Biology subjects

Kwong, G. A.

Publications and source records attributed to Kwong, G. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Multiscale, Survey-and-Target Optical Super-Resolution Histology

Super-resolution microscopy reveals fine subcellular tissue architecture, yet extending this capability to histological screening requires balancing spatial resolution with imaging area, axial access, throughput, and instrumental complexity. Here we present multiscale optical super-resolution histology (MOSH), an integrated optical-computational platform that combines tissue-scale, extended-depth-of-focus super-resolution surveys with coordinate-guided three-dimensional interrogation of selected targets. MOSH leverages stationary multifocal excitation, continuous sample translation, tunable axial refocusing, and parallelized reconstruction within a shared spatial-coordinate framework in a conventional epi-fluorescence setting. This survey-and-target strategy preserves tissue-scale context, directs volumetric acquisition to selected subcellular structures, and generates virtual H&E images that retain resolution-enhanced structural information. Across cultured cells, cytological preparations, conventional tissue sections, and intact unsectioned biopsies, MOSH achieved approximately twofold resolution enhancement, continuous lateral coverage beyond the native field of view, and tunable axial survey ranges up to 20 m. Bridging tissue-scale histological screening and targeted, quantitative nanoscale interrogation, MOSH establishes a scalable framework for multiscale analysis of biological and clinical specimens.

bioengineering↗

bioRxiv2026

Supervised 3D Virtual Fluorescence and Pseudo-H&E Staining of Fresh Bulk Tissues via Multimodal qOBM and Fluorescence Microscopy

We introduce a multimodal microscope that combines quantitative oblique back-illumination microscopy (qOBM) with wide-field epi-fluorescence deconvolution microscopy to acquire intrinsically co-registered phase and fluorescence volumes of fresh, bulk tissue. Using paired qOBM phase and DAPI fluorescence images of fresh ex vivo mouse brain tissue, we train a supervised conditional generative adversarial network (cGAN) to predict DAPI-like nuclear contrast from the structural information encoded in the phase images. Results not only demonstrate high-fidelity image translation but also show improved imaging depth and more homogeneous staining. Additionally, we combine the qOBM phase contrast with captured or virtual fluorescence nuclear images to produce volumetric pseudo-H&E representations of fresh tissue, enabling a supervised approach for achieving robust virtual histological staining. This work paves the way for virtual fluorescence imaging of many more structural targets and supervised virtual histological staining of thick, unaltered, bulk tissues ex vivo and potentially in vivo. Such capabilities could have significant implications for biological research as well as for point-of-care, intraoperative, and other clinical applications.

bioengineering↗

bioRxiv2026

Visualizing immunoreceptor forces and their effects in vivo

Immunoreceptors experience forces that modulate their activities; however, demonstrating this in vivo has been limited by technical challenges. As a first step toward meeting this challenge, we adapted a synthetic Notch (SynNotch) receptor system to report force transmission through immunoreceptors in vivo by replacing the native ligand-binding domain with a receptor-specific antibody and rewiring Notch signaling to drive EGFP or luciferase expression. We expressed SynNotch on Jurkat cells targeting CD40 or T cell receptor (TCR) and characterized their activation in coculture with B or T cells, defining the requirements, optimal conditions, and kinetics of activation. Using complementary mechanobiology approaches, we quantified the exogenous force required for reporter activation and verified that activation depends on forces generated by receptor-expressing sender cells rather than SynNotch-expressing receiver cells. By implanting sensors and targets into immunocompromised mice, we visualized mechanically activated reporter expression on CD40 and TCR-targeting SynNotch cells in vivo. Furthermore, CD40 and TCR signaling was amplified when the receptor bore force against mechanical support from immobilized ligand, indicating that force functions as biologically relevant co-stimulus. Together, our results establish mechanically activated SynNotch reporters as a useful strategy for detecting receptor-associated mechanical signaling across 2D coculture, 3D organoid, and in vivo systems.

