John Hickey

All Publications

• Annotation of spatially resolved single-cell data with STELLAR. Nature methods Brbic, M., Cao, K., Hickey, J. W., Tan, Y., Snyder, M. P., Nolan, G. P., Leskovec, J. 2022

  Abstract

  Accurate cell-type annotation from spatially resolved single cells is crucial to understand functional spatial biology that is the basis of tissue organization. However, current computational methods for annotating spatially resolved single-cell data are typically based on techniques established for dissociated single-cell technologies and thus do not take spatial organization into account. Here we present STELLAR, a geometric deep learning method for cell-type discovery and identification in spatially resolved single-cell datasets. STELLAR automatically assigns cells to cell types present in the annotated reference dataset and discovers novel cell types and cell states. STELLAR transfers annotations across different dissection regions, different tissues and different donors, and learns cell representations that capture higher-order tissue structures. We successfully applied STELLAR to CODEX multiplexed fluorescent microscopy data and multiplexed RNA imaging datasets. Within the Human BioMolecular Atlas Program, STELLAR has annotated 2.6million spatially resolved single cells with dramatic time savings.

  View details for DOI 10.1038/s41592-022-01651-8

  View details for PubMedID 36280720

• Multicellular modules as clinical diagnostic and therapeutic targets. Trends in cancer Baertsch, M., Nolan, G. P., Hickey, J. W. 2021

  Abstract

  The complex determinants of health and disease can be determined when approached as a system of interactions of biological agents at different scales. Similar to the physicochemical properties that govern nucleic acids and proteins, there should be a finite set of rules that dictate the behavior of cells to form tissues. Thus, the occurrence of disease can be seen as flaws in processes that are governed by rules pertaining to multicellular structures. Multiplexed imaging is a technology that connects information that bridges multiple biological scales (i.e., molecules, cells, and tissues) and enables elucidation of rules associated with the formation of multicellular structures. Uncovering important multicellular structures associated with disease will propel a wave of development of new categories of diagnostics and therapeutics.

  View details for DOI 10.1016/j.trecan.2021.11.004

  View details for PubMedID 34872889

• Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging. Nature methods Hickey, J. W., Neumann, E. K., Radtke, A. J., Camarillo, J. M., Beuschel, R. T., Albanese, A., McDonough, E., Hatler, J., Wiblin, A. E., Fisher, J., Croteau, J., Small, E. C., Sood, A., Caprioli, R. M., Angelo, R. M., Nolan, G. P., Chung, K., Hewitt, S. M., Germain, R. N., Spraggins, J. M., Lundberg, E., Snyder, M. P., Kelleher, N. L., Saka, S. K. 2021

  Abstract

  Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.

  View details for DOI 10.1038/s41592-021-01316-y

  View details for PubMedID 34811556

• CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nature protocols Black, S., Phillips, D., Hickey, J. W., Kennedy-Darling, J., Venkataraaman, V. G., Samusik, N., Goltsev, Y., Schurch, C. M., Nolan, G. P. 2021

  Abstract

  Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.

  View details for DOI 10.1038/s41596-021-00556-8

  View details for PubMedID 34215862

• Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction. European journal of immunology Kennedy-Darling, J. n., Bhate, S. S., Hickey, J. W., Black, S. n., Barlow, G. L., Vazquez, G. n., Venkataraaman, V. G., Samusik, N. n., Goltsev, Y. n., Schürch, C. M., Nolan, G. P. 2021

  Abstract

  Multiparameter tissue imaging enables analysis of cell-cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO-Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a lymph node. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single-cell analysis on the data, including unsupervised clustering that revealed 31 cell types across all tissues. We compared cell-type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell-cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre-existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/eji.202048891

  View details for PubMedID 33548142

• Strategies for Accurate Cell Type Identification in CODEX Multiplexed Imaging Data. Frontiers in immunology Hickey, J. W., Tan, Y., Nolan, G. P., Goltsev, Y. 2021; 12: 727626

  Abstract

  Multiplexed imaging is a recently developed and powerful single-cell biology research tool. However, it presents new sources of technical noise that are distinct from other types of single-cell data, necessitating new practices for single-cell multiplexed imaging processing and analysis, particularly regarding cell-type identification. Here we created single-cell multiplexed imaging datasets by performing CODEX on four sections of the human colon (ascending, transverse, descending, and sigmoid) using a panel of 47 oligonucleotide-barcoded antibodies. After cell segmentation, we implemented five different normalization techniques crossed with four unsupervised clustering algorithms, resulting in 20 unique cell-type annotations for the same dataset. We generated two standard annotations: hand-gated cell types and cell types produced by over-clustering with spatial verification. We then compared these annotations at four levels of cell-type granularity. First, increasing cell-type granularity led to decreased labeling accuracy; therefore, subtle phenotype annotations should be avoided at the clustering step. Second, accuracy in cell-type identification varied more with normalization choice than with clustering algorithm. Third, unsupervised clustering better accounted for segmentation noise during cell-type annotation than hand-gating. Fourth, Z-score normalization was generally effective in mitigating the effects of noise from single-cell multiplexed imaging. Variation in cell-type identification will lead to significant differential spatial results such as cellular neighborhood analysis; consequently, we also make recommendations for accurately assigning cell-type labels to CODEX multiplexed imaging.

