of the human epidermal growth factor 2 (HER2) receptor in the plasma membrane of SKBR3 and HCC1954 breast cancer cells was studied. The receptor was labeled with quantum dot nanoparticles, and fixed whole cells were imaged in their native liq. state with environmental SEM using scanning transmission electron microscopy detection. The locations of individual HER2 positions were detd. in a total plasma membrane area of 991 μm2 for several SKBR3 cells and 1062 μm2 for HCC1954 cells. Some of the HER2 receptors were arranged in a linear chain with interlabel distances of 40 ± 7 and 32 ± 10 nm in SKBR3 and HCC1954 cells, resp. The finding was tested against randomly occurring linear chains of six or more positions, from which it was concluded that the exptl. finding is significant and did not arise from random label distributions. Because the measured interlabel distance in the HER2 chains is similar to the 36-nm helix-repetition distance of actin filaments, it is proposed that a linking mechanism between HER2 and actin filaments leads to linearly aligned oligomers.
- 46Bila, H.; Paloja, K.; Caroprese, V.; Kononenko, A.; Bastings, M. M. C. Multivalent Pattern Recognition through Control of Nano-Spacing in Low-Valency Super-Selective Materials. J. Am. Chem. Soc. 2022, 144 (47), 21576– 21586, DOI: 10.1021/jacs.2c0852946https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XivVOksbnM&md5=b978e943b726b821bb876b2a28b5a419Multivalent Pattern Recognition through Control of Nano-Spacing in Low-Valency Super-Selective MaterialsBila, Hale; Paloja, Kaltrina; Caroprese, Vincenzo; Kononenko, Artem; Bastings, Maartje M. C.Journal of the American Chemical Society (2022), 144 (47), 21576-21586CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Super-selective multivalent ligand-receptor interactions display a signature step-like onset in binding when meeting a characteristic d. of target receptors. Materials engineered for super-selective binding generally display a high no. of flexible ligands to enhance the systems' avidity. In many biol. processes, however, ligands are present in moderate copy nos. and arranged in spatio-temporal patterns. In this low-valency regime, the rigidity of the ligand-presenting architecture plays a crit. role in the selectivity of the multivalent complex through decrease of the entropic penalty of binding. Exploiting the precision in spatial design inherent to the DNA nanotechnol., we engineered a library of rigid architectures to explore how valency, affinity, and nano-spacing control the presence of super-selectivity in multivalent binding. A micromolar monovalent affinity was required for super-selective binding to be obsd. within low-valency systems, and the transition point for stable interactions was measured at hexavalent ligand presentation, setting the limits of the low-valency regime. Super-selective binding was obsd. for all hexavalent architectures, and, more strikingly, the ligand pattern detd. the selectivity onset. Hereby, we demonstrate for the first time that nano-control of geometric patterns can be used to discriminate between receptor densities in a super-selective manner. Materials that were indistinguishable in their mol. compn. and ligand valency bound with various efficacies on surfaces with const. receptor densities. We define this new phenomenon in super-selective binding as multivalent pattern recognition.