bioengineering↗

bioRxiv2026

Programming T cells for Early Cancer Detection with Customized Protease-Activatable Receptors

Early cancer detection has the potential to reduce cancer mortality, yet endogenous tumor-shed biomarkers lack sensitivity for early-stage disease. We report OncoSCOUT, a cancer detection strategy using T cells engineered with protease-activatable receptors (PARs) that conditionally recognize tumor cells and release a synthetic biomarker for detection in urine. These PARs comprise masked synthetic Notch receptors in which antigen binding is blocked by a peptide mimotope tethered via a protease-cleavable linker. We demonstrate that requiring both extracellular protease activity and tumor antigen recognition improves spatial specificity and minimizes off-tumor activation of PAR T cells in vivo. To identify tumor-selective PARs, we adoptively transferred a HER2-targeted PAR library displaying [~]160,000 unique 4-mer amino acid linkers and discovered multiple variants significantly enriched in a HER2-positive cancer xenograft model. Using a single customized PAR, we show that OncoSCOUT can detect total tumor burdens as small as 10-30 mm3 with significantly improved sensitivity than the protein biomarker CA 15-3 or a 20-plex circulating tumor DNA (ctDNA) assay.

bioengineering↗

bioRxiv2026

Label- and slide-free multispectral quantitative epi-illumination deep-UV microscopy

Label-free and slide-free imaging is highly desired in clinical pathology because it holds the potential to avoid time- and labor-intensive tissue processing and chemical staining while preserving molecular information for downstream analyses. Deep-ultraviolet (UV) microscopy offers high-resolution, label-free molecular contrast via short wavelengths and intrinsic biomolecular absorption, but prior implementations have been limited to the analysis of thin sections and cell monolayers. Here, we present a fast, low-cost, LED-based, epi-illumination deep-UV microscope (epi-DUV) for label- and slide-free imaging of fresh, thick tissues. Using 255 nm and 280 nm absorption images, and tryptophan autofluorescence, the method yields quantitative maps of nucleic acid mass, protein mass, and quantum yield. Moreover, H&E-like contrast can be generated using native 255-nm absorption images. The system achieves 0.5 {micro}m lateral resolution with an effective slice thickness of [~]6 {micro}m across a 707 {micro}m x 707 {micro}m field of view and uses [~]330-ms exposure. To the best of our knowledge, this is the first demonstration of quantitative deep-UV molecular imaging of fresh, unlabeled thick tissues. Epi-DUV has significant potential to streamline the histopathology workflow while adding objective molecular readouts, enabling point-of-care assessment of unprocessed specimens (e.g., rapid intraoperative evaluation).

bioengineering↗

bioRxiv2025

Non-human primate LIBRA-Seq accelerates neutralizing antibody discovery in RM vaccinated against HIV-1