  View details for DOI 10.3389/fimmu.2021.727626

  View details for PubMedID 34484237

• Adaptive Nanoparticle Platforms for High Throughput Expansion and Detection of Antigen-Specific T cells NANO LETTERS Hickey, J. W., Isser, A., Salathe, S. F., Gee, K. M., Hsiao, M., Shaikh, W., Uzoukwu, N. C., Bieler, J., Mao, H., Schneck, J. P. 2020; 20 (9): 6289–98

  Abstract

  T cells are critical players in disease; yet, their antigen-specificity has been difficult to identify, as current techniques are limited in terms of sensitivity, throughput, or ease of use. To address these challenges, we increased the throughput and translatability of magnetic nanoparticle-based artificial antigen presenting cells (aAPCs) to enrich and expand (E+E) murine or human antigen-specific T cells. We streamlined enrichment, expansion, and aAPC production processes by enriching CD8+ T cells directly from unpurified immune cells, increasing parallel processing capacity of aAPCs in a 96-well plate format, and designing an adaptive aAPC that enables multiplexed aAPC construction for E+E and detection. We applied these adaptive platforms to process and detect CD8+ T cells specific for rare cancer neoantigens, commensal bacterial cross-reactive epitopes, and human viral and melanoma antigens. These innovations dramatically increase the multiplexing ability and decrease the barrier to adopt for investigating antigen-specific T cell responses.

  View details for DOI 10.1021/acs.nanolett.0c01511

  View details for Web of Science ID 000571442000008

  View details for PubMedID 32594746

• Engineering an Artificial T-Cell Stimulating Matrix for Immunotherapy ADVANCED MATERIALS Hickey, J. W., Dong, Y., Chung, J., Salathe, S. F., Pruitt, H. C., Li, X., Chang, C., Fraser, A. K., Bessell, C. A., Ewald, A. J., Gerecht, S., Mao, H., Schneck, J. P. 2019; 31 (23)

  View details for DOI 10.1002/adma.201807359

  View details for Web of Science ID 000474087100001

• Efficient magnetic enrichment of antigen-specific T cells by engineering particle properties BIOMATERIALS Hickey, J. W., Isser, A. Y., Vicente, F. P., Warner, S. B., Mao, H., Schneck, J. P. 2018; 187: 105–16

  Abstract

  Magnetic particles can enrich desired cell populations to aid in understanding cell-type functions and mechanisms, diagnosis, and therapy. As cells are heterogeneous in ligand type, location, expression, and density, careful consideration of magnetic particle design for positive isolation is necessary. Antigen-specific immune cells have low frequencies, which has made studying, identifying, and utilizing these cells for therapy a challenge. Here we demonstrate the importance of magnetic particle design based on the biology of T cells. We create magnetic particles which recognize rare antigen-specific T cells and quantitatively investigate important particle properties including size, concentration, ligand density, and ligand choice in enriching these rare cells. We observe competing optima among particle parameters, with 300 nm particles functionalized with a high density of antigen-specific ligand achieving the highest enrichment and recovery of target cells. In enriching and then activating an endogenous response, 300 nm aAPCs generate nearly 65% antigen-specific T cells with at least 450-fold expansion from endogenous precursors and a 5-fold increase in numbers of antigen-specific cells after only seven days. This systematic study of particle properties in magnetic enrichment provides a case study for the engineering design principles of particles for the isolation of rare cells through biological ligands.

  View details for PubMedID 30312851

  View details for PubMedCentralID PMC6284398

• Biologically Inspired Design of Nanoparticle Artificial Antigen-Presenting Cells for Immunomodulation NANO LETTERS Hickey, J. W., Vicente, F. P., Howard, G. P., Mao, H., Schneck, J. P. 2017; 17 (11): 7045–54

  Abstract

  Particles engineered to engage and interact with cell surface ligands and to modulate cells can be harnessed to explore basic biological questions as well as to devise cellular therapies. Biology has inspired the design of these particles, such as artificial antigen-presenting cells (aAPCs) for use in immunotherapy. While much has been learned about mimicking antigen presenting cell biology, as we decrease the size of aAPCs to the nanometer scale, we need to extend biomimetic design to include considerations of T cell biology-including T-cell receptor (TCR) organization. Here we describe the first quantitative analysis of particle size effect on aAPCs with both Signals 1 and 2 based on T cell biology. We show that aAPCs, larger than 300 nm, activate T cells more efficiently than smaller aAPCs, 50 nm. The 50 nm aAPCs require saturating doses or require artificial magnetic clustering to activate T cells. Increasing ligand density alone on the 50 nm aAPCs did not increase their ability to stimulate CD8+ T cells, confirming the size-dependent phenomenon. These data support the need for multireceptor ligation and activation of T-cell receptor (TCR) nanoclusters of similar sizes to 300 nm aAPCs. Quantitative analysis and modeling of a nanoparticle system provides insight into engineering constraints of aAPCs for T cell immunotherapy applications and offers a case study for other cell-modulating particles.