- 47Reinhardt, S. C. M.; Masullo, L. A.; Baudrexel, I.; Steen, P. R.; Kowalewski, R.; Eklund, A. S.; Strauss, S.; Unterauer, E. M.; Schlichthaerle, T.; Strauss, M. T.; Klein, C.; Jungmann, R. Ångström-Resolution Fluorescence Microscopy. Nature 2023, 617 (7962), 711– 716, DOI: 10.1038/s41586-023-05925-947https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhtVyisr%252FF&md5=9b60751fedd2939b888ccf218e922e8cAngstrom-resolution fluorescence microscopyReinhardt, Susanne C. M.; Masullo, Luciano A.; Baudrexel, Isabelle; Steen, Philipp R.; Kowalewski, Rafal; Eklund, Alexandra S.; Strauss, Sebastian; Unterauer, Eduard M.; Schlichthaerle, Thomas; Strauss, Maximilian T.; Klein, Christian; Jungmann, RalfNature (London, United Kingdom) (2023), 617 (7962), 711-716CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Fluorescence microscopy, with its mol. specificity, is one of the major characterization methods used in the life sciences to understand complex biol. systems. Super-resoln. approaches1-6 can achieve resoln. in cells in the range of 15 to 20 nm, but interactions between individual biomols. occur at length scales below 10 nm and characterization of intramol. structure requires Angstrom resoln. State-of-the-art super-resoln. implementations7-14 have demonstrated spatial resolns. down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolns. do not directly translate to expts. in cells, and Angstrom resoln. has not been demonstrated to date. Here we introdue a DNA-barcoding method, resoln. enhancement by sequential imaging (RESI), that improves the resoln. of fluorescence microscopy down to the Angstrom scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolns. of >15 nm, we demonstrate that single-protein resoln. can be achieved for biomols. in whole intact cells. Furthermore, we exptl. resolve the DNA backbone distance of single bases in DNA origami with Ångstrom resoln. We use our method in a proof-of-principle demonstration to map the mol. arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the mol. mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramol. imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resoln. microscopy and structural biol. studies and thus delivers information key to understanding complex biol. systems.
- 48Magnez, R.; Thiroux, B.; Taront, S.; Segaoula, Z.; Quesnel, B.; Thuru, X. PD-1/PD-L1 Binding Studies Using Microscale Thermophoresis. Sci. Rep 2017, 7 (1), 17623 DOI: 10.1038/s41598-017-17963-148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MzitFemug%253D%253D&md5=d5d0ace31a28ebc7f260cced3fce2f03PD-1/PD-L1 binding studies using microscale thermophoresisMagnez Romain; Thiroux Bryan; Taront Solenne; Segaoula Zacharie; Quesnel Bruno; Thuru Xavier; Magnez Romain; Thiroux Bryan; Taront Solenne; Segaoula Zacharie; Quesnel Bruno; Thuru Xavier; Quesnel BrunoScientific reports (2017), 7 (1), 17623 ISSN:.The characterization of protein interactions has become essential in many fields of life science, especially drug discovery. Microscale thermophoresis (MST) is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. In addition, one of the major advantages of this technique is that no tedious purification step is necessary to access the protein of interest. Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 couple, which is involved in tumour escape processes, without purification of the target protein from cell lysates. The method requires the overexpression of fluorescent proteins in CHO-K1 cells and describes the optimal conditions for determining the dissociation constant. The protocol has a variety of potential applications in studying the interactions of these proteins with small molecules and demonstrates that MST is a valuable method for studying the PD-1/PD-L1 pathway.
- 49Lázár-Molnár, E.; Scandiuzzi, L.; Basu, I.; Quinn, T.; Sylvestre, E.; Palmieri, E.; Ramagopal, U. A.; Nathenson, S. G.; Guha, C.; Almo, S. C. Structure-Guided Development of a High-Affinity Human Programmed Cell Death-1: Implications for Tumor Immunotherapy. EBioMedicine 2017, 17, 30– 44, DOI: 10.1016/j.ebiom.2017.02.00449https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1czgt1ehtw%253D%253D&md5=4bb2f1751befebc4a0a73b51241619dcStructure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapyLazar-Molnar Eszter; Sylvestre Eliezer; Palmieri Edith; Scandiuzzi Lisa; Basu Indranil; Quinn Thomas; Ramagopal Udupi A; Nathenson Stanley G; Guha Chandan; Almo Steven CEBioMedicine (2017), 17 (), 30-44 ISSN:.Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.
