Publications by authors named "Kenneth H Hu"

12 Publications

  • Page 1 of 1

Global absence and targeting of protective immune states in severe COVID-19.

Nature 2021 Jan 25. Epub 2021 Jan 25.

Department of Pathology, University of California San Francisco, San Francisco, CA, USA.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs) across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.
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http://dx.doi.org/10.1038/s41586-021-03234-7DOI Listing
January 2021

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

bioRxiv 2020 Oct 29. Epub 2020 Oct 29.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.

One Sentence Summary: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.
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http://dx.doi.org/10.1101/2020.10.28.359935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605559PMC
October 2020

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

Res Sq 2020 Oct 28. Epub 2020 Oct 28.

Department of Pathology, San Francisco, 513 Parnassus Ave, HSW512, San Francisco, CA 94143-0511, USA.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a wholeblood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferonstimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and autodirected antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.
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http://dx.doi.org/10.21203/rs.3.rs-97042/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605560PMC
October 2020

ZipSeq: barcoding for real-time mapping of single cell transcriptomes.

Nat Methods 2020 08 6;17(8):833-843. Epub 2020 Jul 6.

Department of Pathology, University of California San Francisco, San Francisco, CA, USA.

Spatial transcriptomics seeks to integrate single cell transcriptomic data within the three-dimensional space of multicellular biology. Current methods to correlate a cell's position with its transcriptome in living tissues have various limitations. We developed an approach, called 'ZipSeq', that uses patterned illumination and photocaged oligonucleotides to serially print barcodes ('zipcodes') onto live cells in intact tissues, in real time and with an on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in vitro wound healing, live lymph node sections and a live tumor microenvironment. In all cases, we discovered new gene expression patterns associated with histological structures. In the tumor microenvironment, this demonstrated a trajectory of myeloid and T cell differentiation from the periphery inward. A combinatorial variation of ZipSeq efficiently scales in the number of regions defined, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.
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http://dx.doi.org/10.1038/s41592-020-0880-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891292PMC
August 2020

Biochemical Stimulation of Immune Cells and Measurement of Mechanical Responses Using Atomic Force Microscopy.

Curr Protoc Chem Biol 2019 06 1;11(2):e63. Epub 2019 Feb 1.

Department of Pediatrics, Division of Immunology, and Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California.

This manuscript details methods to ligate cell-surface receptors on live cells with precise spatiotemporal control using an atomic force microscope (AFM) to deliver ligands. This approach can be used to image cellular responses upon activating T cell receptors when the AFM is mounted on an optical microscope. Moreover, the AFM measures forces generated by the cell during the contact. Using AFM to trigger cellular responses adds an important capability to the field of mechanobiology. We describe how to incorporate anti-CD3 antibodies or other molecules onto an AFM cantilever and how to use AFM to activate T cells. © 2019 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpch.63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565469PMC
June 2019

Hyaluronan content governs tissue stiffness in pancreatic islet inflammation.

J Biol Chem 2018 01 28;293(2):567-578. Epub 2017 Nov 28.

the Department of Pediatrics, Division of Immunology, Allergy, and Rheumatology, Stanford University, Stanford, California 94305 and.

We have identified a novel role for hyaluronan (HA), an extracellular matrix polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes of mice and humans is preceded by intraislet accumulation of HA, a highly hygroscopic polymer. Using the double transgenic DO11.10 × RIPmOVA (DORmO) mouse model of type 1 diabetes, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared with controls. Conversely, treatment with 4-methylumbelliferone, a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content, we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.
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http://dx.doi.org/10.1074/jbc.RA117.000148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767862PMC
January 2018

Quantitative Deformability Cytometry: Rapid, Calibrated Measurements of Cell Mechanical Properties.

Biophys J 2017 Oct;113(7):1574-1584

Department of Integrative Biology and Physiology, University of California, Los Angeles, California; Department of Bioengineering, University of California, Los Angeles, California; UCLA Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California; Broad Stem Cell Research Center, University of California, Los Angeles, California; Center for Biological Physics, University of California, Los Angeles, California. Electronic address:

Advances in methods that determine cell mechanical phenotype, or mechanotype, have demonstrated the utility of biophysical markers in clinical and research applications ranging from cancer diagnosis to stem cell enrichment. Here, we introduce quantitative deformability cytometry (q-DC), a method for rapid, calibrated, single-cell mechanotyping. We track changes in cell shape as cells deform into microfluidic constrictions, and we calibrate the mechanical stresses using gel beads. We observe that time-dependent strain follows power-law rheology, enabling single-cell measurements of apparent elastic modulus, E, and power-law exponent, β. To validate our method, we mechanotype human promyelocytic leukemia (HL-60) cells and thereby confirm q-DC measurements of E = 0.53 ± 0.04 kPa. We also demonstrate that q-DC is sensitive to pharmacological perturbations of the cytoskeleton as well as differences in the mechanotype of human breast cancer cell lines (E = 2.1 ± 0.1 and 0.80 ± 0.19 kPa for MCF-7 and MDA-MB-231 cells). To establish an operational framework for q-DC, we investigate the effects of applied stress and cell/pore-size ratio on mechanotype measurements. We show that E increases with applied stress, which is consistent with stress stiffening behavior of cells. We also find that E increases for larger cell/pore-size ratios, even when the same applied stress is maintained; these results indicate strain stiffening and/or dependence of mechanotype on deformation depth. Taken together, the calibrated measurements enabled by q-DC should advance applications of cell mechanotype in basic research and clinical settings.
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http://dx.doi.org/10.1016/j.bpj.2017.06.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627151PMC
October 2017

Cytoskeletal adaptivity regulates T cell receptor signaling.