  View details for PubMedID 28994285

• Physioxia improves the selectivity of hematopoietic stem cell expansion cultures. Blood advances Igarashi, K. J., Kucinski, I., Chan, Y. Y., Tan, T., Khoo, H. M., Kealy, D., Bhadury, J., Hsu, I., Ho, P. Y., Niizuma, K., Hickey, J. W., Nolan, G., Bridge, K. S., Czechowicz, A., Gottgens, B., Nakauchi, H., Wilkinson, A. C. 2023

  Abstract

  Hematopoietic stem cells (HSCs) are a rare hematopoietic cell type that can entirely reconstitute the blood and immune systems following transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases, but remains a high-risk therapy due to potential side effects including poor graft function and graft-vs-host disease (GvHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution from low-cell dose grafts. Here, we demonstrate that we can improve the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures through the use of physioxic culture conditions. Single-cell transcriptomic analysis confirmed inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GvHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, as well as highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT.

  View details for DOI 10.1182/bloodadvances.2023009668

  View details for PubMedID 36809781

• Shape Matters: Biodegradable Anisotropic Nanoparticle Artificial Antigen Presenting Cells for Cancer Immunotherapy. Acta biomaterialia Ben-Akiva, E., Hickey, J. W., Meyer, R. A., Isser, A., Shannon, S. R., Livingston, N. K., Rhodes, K. R., Kosmides, A. K., Warren, T. R., Tzeng, S. Y., Schneck, J. P., Green, J. J. 2023

  Abstract

  Artificial antigen presenting cells are biomimetic particles that recapitulate the signals presented by natural antigen presenting cells in order to stimulate T cells in an antigen-specific manner using an acellular platform. We have engineered an enhanced nanoscale biodegradable artificial antigen presenting cell by modulating particle shape to achieve a nanoparticle geometry that allows for increased radius of curvature and surface area for T cell contact. The non-spherical nanoparticle artificial antigen presenting cells developed here have reduced nonspecific uptake and improved circulation time compared both to spherical nanoparticles and to traditional microparticle technologies. Additionally, the anisotropic nanoparticle artificial antigen presenting cells efficiently engage with and activate T cells, ultimately leading to a marked anti-tumor effect in a mouse melanoma model that their spherical counterparts were unable to achieve. STATEMENT OF SIGNIFICANCE: Artificial antigen presenting cells (aAPC) can activate antigen-specific CD8+ T cells but have largely been limited to microparticle-based platforms and ex vivo T cell expansion. Although more amenable to in vivo use, nanoscale aAPC have traditionally been ineffective due to limited surface area available for T cell interaction. In this work, we engineered non-spherical biodegradable nanoscale aAPC to investigate the role of particle geometry and develop a translatable platform for T cell activation. The non-spherical aAPC developed here have increased surface area and a flatter surface for T cell engagement and, therefore, can more effectively stimulate antigen-specific T cells, resulting in anti-tumor efficacy in a mouse melanoma model.

  View details for DOI 10.1016/j.actbio.2023.02.023

  View details for PubMedID 36812956

• IN VIVO EXPANSION OF ENDOGENOUS ANTIGENSPECIFIC CD8+T CELLS USING ARTIFICIAL T-CELL STIMULATING MICROPARTICLES Livingston, N., Hickey, J., Sim, H., Mao, H., Schneck, J. BMJ PUBLISHING GROUP. 2022: A271

  View details for Web of Science ID 000919423400252

• A real-time GPU-accelerated parallelized image processor for large-scale multiplexed fluorescence microscopy data. Frontiers in immunology Lu, G., Baertsch, M. A., Hickey, J. W., Goltsev, Y., Rech, A. J., Mani, L., Forgo, E., Kong, C., Jiang, S., Nolan, G. P., Rosenthal, E. L. 2022; 13: 981825

  Abstract

  Highly multiplexed, single-cell imaging has revolutionized our understanding of spatial cellular interactions associated with health and disease. With ever-increasing numbers of antigens, region sizes, and sample sizes, multiplexed fluorescence imaging experiments routinely produce terabytes of data. Fast and accurate processing of these large-scale, high-dimensional imaging data is essential to ensure reliable segmentation and identification of cell types and for characterization of cellular neighborhoods and inference of mechanistic insights. Here, we describe RAPID, a Real-time, GPU-Accelerated Parallelized Image processing software for large-scale multiplexed fluorescence microscopy Data. RAPID deconvolves large-scale, high-dimensional fluorescence imaging data, stitches and registers images with axial and lateral drift correction, and minimizes tissue autofluorescence such as that introduced by erythrocytes. Incorporation of an open source CUDA-driven, GPU-assisted deconvolution produced results similar to fee-based commercial software. RAPID reduces data processing time and artifacts and improves image contrast and signal-to-noise compared to our previous image processing pipeline, thus providing a useful tool for accurate and robust analysis of large-scale, multiplexed, fluorescence imaging data.