Broadly neutralizing antibodies (bNAbs) exhibit protective efficacy against HIV-1 infection making them an ideal archetype for HIV-1 vaccine design. Presently, no vaccine candidate has induced antibody responses capable of meaningful protection against the swathe of circulating, difficult to neutralize tier 2 HIV-1 viruses. However, the development of stabilized, native-like envelope (Env) trimers such as BG505.SOSIP.664.T332N (BG505 SOSIP) has marked a significant advancement in vaccine design, due to their ability to elicit NAbs that neutralize tier 2 viruses in rhesus macaques (RM). NAb development following envelope trimer immunization in RM remains poorly understood, with hypothesized contributions from genetic variation at the IG loci, naive B cell repertoire, and differential gene expression in B cell lineages. To address these knowledge gaps, we have developed a set of BG505 SOSIP probes capable of recovering paired clonotype identity, antigen specificity, and gene expression of B cells in a high throughput fashion. These probes were constructed by conjugating biotinylated BG505 SOSIP to streptavidin covalently linked to both sc-RNA-Seq compatible DNA oligonucleotides and flow cytometry compatible fluorophores. Using these reagents, we isolated and sequenced BG505 SOSIP specific memory B cells from the PBMCs of an RM that developed high titers of neutralizing antibodies. To benchmark the accuracy of our technology, we compared our recovered heavy and light chain sequences to those identified from the same animal using conventional methodology and recovered 100% of previously identified NAbs. We then applied this technology to recover BG505 SOSIP specific memory B cells from five additional vaccinated RMs, cloned 34 antibodies for functional characterization, and identified ten antibodies with autologous neutralizing activity. Author SummaryUnderstanding how effective antibodies arise after HIV vaccination is essential for developing a protective vaccine, yet studying these responses in non-human primates has been limited by low- throughput methods. In this study, we adapted a high-throughput single-cell sequencing approach to identify HIV envelope-specific antibodies from vaccinated rhesus macaques. This method allowed us to recover paired antibody sequences together with their antigen specificity from thousands of individual B cells. We successfully identified known neutralizing antibodies and discovered additional antibodies capable of neutralizing HIV across multiple animals. Our analysis revealed that vaccine-elicited antibody responses were dominated by a small number of expanded lineages, with shared genetic features among animals with stronger neutralization. These findings demonstrate that this approach can efficiently define the antibody repertoires generated by HIV vaccines and provide a powerful tool for evaluating and improving immunogens in preclinical vaccine studies.

immunology↗

bioRxiv2025

Non-destructive, high-resolution T cell characterization and subtyping via deep-ultraviolet microscopy

T cell characterization is critical for understanding immune function, monitoring disease progression, and optimizing cell-based therapies. Current technologies to characterize T cells, such as flow cytometry, require fluorescent labeling and typically are destructive endpoint measurements. Non-destructive, label-free imaging methods have been proposed, but face limitations with throughput, specificity, and system complexity. Here we demonstrate deep-ultraviolet (UV) microscopy as a label-free, non-destructive, fast and simple imaging approach for assessing T cell viability, activation state, and subtype with high accuracy. Using static deep-UV images, we characterize T cell viability and activation state, demonstrating excellent agreement with flow cytometry measurements. We further apply dynamic deep-UV imaging to quantify intracellular activity, enabling fast and accurate subtyping of CD4+ and CD8+ T cells. These results corroborate recent studies on metabolic activity differences between these subtypes, but now with deep-UV microscopy they are enabled by a non-destructive, fast, low-cost and simple approach. Together, our results demonstrate deep-UV microscopy as a powerful tool for high-throughput immune cell characterization, with broad applications in immunology re-search, immune monitoring, and development of emerging cell-based therapies.

immunology↗

bioRxiv2025

Label-Free In-Line Characterization of Immune Cell Culture using Quantitative Phase Imaging

Cell therapies, including T cell immunotherapies, offer promising treatments for previously untreatable diseases, but their widespread use is hindered by challenges in monitoring therapeutic cells during culture--impacting consistency, potency, and cost. This work demonstrates the use of quantitative phase imaging (QPI), specifically a compact, non-interferometric form called quantitative oblique back illumination microscopy (qOBM), for non-destructive, label-free, in-line assessment of T cell cultures. qOBM enables near real-time feedback on culture growth, contamination, and cell status (viability and activation), comparable to flow cytometry. We further apply this method to characterize genetically modified CAR T cells and explore its potential for advanced T cell phenotyping. Analysis of data from over 50 independent donors shows strong correlation between qOBM metrics and traditional destructive at-line assays. Overall, qOBM provides a powerful tool for continuous, in-line monitoring of therapeutic cell cultures, which can be transformative for improving reproducibility, reducing costs, and advancing the development of cell-based therapies.

immunology↗

bioRxiv2025

Closed-loop sonothermogenetic control of CAR T cells for metronomic brain cancer therapy