- 50Butte, M. J.; Keir, M. E.; Phamduy, T. B.; Sharpe, A. H.; Freeman, G. J. Programmed Death-1 Ligand 1 Interacts Specifically with the B7–1 Costimulatory Molecule to Inhibit T Cell Responses. Immunity 2007, 27 (1), 111– 122, DOI: 10.1016/j.immuni.2007.05.01650https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXosFeqtrk%253D&md5=8fabef0e1244a1d356378867d92a248eProgrammed Death-1 Ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responsesButte, Manish J.; Keir, Mary E.; Phamduy, Theresa B.; Sharpe, Arlene H.; Freeman, Gordon J.Immunity (2007), 27 (1), 111-122CODEN: IUNIEH; ISSN:1074-7613. (Cell Press)Pathways in the B7:CD28 family of costimulatory mols. regulate T cell activation and tolerance. B7-dependent responses in Cd28-/- Ctla4-/- T cells together with reports of stimulatory and inhibitory functions for Programmed Death-1 Ligand 1 or 2 mols. (PD-L1 or PD-L2) have suggested addnl. receptors for these B7 family members. The authors show that B7-1 and PD-L1 interacted with affinity intermediate to that of B7-1:CD28 and B7-1:CTLA-4. The PD-L1:B7-1 interface overlapped with the B7-1:CTLA-4 and PD-L1:PD-1 (Programmed Death-1) interfaces. T cell activation and cytokine prodn. were inhibited by the interaction of B7-1 with PD-L1. The responses of PD-1-deficient vs. PD-1,B7-1 double-deficient T cells to PD-L1 and of CD28,CTLA-4 double-deficient vs. CD28,CTLA-4,PD-L1 triple-deficient T cells to B7-1 demonstrated that PD-L1 and B7-1 interact specifically to inhibit T cell activation. The authors' findings point to a substantial bidirectional inhibitory interaction between B7-1 and PD-L1 and add an addnl. dimension to immunoregulatory functions of the B7:CD28 family.
- 51Ponnuswamy, N.; Bastings, M. M. C.; Nathwani, B.; Ryu, J. H.; Chou, L. Y. T.; Vinther, M.; Li, W. A.; Anastassacos, F. M.; Mooney, D. J.; Shih, W. M. Oligolysine-Based Coating Protects DNA Nanostructures from Low-Salt Denaturation and Nuclease Degradation. Nat. Commun. 2017, 8 (1), 15654 DOI: 10.1038/ncomms1565451https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXovFCls7Y%253D&md5=253bfcf6f3cb7cd9632092bad7624b0eOligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradationPonnuswamy, Nandhini; Bastings, Maartje M. C.; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y. T.; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M.; Mooney, David J.; Shih, William M.Nature Communications (2017), 8 (), 15654CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviors. However, their utility in biol. fluids can be compromised through denaturation induced by physiol. salt concns. and degrdn. mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.
- 52Ueno, H.; Klechevsky, E.; Morita, R.; Aspord, C.; Cao, T.; Matsui, T.; Di Pucchio, T.; Connolly, J.; Fay, J. W.; Pascual, V.; Palucka, A. K.; Banchereau, J. Dendritic Cell Subsets in Health and Disease. Immunological Reviews 2007, 219 (1), 118– 142, DOI: 10.1111/j.1600-065X.2007.00551.x52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1Cnt7zI&md5=0231ed1978d62f49a2aa5ba959d92d30Dendritic cell subsets in health and diseaseUeno, Hideki; Klechevsky, Eynav; Morita, Rimpei; Aspord, Caroline; Cao, Tinghua; Matsui, Toshimichi; Di Pucchio, Tiziana; Connolly, John; Fay, Joseph W.; Pascual, Virginia; Palucka, A. Karolina; Banchereau, JacquesImmunological Reviews (2007), 219 (), 118-142CODEN: IMRED2; ISSN:0105-2896. (Blackwell Publishing Ltd.)A review. The dendritic cell (DC) system of antigen-presenting cells controls immunity and tolerance. DCs initiate and regulate immune responses in a manner that depends on signals they receive from microbes and their cellular environment. They allow the immune system to make qual. distinct responses against different microbial infections. DCs are composed of subsets that express different microbial receptors and express different surface mols. and cytokines. Our studies lead us to propose that interstitial (dermal) DCs preferentially activate humoral immunity, whereas Langerhans cells preferentially induce cellular immunity. Alterations of the DC system result in diseases such as autoimmunity, allergy, and cancer. Conversely, DCs can be exploited for vaccination, and novel vaccines that directly target DCs in vivo are being designed.