  View details for DOI 10.3389/fimmu.2022.981825

  View details for PubMedID 36211386

• Rhesus Macaque CODEX Multiplexed Immunohistochemistry Panel for Studying Immune Responses During Ebola Infection FRONTIERS IN IMMUNOLOGY Jiang, S., Mukherjee, N., Bennett, R. S., Chen, H., Logue, J., Dighero-Kemp, B., Kurtz, J. R., Adams, R., Phillips, D., Schuerch, C. M., Goltsev, Y., Hickey, J. W., McCaffrey, E. F., Delmastro, A., Chu, P., Reader, J., Keesler, R., Galvan, J. A., Zlobec, I., Van Rompay, K. A., Liu, D. X., Hensley, L. E., Nolan, G. P., McIlwain, D. R. 2021; 12: 729845

  Abstract

  Non-human primate (NHP) animal models are an integral part of the drug research and development process. For some biothreat pathogens, animal model challenge studies may offer the only possibility to evaluate medical countermeasure efficacy. A thorough understanding of host immune responses in such NHP models is therefore vital. However, applying antibody-based immune characterization techniques to NHP models requires extensive reagent development for species compatibility. In the case of studies involving high consequence pathogens, further optimization for use of inactivated samples may be required. Here, we describe the first optimized CO-Detection by indEXing (CODEX) multiplexed tissue imaging antibody panel for deep profiling of spatially resolved single-cell immune responses in rhesus macaques. This 21-marker panel is composed of a set of 18 antibodies that stratify major immune cell types along with a set three Ebola virus (EBOV)-specific antibodies. We validated these two sets of markers using immunohistochemistry and CODEX in fully inactivated Formalin-Fixed Paraffin-Embedded (FFPE) tissues from mock and EBOV challenged macaques respectively and provide an efficient framework for orthogonal validation of multiple antibody clones using CODEX multiplexed tissue imaging. We also provide the antibody clones and oligonucleotide tag sequences as a valuable resource for other researchers to recreate this reagent set for future studies of tissue immune responses to EBOV infection and other diseases.

  View details for DOI 10.3389/fimmu.2021.729845

  View details for Web of Science ID 000732063400001

  View details for PubMedID 34938283

  View details for PubMedCentralID PMC8685521

• T CELL PHENOTYPE DRIVES RESTRUCTURING OF TUMOR MICROENVIRONMENT TO BALANCE T CELL LONGEVITY AND TUMOR CONTROL: INSIGHTS FROM MULTIPLEXED IMAGING AND MULTI-SCALE AGENT BASED MODELING Hickey, J., Nolan, G., Covert, M., Agmon, E., Horowitz, N., Sunwoo, J. BMJ PUBLISHING GROUP. 2021: A192

  View details for DOI 10.1136/jitc-2021-SITC2021.180

  View details for Web of Science ID 000774877500174

• Anatomical structures, cell types and biomarkers of the Human Reference Atlas. Nature cell biology Borner, K., Teichmann, S. A., Quardokus, E. M., Gee, J. C., Browne, K., Osumi-Sutherland, D., Herr, B. W., Bueckle, A., Paul, H., Haniffa, M., Jardine, L., Bernard, A., Ding, S., Miller, J. A., Lin, S., Halushka, M. K., Boppana, A., Longacre, T. A., Hickey, J., Lin, Y., Valerius, M. T., He, Y., Pryhuber, G., Sun, X., Jorgensen, M., Radtke, A. J., Wasserfall, C., Ginty, F., Ho, J., Sunshine, J., Beuschel, R. T., Brusko, M., Lee, S., Malhotra, R., Jain, S., Weber, G. 2021; 23 (11): 1117-1128

  Abstract

  The Human Reference Atlas (HRA) aims to map all of the cells of the human body to advance biomedical research and clinical practice. This Perspective presents collaborative work by members of 16 international consortia on two essential and interlinked parts of the HRA: (1) three-dimensional representations of anatomy that are linked to (2) tables that name and interlink major anatomical structures, cell types, plus biomarkers (ASCT+B). We discuss four examples that demonstrate the practical utility of the HRA.