Achieving durable CAR T cell responses against primary brain tumors and metastases requires strategies that enable intracranial control of therapy to overcome the barriers of solid tumor treatment without compromising safety. Here, we show that closed-loop sonothermogenetics enables remote regulation of CAR T cell therapeutic activity through the intact skull. Using MR-guided focused ultrasound with closed-loop temperature feedback, we modulate CAR T cells engineered with a genetically encoded thermal bioswitch to achieve metronomic activation in the brain without lasting adverse effects on healthy brain tissue. In murine models of brain cancer, metronomic production of NKG2D T cell engagers by intratumoral CAR T cells overcomes antigen heterogeneity in breast cancer brain metastasis and myeloid-derived immunosuppression in glioblastoma to drive antitumor responses. Our findings support the use of closed-loop sonothermogenetics for spatial and temporal control of CAR T cell therapies targeting solid brain tumors.

bioengineering↗

bioRxiv2024

Antigen-specific T cell immunotherapy by in vivo mRNA delivery

Immunotherapy has shown promise for treating patients with autoimmune diseases or cancer, yet treatment is associated with adverse effects associated with global activation or suppression of T cell immunity. Here, we developed antigen-presenting nanoparticles (APNs) to selectively engineer disease antigen (Ag)-specific T cells by in vivo mRNA delivery. APNs consist of a lipid nanoparticle core functionalized with peptide-major histocompatibility complexes (pMHCs), facilitating antigen-specific T cell transfection through cognate T cell receptor-mediated endocytosis. In mouse models of type 1 diabetes and multiple myeloma, APNs selectively deplete autoreactive T cells leading to durable control of glycemia, and engineer virus-specific T cells with anti-cancer chimeric antigen receptors (CARs), achieving comparable therapeutic outcome as virally transduced ex vivo CAR. Overall, our work supports the use of APNs to engineer disease-relevant T cells in vivo as Ag-specific immunotherapy for autoimmune disorders and cancer.

bioengineering↗

bioRxiv2023

Spatiotemporal Control of Genomics and Epigenomics by Ultrasound

CRISPR (clustered regularly interspaced short palindromic repeats) is a revolutionary technology for genome editing. Its derived technologies such as CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) further allow transcriptional and epigenetic modulations. Focused ultrasound (FUS) can penetrate deep in biological tissues and induce mild hyperthermia in a confined region to activate heat-sensitive genes. Here we engineer a set of CRISPR(a/i) tools containing heat-sensitive genetic modules controllable by FUS for the regulation of genome and epigenome in live cells and animals. We demonstrated the capabilities of FUS-inducible CRISPRa, CRISPRi, and CRISPR (FUS-CRISPR(a/i)) to upregulate, repress, and knockout exogenous and/or endogenous genes, respectively, in different cell types. We further targeted FUS-CRISPR to telomeres in tumor cells to induce telomere disruption, inhibiting tumor growth and enhancing tumor susceptibility to killing by chimeric antigen receptor (CAR)-T cells. FUS-CRISPR-mediated telomere disruption for tumor priming combined with CAR-T therapy demonstrated synergistic therapeutic effects in xenograft mouse models. The FUS-CRISPR(a/i) toolbox allows the remote, noninvasive, and spatiotemporal control of genomic and epigenomic reprogramming in vivo, with extended applications in cancer treatment.

bioengineering↗

bioRxiv2022

Embracing enzyme promiscuity with activity-based compressed biosensing

Genome-scale activity-based profiling of proteases requires identifying substrates that are specific to each individual protease. However, this process becomes increasingly difficult as the number of target proteases increases because most substrates are promiscuously cleaved by multiple proteases. We introduce a method - Substrate Libraries for Compressed sensing of Enzymes (SLICE) - for selecting complementary sets of promiscuous substrates to compile libraries that classify complex protease samples (1) without requiring deconvolution of the compressed signals and (2) without the use of highly specific substrates. SLICE ranks substrate libraries according to two features: substrate orthogonality and protease coverage. To quantify these features, we design a compression score that was predictive of classification accuracy across 140 in silico libraries (Pearson r = 0.71) and 55 in vitro libraries (Pearson r = 0.55) of protease substrates. We demonstrate that a library comprising only two protease substrates selected with SLICE can accurately classify twenty complex mixtures of 11 enzymes with perfect accuracy. We envision that SLICE will enable the selection of peptide libraries that capture information from hundreds of enzymes while using fewer substrates for applications such as the design of activity-based sensors for imaging and diagnostics.