- 53Domogalla, M. P.; Rostan, P. V.; Raker, V. K.; Steinbrink, K. Tolerance through Education: How Tolerogenic Dendritic Cells Shape Immunity. Front. Immunol. 2017, DOI: 10.3389/fimmu.2017.01764There is no corresponding record for this reference.
- 54Iberg, C. A.; Hawiger, D. Natural and Induced Tolerogenic Dendritic Cells. J. Immunol. 2020, 204 (4), 733– 744, DOI: 10.4049/jimmunol.190112154https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXks1SgsL0%253D&md5=eaf2afc58e975a8879c3da4b741f7398Natural and induced tolerogenic dendritic cellsIberg, Courtney A.; Hawiger, DanielJournal of Immunology (2020), 204 (4), 733-744CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)A review. Dendritic cells (DCs) are highly susceptible to extrinsic signals that modify the functions of these crucial APCs. Maturation of DCs induced by diverse proinflammatory conditions promotes immune responses, but certain signals also induce tolerogenic functions in DCs. These "induced tolerogenic DCs" help to moderate immune responses such as those to commensals present at specific anatomical locations. However, also under steady-state conditions, some DCs are characterized by inherent tolerogenic properties. The immunomodulatory mechanisms constitutively present in such "natural tolerogenic DCs" help to promote tolerance to peripheral Ags. By extending tolerance initially established in the thymus, these functions of DCs help to regulate autoimmune and other immune responses. In this review we will discuss the mechanisms and functions of natural and induced tolerogenic DCs and offer further insight into how their possible manipulations may ultimately lead to more precise treatments for various immune-mediated conditions and diseases.
- 55Koga, M. M.; Engel, A.; Pigni, M.; Lavanchy, C.; Stevanin, M.; Laversenne, V.; Schneider, B. L.; Acha-Orbea, H. IL10- and IL35-Secreting MutuDC Lines Act in Cooperation to Inhibit Memory T Cell Activation Through LAG-3 Expression. Front. Immunol. 2021, DOI: 10.3389/fimmu.2021.607315There is no corresponding record for this reference.
- 56Zhao, Y.; Lee, C. K.; Lin, C.-H.; Gassen, R. B.; Xu, X.; Huang, Z.; Xiao, C.; Bonorino, C.; Lu, L.-F.; Bui, J. D.; Hui, E. PD-L1:CD80 Cis-Heterodimer Triggers the Co-Stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways. Immunity 2019, 51 (6), 1059– 1073, DOI: 10.1016/j.immuni.2019.11.00356https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFylsL%252FE&md5=bc7d76d40f9923ffaaafb12934663e7aPD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 PathwaysZhao, Yunlong; Lee, Calvin K.; Lin, Chia-Hao; Gassen, Rodrigo B.; Xu, Xiaozheng; Huang, Zhe; Xiao, Changchun; Bonorino, Cristina; Lu, Li-Fan; Bui, Jack D.; Hui, EnfuImmunity (2019), 51 (6), 1059-1073.e9CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)Combined immunotherapy targeting the immune checkpoint receptors cytotoxic T-lymphocyte-assocd. protein 4 (CTLA-4) and programmed cell death 1 (PD-1), or CTLA-4 and the PD-1 ligand (PD-L1) exhibits superior anti-tumor responses compared with single-agent therapy. Here, we examd. the mol. basis for this synergy. Using reconstitution assays with fluorescence readouts, we found that PD-L1 and the CTLA-4 ligand CD80 heterodimerize in cis but not trans. Quant. biochem. and cell biol. assays revealed that PD-L1:CD80 cis-heterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the T cell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.