  View details for DOI 10.1038/s41556-021-00788-6

  View details for PubMedID 34750582

• INTRAEPITHELIAL GROUP 1 INNATE LYMPHOID CELLS GENERATED IN VITRO EXHIBIT ENHANCED CYTOTOXICITY AND INFILTRATION INTO SOLID TUMOROIDS Horowitz, N., Hickey, J., Sunwoo, J. BMJ PUBLISHING GROUP. 2021: A193

  View details for DOI 10.1136/jitc-2021-SITC2021.181

  View details for Web of Science ID 000774877500175

• Application of machine learning in understanding atherosclerosis: Emerging insights. APL bioengineering Munger, E., Hickey, J. W., Dey, A. K., Jafri, M. S., Kinser, J. M., Mehta, N. N. 2021; 5 (1): 011505

  Abstract

  Biological processes are incredibly complex-integrating molecular signaling networks involved in multicellular communication and function, thus maintaining homeostasis. Dysfunction of these processes can result in the disruption of homeostasis, leading to the development of several disease processes including atherosclerosis. We have significantly advanced our understanding of bioprocesses in atherosclerosis, and in doing so, we are beginning to appreciate the complexities, intricacies, and heterogeneity atherosclerosi. We are also now better equipped to acquire, store, and process the vast amount of biological data needed to shed light on the biological circuitry involved. Such data can be analyzed within machine learning frameworks to better tease out such complex relationships. Indeed, there has been an increasing number of studies applying machine learning methods for patient risk stratification based on comorbidities, multi-modality image processing, and biomarker discovery pertaining to atherosclerotic plaque formation. Here, we focus on current applications of machine learning to provide insight into atherosclerotic plaque formation and better understand atherosclerotic plaque progression in patients with cardiovascular disease.

  View details for DOI 10.1063/5.0028986

  View details for PubMedID 33644628

• Improving Biomedical Engineering Undergraduate Learning Through Use of Online Graduate Engineering Courses During the COVID-19 Pandemic. Biomedical engineering education Nesmith, J. E., Hickey, J. W., Haase, E. 2021: 1–8

  Abstract

  In order to provide undergraduate students with a full, rich online learning experience we adapted pre-existing online content including graduate courses from Johns Hopkins University Engineering for Professionals (JHU EP) program. These online courses were designed using published methodologies and held to a high level of rigor of a Masters-level curriculum. Adapting pre-existing online course material enabled us to more rapidly adapt to the COVID-19 shutdown of in-person education. We adapted content to meet the majority of lab-based learning objectives rather than generating self-recorded lecture material and allowing us to focus faculty time on addressing student needs. Here we discuss benefits, challenges, and methods for replicating these courses, and lessons to be applied in future offerings from this experience.

  View details for DOI 10.1007/s43683-020-00041-w

  View details for PubMedID 33554220

• Biodegradable Cationic Polymer Blends for Fabrication of Enhanced Artificial Antigen Presenting Cells to Treat Melanoma. ACS applied materials & interfaces Rhodes, K. R., Isser, A. n., Hickey, J. W., Ben-Akiva, E. n., Meyer, R. A., Kosmides, A. K., Livingston, N. K., Tzeng, S. Y., Schneck, J. P., Green, J. J. 2021

  Abstract

  Biomimetic biomaterials are being actively explored in the context of cancer immunotherapy because of their ability to directly engage the immune system to generate antitumor responses. Unlike cellular therapies, biomaterial-based immunotherapies can be precisely engineered to exhibit defined characteristics including biodegradability, physical size, and tuned surface presentation of immunomodulatory signals. In particular, modulating the interface between the biomaterial surface and the target biological cell is key to enabling biological functions. Synthetic artificial antigen presenting cells (aAPCs) are promising as a cancer immunotherapy but are limited in clinical translation by the requirement of ex vivo cell manipulation and adoptive transfer of antigen-specific CD8+ T cells. To move toward acellular aAPC technology for in vivo use, we combine poly(lactic-co-glycolic acid) (PLGA) and cationic poly(beta-amino-ester) (PBAE) to form a biodegradable blend based on the hypothesis that therapeutic aAPCs fabricated from a cationic blend may have improved functions. PLGA/PBAE aAPCs demonstrate enhanced surface interactions with antigen-specific CD8+ T cells that increase T cell activation and expansion ex vivo, associated with significantly increased conjugation efficiency of T cell stimulatory signals to the aAPCs. Critically, these PLGA/PBAE aAPCs also expand antigen-specific cytotoxic CD8+ T cells in vivo without the need of adoptive transfer. Treatment with PLGA/PBAE aAPCs in combination with checkpoint therapy decreases tumor growth and extends survival in a B16-F10 melanoma mouse model. These results demonstrate the potential of PLGA/PBAE aAPCs as a biocompatible, directly injectable acellular therapy for cancer immunotherapy.