bioengineering↗

bioRxiv2021

Sensitizing solid tumors to CAR-mediated cytotoxicity using synthetic antigens

CAR T cell immunotherapy relies on CAR targeting of tumor-associated antigens, yet heterogenous antigen expression, interpatient variation, and off-tumor expression by healthy cells remain barriers. Here, we develop synthetic antigens to sensitize solid tumors for recognition and elimination by CAR T cells. Unlike tumor-associated antigens, we design synthetic antigens that are orthogonal to endogenous proteins to eliminate off-tumor targeting and that have a small genetic footprint to facilitate efficient tumor delivery to tumors by viral vectors. Using the RSV-F camelid single-domain antibody (VHH) as a synthetic antigen, we show that adoptive transfer of VHH CAR T cells to mice bearing VHH expressing tumors reduced tumor burden in multiple syngeneic mouse models of cancer, improved survival, induced epitope spread, and protected against tumor rechallenge. Our work supports in situ delivery of synthetic antigens to treat antigen low or negative tumors with CAR T cells.

bioengineering↗

bioRxiv2021

In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles

Simultaneous delivery of mRNA to multiple populations of antigen (Ag)-specific CD8+ T cells is challenging given the diversity of peptide epitopes and polymorphism of class I major histocompatibility complexes (MHCI). We developed Ag-presenting nanoparticles (APNs) for mRNA delivery using pMHCI molecules that were refolded with photocleavable peptides to allow rapid ligand exchange by UV light and site-specifically conjugated with a lipid tail for post-insertion into preformed mRNA lipid nanoparticles. Across different TCR transgenic mouse models (P14, OT-1, Pmel), UV-exchanged APNs bound and transfected their cognate Ag-specific CD8+ T cells equivalent to APNs produced using conventionally refolded pMHCI molecules. In mice infected with PR8 influenza, multiplexed delivery of UV-exchanged APNs against three immunodominant epitopes led to ~50% transfection of a VHH mRNA reporter in cognate Ag-specific CD8+ T cells. Our data shows that UV-mediated peptide exchange can be used to rapidly produce APNs for mRNA delivery to multiple populations of Ag-specific T cells in vivo. TeaserLight-induced rapid production of antigen-presenting nanoparticles for mRNA delivery to multiple virus-specific T cell populations.

bioengineering↗

bioRxiv2021

Synthetic Antigen-Presenting Cells for Adoptive T Cell Therapy

Adoptive T cell therapies are transforming the treatment of solid and liquid tumors, yet their widespread adoption is limited in part by the challenge of generating functional cells. T cell activation and expansion using conventional antigen-presenting cells (APCs) is unreliable due to the variable quality of donor-derived APCs. As a result, engineered approaches using nanomaterials presenting T cell activation signals are a promising alternative due to their ability to be robustly manufactured with precise control over stimulation cues. In this work, we design synthetic APCs that consist of liposomes surface-functionalized with peptide-major histocompatibility complexes (pMHC). Synthetic APCs selectively target and activate antigen-specific T cell populations to levels similar to conventional protocols using non-specific CD3 and CD28 antibodies without the need for costimulation signals. T cells treated with synthetic APCs produce effector cytokines and demonstrate cytotoxic activity when co-cultured with tumor cells presenting target antigen in vitro. Following adoptive transfer into tumor-bearing mice, activated cells control tumor growth and improve overall survival compared to untreated mice. Synthetic APCs could potentially be used in the future to improve the accessibility of adoptive T cell therapies by removing the need for conventional APCs during manufacturing.

bioengineering↗

bioRxiv2020

Activity-based urinary biomarkers of response and resistance to checkpoint blockade immunotherapy