- 57Sansom, D. M.; Walker, L. S. K. Dimers Aren’t Forever: CD80 Breaks up with PD-L1. Immunity 2019, 51 (6), 972– 974, DOI: 10.1016/j.immuni.2019.11.01157https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtVGlsbzL&md5=429f5e91cd86fcd2cb44adb66b54a75dDimers Aren't Forever: CD80 Breaks up with PD-L1Sansom, David M.; Walker, Lucy S. K.Immunity (2019), 51 (6), 972-974CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)A review. Targeting the CTLA-4 and PD-1 "checkpoints" is an effective treatment for a no. of cancers. In this issue of Immunity, Hui et al. reveal that interaction between a CTLA-4 ligand, CD80, and its counterpart in the PD-1 pathway, PD-L1, affects both PD-1 and CTLA-4 function, raising new questions about the biol. effects of using checkpoint inhibitors alone and in combination.
- 58Martinez-Veracoechea, F. J.; Frenkel, D. Designing Super Selectivity in Multivalent Nano-Particle Binding. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (27), 10963– 10968, DOI: 10.1073/pnas.110535110858https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptV2htrY%253D&md5=79356231cc82dd3344d53f2a85e5daadDesigning super selectivity in multivalent nano-particle bindingMartinez-Veracoechea, Francisco J.; Frenkel, DaanProceedings of the National Academy of Sciences of the United States of America (2011), 108 (27), 10963-10968, S10963/1-S10963/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A key challenge in nano-science is to design ligand-coated nano-particles that can bind selectively to surfaces that display the cognate receptors above a threshold (surface) concn. Nano-particles that bind monovalently to a target surface do not discriminate sharply between surfaces with high and low receptor coverage. In contrast, "multivalent" nano-particles that can bind to a larger no. of ligands simultaneously, display regimes of "super selectivity" where the fraction of bound particles varies sharply with the receptor concn. We present numerical simulations that show that multivalent nano-particles can be designed such that they approach the 'on-off" binding behavior ideal for receptor-concn. selective targeting. We propose a simple anal. model that accounts for the super selective behavior of multivalent nano-particles. The model shows that the super selectivity is due to the fact that the no. of distinct ligand-receptor binding arrangements increases in a highly nonlinear way with receptor coverage. Somewhat counterintuitively, our study shows that selectivity can be improved by making the individual ligand-receptor bonds weaker. We propose a simple rule of thumb to predict the conditions under which super selectivity can be achieved. We validate our model predictions against the Monte Carlo simulations.
- 59Curk, T.; Dobnikar, J.; Frenkel, D. Optimal Multivalent Targeting of Membranes with Many Distinct Receptors. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (28), 7210– 7215, DOI: 10.1073/pnas.170422611459https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhsrnI&md5=588b429ecc50b542e70dda1ef2e56826Optimal multivalent targeting of membranes with many distinct receptorsCurk, Tine; Dobnikar, Jure; Frenkel, DaanProceedings of the National Academy of Sciences of the United States of America (2017), 114 (28), 7210-7215CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Cells can often be recognized by the concns. of receptors expressed on their surface. For better (targeted drug treatment) or worse (targeted infection by pathogens), it is clearly important to be able to target cells selectively. A good targeting strategy would result in strong binding to cells with the desired receptor profile and barely binding to other cells. Using a simple model, the authors formulate optimal design rules for multivalent particles that allow them to distinguish target cells based on their receptor profile. The authors found the following: (1) It is not a good idea to aim for very strong binding between the individual ligands on the guest (delivery vehicle) and the receptors on the host (cell). Rather, one should exploit multivalency: High sensitivity to the receptor d. on the host can be achieved by coating the guest with many ligands that bind only weakly to the receptors on the cell surface. (2) The concn. profile of the ligands on the guest should closely match the compn. of the cognate membrane receptors on the target surface. And (3) irresp. of all details, the effective strength of the ligand-receptor interaction should be of the order of the thermal energy kBT, where T is the abs. temp. and kB is Boltzmann's const. The authors present simulations that support the theor. predictions. The authors speculate that, using the above design rules, it should be possible to achieve targeted drug delivery with a greatly reduced incidence of side effects.