  View details for DOI 10.1021/acsami.0c19955

  View details for PubMedID 33573372

• Commensal bacteria stimulate antitumor responses via T cell cross-reactivity. JCI insight Bessell, C. A., Isser, A., Havel, J. J., Lee, S., Bell, D. R., Hickey, J. W., Chaisawangwong, W., Glick Bieler, J., Srivastava, R., Kuo, F., Purohit, T., Zhou, R., Chan, T. A., Schneck, J. P. 2020; 5 (8)

  Abstract

  Recent studies show gut microbiota modulate antitumor immune responses; one proposed mechanism is cross-reactivity between antigens expressed in commensal bacteria and neoepitopes. We found that T cells targeting an epitope called SVYRYYGL (SVY), expressed in the commensal bacterium Bifidobacterium breve (B. breve), cross-react with a model neoantigen, SIYRYYGL (SIY). Mice lacking B. breve had decreased SVY-reactive T cells compared with B. breve-colonized mice, and the T cell response was transferable by SVY immunization or by cohousing mice without Bifidobacterium with ones colonized with Bifidobacterium. Tumors expressing the model SIY neoantigen also grew faster in mice lacking B. breve compared with Bifidobacterium-colonized animals. B. breve colonization also shaped the SVY-reactive TCR repertoire. Finally, SVY-specific T cells recognized SIY-expressing melanomas in vivo and led to decreased tumor growth and extended survival. Our work demonstrates that commensal bacteria can stimulate antitumor immune responses via cross-reactivity and how bacterial antigens affect the T cell landscape.

  View details for DOI 10.1172/jci.insight.135597

  View details for PubMedID 32324171

• Collagen fiber structure guides 3D motility of cytotoxic T lymphocytes MATRIX BIOLOGY Pruitt, H. C., Lewis, D., Ciccaglione, M., Connor, S., Smith, Q., Hickey, J. W., Schneck, J. P., Gerecht, S. 2020; 85-86: 147–59

  View details for DOI 10.1016/j.matbio.2019.02.003

  View details for Web of Science ID 000528055100010

• Collagen fiber structure guides 3D motility of cytotoxic T lymphocytes. Matrix biology : journal of the International Society for Matrix Biology Pruitt, H. C., Lewis, D. n., Ciccaglione, M. n., Connor, S. n., Smith, Q. n., Hickey, J. W., Schneck, J. P., Gerecht, S. n. 2019

  Abstract

  Lymphocyte motility is governed by a complex array of mechanisms, and highly dependent on external microenvironmental cues. Tertiary lymphoid sites in particular have unique physical structure such as collagen fiber alignment, due to matrix deposition and remodeling. Three dimensional studies of human lymphocytes in such environments are lacking. We hypothesized that aligned collagenous environment modulates CD8+ T cells motility. We encapsulated activated CD8+ T cells in collagen hydrogels of distinct fiber alignment, a characteristic of tumor microenvironments. We found that human CD8+ T cells move faster and more persistently in aligned collagen fibers compared with nonaligned collagen fibers. Moreover, CD8+ T cells move along the axis of collagen alignment. We showed that myosin light chain kinase (MLCK) inhibition could nullify the effect of aligned collagen on CD8+ T cell motility patterns by decreasing T cell turning in unaligned collagen fiber gels. Finally, as an example of a tertiary lymphoid site, we found that xenograft prostate tumors exhibit highly aligned collagen fibers. We observed CD8+ T cells alongside aligned collagen fibers, and found that they are mostly concentrated in the periphery of tumors. Overall, using an in vitro controlled hydrogel system, we show that collagen fiber organization modulates CD8+ T cells movement via MLCK activation thus providing basis for future studies into relevant therapeutics.

  View details for PubMedID 30776427

• Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Hickey, J. W., Schneck, J. P. 2018

  Abstract

  We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

  View details for PubMedID 30507913

• Separating T Cell Targeting Components onto Magnetically Clustered Nanoparticles Boosts Activation NANO LETTERS Kosmides, A. K., Necochea, K., Hickey, J. W., Schneck, J. P. 2018; 18 (3): 1916–24

  Abstract

  T cell activation requires the coordination of a variety of signaling molecules including T cell receptor-specific signals and costimulatory signals. Altering the composition and distribution of costimulatory molecules during stimulation greatly affects T cell functionality for applications such as adoptive cell therapy (ACT), but the large diversity in these molecules complicates these studies. Here, we develop and validate a reductionist T cell activation platform that enables streamlined customization of stimulatory conditions. This platform is useful for the optimization of ACT protocols as well as the more general study of immune T cell activation. Rather than decorating particles with both signal 1 antigen and signal 2 costimulus, we use distinct, monospecific, paramagnetic nanoparticles, which are then clustered on the cell surface by a magnetic field. This allows for rapid synthesis and characterization of a small number of single-signal nanoparticles which can be systematically combined to explore and optimize T cell activation. By increasing cognate T cell enrichment and incorporating additional costimulatory molecules using this platform, we find significantly higher frequencies and numbers of cognate T cells stimulated from an endogenous population. The magnetic field-induced association of separate particles thus provides a tool for optimizing T cell activation for adoptive immunotherapy and other immunological studies.