Immune checkpoint blockade (ICB) therapy has transformed cancer treatment, yet most patients do not derive clinical benefit and responders can acquire resistance to therapy. Noninvasive biomarkers are needed to indicate early on-treatment response and resistance mechanisms. Here we developed ImmuNe Sensors for monItorinG cHeckpoint blockade Therapy (INSIGHT), which comprises a library of mass-barcoded peptide substrates conjugated to PD1 antibodies, as therapeutic sensors. Following systemic administration, INSIGHT carries out the dual role of reinvigorating T cell function and profiling T cell and tumor proteases by the release of cleaved peptides into urine for noninvasive detection by mass spectrometry. We show that an PD1 therapeutic sensor for Granzyme B discriminates early treatment responses before tumor volumes significantly diverge from isotype controls in murine models of colorectal cancer. To differentiate mechanisms of resistance by multivariate analysis, we design a mass-barcoded, 14-plex INSIGHT library to profile proteases differentially expressed by tumors harboring B2m or Jak1 loss-of-function mutations. We find that binary classifiers trained on urine samples indicate response to PD-1 therapy as early as the start of the second dose, and discriminate B2m from Jak1 resistance with high sensitivity and specificity (AUROCs > 0.9). Our data supports the use of activity-based biomarkers for early on-treatment response assessment and classification of refractory tumors based on resistance mechanisms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/420265v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@12ce799org.highwire.dtl.DTLVardef@118fcc5org.highwire.dtl.DTLVardef@17b227eorg.highwire.dtl.DTLVardef@115a7fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Remote control of CAR T cell therapies by thermal targeting

The limited ability to control anti-tumor activity within tumor sites contributes to poor CAR T cell responses against solid malignancies. Systemic delivery of biologic drugs such as cytokines can augment CAR T cell activity despite off-target toxicity in healthy tissues that narrows their therapeutic window. Here we develop a platform for remote control of CAR T therapies by thermal targeting. To enable CAR T cells to respond to heat, we construct synthetic thermal gene switches that trigger expression of transgenes in response to mild elevations in local temperature (40-42 {degrees}C) but not to orthogonal cellular stresses such as hypoxia. We show that short pulses of heat (15-30 min) lead to more than 60-fold increases in gene expression without affecting key T cell functions including proliferation, migration, and cytotoxicity. We demonstrate thermal control of broad classes of immunostimulatory agents including CARs, Bispecific T cell Engagers (BiTEs), and cytokine superagonists to enhance proliferation and cell targeting. In mouse models of adoptive transfer, photothermal targeting of intratumoral CAR T cells to control the production of an IL-15 superagonist significantly enhances anti-tumor activity and overall survival. We envision that thermal targeting could improve the safety and efficacy of next-generation therapies by allowing remote control of CAR T cell activity.

bioengineering↗

bioRxiv2019

Proteases as Biological Bits for Programmable Medicine

Engineered biocircuits that interface with living systems as plug-and-play constructs may enable new applications for programmable therapies and diagnostics. We create biological bits (bbits) using proteases - a family of pleiotropic, promiscuous enzymes - to construct the biological equivalent of Boolean logic gates, comparators and analog-to-digital converters. We use these modules to write a cell-free bioprogram that can combine with bacteria-infected blood, quantify infection burden, and then calculate and unlock a selective drug dose. Inspired by probabilistic computing, we leverage multi- and common-target protease promiscuity as the biological analog of superposition to program three probabilistic bbits that solve all implementations of the two-bit oracle problem, Learning Parity with Noise. Treating a network of dysregulated proteases in a living animal as an oracle, we use this algorithm to resolve the probability distribution of coagulation proteases in vivo, allowing diagnosis of pulmonary embolism with high sensitivity and specificity (AUROC = 0.92) in a mouse model of thrombosis. Our results demonstrate that protease activity can be programmed in cell-free systems to carry out classical and probabilistic algorithms for programmable medicine.

bioengineering↗