- 60Dubacheva, G. V.; Curk, T.; Richter, R. P. Determinants of Superselectivity─Practical Concepts for Application in Biology and Medicine. Acc. Chem. Res. 2023, 56 (7), 729– 739, DOI: 10.1021/acs.accounts.2c0067260https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXkvFyktr4%253D&md5=82e46f198147e0ce1021a8fd4223c987Determinants of Superselectivity-Practical Concepts for Application in Biology and MedicineDubacheva, Galina V.; Curk, Tine; Richter, Ralf P.Accounts of Chemical Research (2023), 56 (7), 729-739CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)Multivalent interactions are common in biol. systems and are also widely deployed for targeting applications in biomedicine. A unique feature of multivalent binding is "superselectivity". Superselectivity refers to the sharp discrimination of surfaces (e.g., on cells or cell compartments) by their comparative surface densities of a given receptor. This feature is different from the conventional "type" selectivity, which discriminates surfaces by their distinct receptor types. In a broader definition, a probe is superselective if it converts a gradual change in any one interaction parameter into a sharp on/off dependency in probe binding. This Account describes our systematic exptl. and theorotical efforts over the past decade to analyze the determinants of superselective binding. It aims to offer chem. biologists, biophysicists, biologists, and biomedical scientists a set of guidelines for the interpretation of multivalent binding data, and design rules for tuning superselective targeting. We first provide a basic introduction that identifies multiple low-affinity interactions and combinatorial entropy as the minimal set of conditions required for superselective recognition. We then introduce the main exptl. and theteoritical tools and analyze how salient features of the multivalent probes (i.e., their concn., size, ligand valency, and scaffold type), of the surface receptors (i.e., their affinity for ligands, surface d., and mobility), and of competitors and cofactors (i.e., their concn. and affinity for the ligands and/or receptors) influence the sharpness and the position of the threshold for superselective recognition. Emerging from this work are a set of relatively simple yet quantative data anal. guidelines and superselectivity design rules that apply to a broad range of probe types and interaction systems. The key finding is the scaling variable xS which faithfully predicts the influence of the surface receptor d., probe ligand valency, receptor-ligand affinity, and competitor/cofactor concns. and affinities on superselective recognition. The scaling variable is a simple yet versatile tool to quant. tune the on/off threshold of superselective probes. We exemplify its application by reviewing and reinterpreting literature data for selected biol. and biomedical interaction systems where superselectivity clearly is important. Our guidelines can be deployed to generate a new mechanistic understanding of multivalent recognition events inside and outside cells and the downstream physiol./pathol. implications. Moreover, the design rules can be harnessed to develop novel superselective probes for anal. purposes in the life sciences and for diagnostic/therapeutic intervention in biomedicine.
- 61Strauss, S.; Jungmann, R. Up to 100-Fold Speed-up and Multiplexing in Optimized DNA-PAINT. Nat. Methods 2020, 17 (8), 789– 791, DOI: 10.1038/s41592-020-0869-x61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1yjtbfO&md5=14599350694abb5936e9b187d8ce7c15Up to 100-fold speed-up and multiplexing in optimized DNA-PAINTStrauss, Sebastian; Jungmann, RalfNature Methods (2020), 17 (8), 789-791CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)DNA-PAINT's imaging speed has recently been significantly enhanced by optimized sequence design and buffer conditions. However, this implementation has not reached an ultimate speed limit and is only applicable to imaging of single targets. To further improve acquisition speed, we introduce concatenated, periodic DNA sequence motifs, yielding up to 100-fold-faster sampling in comparison to traditional DNA-PAINT. We extend this approach to six orthogonal sequence motifs, now enabling speed-optimized multiplexed imaging.