  View details for DOI 10.1021/acs.nanolett.7b05284

  View details for Web of Science ID 000427910600050

  View details for PubMedID 29488768

• Engineering Platforms for T Cell Modulation BIOLOGY OF T CELLS, PT A Hickey, J. W., Kosmides, A. K., Schneck, J. P., Galluzzi, L., Rudqvist, N. P. 2018; 341: 277–362

  Abstract

  T cells are crucial contributors to mounting an effective immune response and increasingly the focus of therapeutic interventions in cancer, infectious disease, and autoimmunity. Translation of current T cell immunotherapies has been hindered by off-target toxicities, limited efficacy, biological variability, and high costs. As T cell therapeutics continue to develop, the application of engineering concepts to control their delivery and presentation will be critical for their success. Here, we outline the engineer's toolbox and contextualize it with the biology of T cells. We focus on the design principles of T cell modulation platforms regarding size, shape, material, and ligand choice. Furthermore, we review how application of these design principles has already impacted T cell immunotherapies and our understanding of T cell biology. Recent, salient examples from protein engineering, synthetic particles, cellular and genetic engineering, and scaffolds and surfaces are provided to reinforce the importance of design considerations. Our aim is to provide a guide for immunologists, engineers, clinicians, and the pharmaceutical sector for the design of T cell-targeting platforms.

  View details for PubMedID 30262034

• Biomimetic Artificial Antigen Presenting Cells Synergize with Anti-PD1 in the Treatment of Melanoma Meyer, R. A., Kosmides, A. K., Hickey, J. W., Aje, K., Cheung, K., Schneck, J. P., Green, J. J. CELL PRESS. 2017: 269–70

  View details for Web of Science ID 000401083600580

• Biomimetic biodegradable artificial antigen presenting with PD-1 blockade to treat melanoma cells synergize BIOMATERIALS Kosmides, A. K., Meyer, R. A., Hickey, J. W., Aje, K., Cheung, K. N., Green, J. J., Schneck, J. P. 2017; 118: 16–26

  Abstract

  Biomimetic materials that target the immune system and generate an anti-tumor responses hold promise in augmenting cancer immunotherapy. These synthetic materials can be engineered and optimized for their biodegradability, physical parameters such as shape and size, and controlled release of immune-modulators. As these new platforms enter the playing field, it is imperative to understand their interaction with existing immunotherapies since single-targeted approaches have limited efficacy. Here, we investigate the synergy between a PLGA-based artificial antigen presenting cell (aAPC) and a checkpoint blockade molecule, anti-PD1 monoclonal antibody (mAb). The combination of antigen-specific aAPC-based activation and anti-PD-1 mAb checkpoint blockade induced the greatest IFN-γ secretion by CD8+ T cells in vitro. Combination treatment also acted synergistically in an in vivo murine melanoma model to result in delayed tumor growth and extended survival, while either treatment alone had no effect. This was shown mechanistically to be due to decreased PD-1 expression and increased antigen-specific proliferation of CD8+ T cells within the tumor microenvironment and spleen. Thus, biomaterial-based therapy can synergize with other immunotherapies and motivates the translation of biomimetic combinatorial treatments.

  View details for DOI 10.1016/j.biomaterials.2016.11.038

  View details for Web of Science ID 000393254700002

  View details for PubMedID 27940380

  View details for PubMedCentralID PMC5207804

• Control of polymeric nanoparticle size to improve therapeutic delivery JOURNAL OF CONTROLLED RELEASE Hickey, J. W., Santos, J., Williford, J., Mao, H. 2015; 219: 536–47

  Abstract

  As nanoparticle (NP)-mediated drug delivery research continues to expand, understanding parameters that govern NP interactions with the biological environment becomes paramount. The principles identified from the study of these parameters can be used to engineer new NPs, impart unique functionalities, identify novel utilities, and improve the clinical translation of NP formulations. One key design parameter is NP size. New methods have been developed to produce NPs with increased control of NP size between 10 and 200nm, a size range most relevant to physical and biochemical targeting through both intravascular and site-specific deliveries. Three notable techniques best suited for generating polymeric NPs with narrow size distributions are highlighted in this review: self-assembly, microfluidics-based preparation, and flash nanoprecipitation. Furthermore, the effect of NP size on the biological fate and transport properties at the molecular scale (protein-NP interactions) and the tissue and systemic scale (convective and diffusive transport of NPs) are analyzed here. These analyses underscore the importance of NP size control in considering clinical translation and assessment of therapeutic outcomes of NP delivery vehicles.