Sci Signal 2017 Mar 7;10(469). Epub 2017 Mar 7.

Stanford Immunology, Stanford University, Stanford, CA 94305, USA.

The factors that govern T cell activation control the initiation and progression of adaptive immune responses. T cells recognize their cognate antigen on the surface of antigen-presenting cells (APCs) through the T cell receptor, which results in the formation of a contact region (immune synapse) between the two cells and the activation of the T cells. Activated T cells proliferate and differentiate into effector T cells that secrete cytokines, provide help to B cells, and kill target cells. We asked whether the actin cytoskeleton governs differences in signaling in effector T cells versus naïve (unstimulated) T cells. Using atomic force microscopy and quantitative confocal microscopy, we found that naïve T cells had a mechanically stiffer cortical cytoskeleton than that of effector cells, which resulted in naïve cells forming smaller immune synapses with APCs. This suggests that the cytoskeletal stiffness of the T cell before it undergoes antigen stimulation predicts its subsequent dynamic engagement with APCs and its activation potential. Cytoskeletal rigidity depended on the activity of the actin-severing enzyme cofilin through a pathway requiring the small guanosine triphosphatase RhoA and the kinases ROCK (Rho-activated kinase) and LIMK. These findings suggest that the baseline cytoskeletal state controls T cell responses and that the underlying pathway could be a therapeutic target for modulating adaptive immunity.
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http://dx.doi.org/10.1126/scisignal.aah3737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854469PMC
March 2017

YAP-dependent mechanotransduction is required for proliferation and migration on native-like substrate topography.

Biomaterials 2017 01 16;115:155-166. Epub 2016 Nov 16.

Department of Materials Science and Engineering, Stanford University, Stanford, CA, 94305, USA. Electronic address:

Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials.
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http://dx.doi.org/10.1016/j.biomaterials.2016.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572766PMC
January 2017

T cell activation requires force generation.

J Cell Biol 2016 06 30;213(5):535-42. Epub 2016 May 30.

Stanford Biophysics, Stanford University, Stanford, CA 94305 Department of Pediatrics, Division of Allergy, Immunology, and Rheumatology, Stanford University, Stanford, CA 94305

Triggering of the T cell receptor (TCR) integrates both binding kinetics and mechanical forces. To understand the contribution of the T cell cytoskeleton to these forces, we triggered T cells using a novel application of atomic force microscopy (AFM). We presented antigenic stimulation using the AFM cantilever while simultaneously imaging with optical microscopy and measuring forces on the cantilever. T cells respond forcefully to antigen after calcium flux. All forces and calcium responses were abrogated upon treatment with an F-actin inhibitor. When we emulated the forces of the T cell using the AFM cantilever, even these actin-inhibited T cells became activated. Purely mechanical stimulation was not sufficient; the exogenous forces had to couple through the TCR. These studies suggest a mechanical-chemical feedback loop in which TCR-triggered T cells generate forceful contacts with antigen-presenting cells to improve access to antigen.
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http://dx.doi.org/10.1083/jcb.201511053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896056PMC
June 2016

Strain-enhanced stress relaxation impacts nonlinear elasticity in collagen gels.

Proc Natl Acad Sci U S A 2016 May 2;113(20):5492-7. Epub 2016 May 2.

Department of Mechanical Engineering, Stanford University, Stanford, CA 94305;

The extracellular matrix (ECM) is a complex assembly of structural proteins that provides physical support and biochemical signaling to cells in tissues. The mechanical properties of the ECM have been found to play a key role in regulating cell behaviors such as differentiation and malignancy. Gels formed from ECM protein biopolymers such as collagen or fibrin are commonly used for 3D cell culture models of tissue. One of the most striking features of these gels is that they exhibit nonlinear elasticity, undergoing strain stiffening. However, these gels are also viscoelastic and exhibit stress relaxation, with the resistance of the gel to a deformation relaxing over time. Recent studies have suggested that cells sense and respond to both nonlinear elasticity and viscoelasticity of ECM, yet little is known about the connection between nonlinear elasticity and viscoelasticity. Here, we report that, as strain is increased, not only do biopolymer gels stiffen but they also exhibit faster stress relaxation, reducing the timescale over which elastic energy is dissipated. This effect is not universal to all biological gels and is mediated through weak cross-links. Mechanistically, computational modeling and atomic force microscopy (AFM) indicate that strain-enhanced stress relaxation of collagen gels arises from force-dependent unbinding of weak bonds between collagen fibers. The broader effect of strain-enhanced stress relaxation is to rapidly diminish strain stiffening over time. These results reveal the interplay between nonlinear elasticity and viscoelasticity in collagen gels, and highlight the complexity of the ECM mechanics that are likely sensed through cellular mechanotransduction.
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http://dx.doi.org/10.1073/pnas.1523906113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878492PMC
May 2016

The sigma(E) stress response is required for stress-induced mutation and amplification in Escherichia coli.

Mol Microbiol 2010 Jul 19;77(2):415-30. Epub 2010 May 19.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030-3411, USA.

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by 'point' mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, sigma(E) acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of sigma(32), which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that sigma(E) promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by sigma(E), and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.
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http://dx.doi.org/10.1111/j.1365-2958.2010.07213.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909356PMC
July 2010