- 62Fabricius, V.; Lefèbre, J.; Geertsema, H.; Marino, S. F.; Ewers, H. Rapid and Efficient C-Terminal Labeling of Nanobodies for DNA-PAINT. J. Phys. D: Appl. Phys. 2018, 51 (47), 474005 DOI: 10.1088/1361-6463/aae0e262https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlWrtrrO&md5=e4e74321222ca126b2005abad174b4f6Rapid and efficient C-terminal labeling of nanobodies for DNA-PAINTFabricius, Valentin; Lefebre, Jonathan; Geertsema, Hylkje; Marino, Stephen F.; Ewers, HelgeJournal of Physics D: Applied Physics (2018), 51 (47), 474005/1-474005/8CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)Single mol. localization-based approaches to super-resoln. microscopy (SMLM) create images that resolve features smaller than the diffraction limit of light by rendering them from the sequentially measured positions of thousands of individual mols. New SMLM approaches based on the transient binding of very bright dyes via DNA-DNA interaction (DNA-PAINT) allow the resoln. of dyes only a few nanometers apart in vitro. This imaging of cellular structures requires the specific assocn. of dyes to their targets, which results in an addnl. 'linkage error'. This error can be minimized by using extremely small, single-domain antibody-based binders such as nanobodies, but the DNA-oligomers used in DNA-PAINT are of significant size in comparison to nanobodies and may interfere with binding. We have developed an optimized procedure based on enzymic labeling and click-chem. for the coupling of DNA oligomers to the nanobody C-terminus, which is located on the opposite side of the epitope-binding domain. Our approach allows for straightforward labeling, purifn. and DNA-PAINT imaging. We performed high efficiency labeling of two different nanobodies and show dual color multiplexed SMLM to demonstrate the general applicability of our labeling scheme.
- 63Eklund, A. S.; Ganji, M.; Gavins, G.; Seitz, O.; Jungmann, R. Peptide-PAINT Super-Resolution Imaging Using Transient Coiled Coil Interactions. Nano Lett. 2020, 20 (9), 6732– 6737, DOI: 10.1021/acs.nanolett.0c0262063https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVKntLrM&md5=541b25b2df246c513591ead462186ea5Peptide-PAINT Super-Resolution Imaging Using Transient Coiled Coil InteractionsEklund, Alexandra S.; Ganji, Mahipal; Gavins, Georgina; Seitz, Oliver; Jungmann, RalfNano Letters (2020), 20 (9), 6732-6737CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Super-resoln. microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale. DNA-PAINT is a relatively easy-to-implement single-mol.-based technique, which uses the programmable and transient interaction of dye-labeled oligonucleotides with their complements for super-resoln. imaging. However, similar to many imaging approaches, it is still hampered by the subpar performance of labeling probes in terms of their large size and limited labeling efficiency. To overcome this, the authors here translate the programmability and transient binding nature of DNA-PAINT to coiled coil interactions of short peptides and introduce Peptide-PAINT. The authors benchmark and optimize its binding kinetics in a single-mol. assay and demonstrate its super-resoln. capability using self-assembled DNA origami structures. Peptide-PAINT outperforms classical DNA-PAINT in terms of imaging speed and efficiency. Finally, the authors prove the suitability of Peptide-PAINT for cellular super-resoln. imaging by visualizing the microtubule and vimentin network in fixed cells.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.3c06552.
PD-L1 density on DCs measured by DNA-PAINT; DNA origami designs and characterization by AGE; PD-1-DNA conjugate characterization by PAGE; AGE analysis of PD-1 DNA disks; DNA-PAINT overview images of hexavalent PD-1 DNA disk pattern library and controls; SPR binding sensorgrams of PD-1 and PD-1-DNA conjugate; control DC staining experiments for PD-1 DNA disks and antibody DNA disks; characterization of immune markers on WT DC, IL-10 DC, and B16 by flow cytometry; additional data on cytokine production in immune checkpoint blockade assay; PD-1 and SrtA purification data; K10-PEG1K DNA disk coating characterization by AGE; sequences for DNA origami disk constructs used in this work; imaging parameters used in DNA-PAINT experiments (PDF)
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