  View details for PubMedID 26450667

  View details for PubMedCentralID PMC4656075

• Prevention and Removal of Lipid Deposits by Lens Care Solutions and Rubbing OPTOMETRY AND VISION SCIENCE Tam, N., Pitt, W. G., Perez, K. X., Hickey, J. W., Glenn, A. A., Chinn, J., Liu, X., Maziarz, E. 2014; 91 (12): 1430–39

  Abstract

  Despite the prevalence of silicone hydrogel (SiHy) contact lenses, there are relatively few studies that evaluate the efficacy of multipurpose lens care solutions (MPSs) in reducing lipid deposition on these lenses and the effect of rubbing on the removal. Therefore, we used an in vitro soaking and rubbing model to compare the effectiveness of borate buffered saline (BBS) and two commercial MPSs, PureMoist and Biotrue, in preventing sorption of representative polar and nonpolar lipids.Radiolabeled cholesterol (CH) and dipalmitoylphosphatidylcholine (DPPC) were sorbed on two SiHy lenses (senofilcon A and balafilcon A) from an artificial tear fluid. Deposition and removal were evaluated by quantitative solvent extraction and scintillation counting.The efficiencies of the MPSs in reducing lipid deposition are somewhat dependent on lens material. Both DPPC and CH sorption on senofilcon A are greater when lenses are preconditioned in BBS compared with preconditioning in either MPS (p < 0.05). However, neither MPS affects lipid sorption on balafilcon A lenses (p > 0.05). As for removal of presorbed lipids, neither PureMoist, Biotrue, nor BBS removed CH in the absence of rubbing. When a simulated rubbing protocol was used, minimal but detectible CH was removed (p < 0.05) from senofilcon A and balafilcon A lenses (likely only from the lens surface). These commercial solutions were not substantially better than BBS in removing DPPC, with or without rubbing (p > 0.05).These data suggest that MPSs do not appreciably alter lipid sorption. Rubbing lenses removes a small amount of sorbed lipids. Yet, we recommend that MPSs be used as they may disinfect SiHy lenses and may clean their surfaces of large particles.

  View details for PubMedID 25325760

• The role of multi-purpose solutions in prevention and removal of lipid depositions on contact lenses CONTACT LENS & ANTERIOR EYE Tam, N., Pitt, W. G., Perez, K. X., Handly, E., Glenn, A. A., Hickey, J. W., Larsen, B. G. 2014; 37 (6): 405–14

  Abstract

  The sorption and desorption of radiolabeled dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CH) were measured on 5 types of commercial contact lenses. The lenses were soaked in vitro in an artificial tear fluid for 16h. The effects of borate buffered saline and two commercial multi-purpose lens-care solutions (MPSs) on reducing the lipid (DPPC and CH) sorption and increasing the lipid removal were examined. The results showed that silicone hydrogel (SiHy) lenses accumulated the most lipids, sorbing over an order of magnitude more than polymacon, a conventional hydrogel lens. Pre-soaking the SiHy lenses for 16h in MPSs reduced the DPPC sorption by up to 13% and the CH sorption by up to 11%, compared to controls that were not pre-soaked. However neither these reductions nor those on polymacon were statistically significant (p>0.05). In sorption experiments without presoaking, subsequent exposure to the MPSs removed some DPPC from the lenses (0-3.1% for SiHy lenses and 14-55% for polymacon), but CH removal was 0.0-0.8% for SiHy lenses and 0.6-28% for polymacon lenses. Some of these removals were statistically significant (p<0.05).

  View details for PubMedID 25081521

• Metallization of Branched DNA Origami for Nanoelectronic Circuit Fabrication ACS NANO Liu, J., Geng, Y., Pound, E., Gyawali, S., Ashton, J. R., Hickey, J., Woolley, A. T., Harb, J. N. 2011; 5 (3): 2240-2247

  Abstract

  This work examines the metallization of folded DNA, known as DNA origami, as an enabling step toward the use of such DNA as templates for nanoelectronic circuits. DNA origami, a simple and robust method for creating a wide variety of shapes and patterns, makes possible the increased complexity and flexibility needed for both the design and assembly of useful circuit templates. In addition, selective metallization of the DNA template is essential for circuit fabrication. Metallization of DNA origami presents several challenges over and above those associated with the metallization of other DNA templates such as λ-DNA. These challenges include (1) the stability of the origami in the processes used for metallization, (2) the enhanced selectivity required to metallize small origami structures, (3) the increased difficulty of adhering small structures to the surface so that they will not be removed when subject to multiple metallization steps, and (4) the influence of excess staple strands present with the origami. This paper describes our efforts to understand and address these challenges. Specifically, the influence of experimental conditions on template stability and on the selectivity of metal deposition was investigated for small DNA origami templates. These templates were seeded with Ag and then plated with Au via an electroless deposition process. Both staple strand concentration and the concentration of ions in solution were found to have a significant impact. Selective continuous metal deposition was achieved, with an average metallized height as small as 32 nm. The shape of branched origami was also retained after metallization. These results represent important progress toward the realization of DNA-templated nanocircuits.

  View details for DOI 10.1021/nn1035075

  View details for Web of Science ID 000288570600083

  View details for PubMedID 21323323