Faculty Bibliography

Cancer cells alter their mechanical properties in response to the rigidity of their environment. Here, we explored the implications of this environmental mechanosensing for anti-tumor immunosurveillance using single cell biophysical profiling and metastasis models. Cancer cells stiffened in more rigid environments, a biophysical change that sensitized them to cytotoxic lymphocytes. In immunodeficient mice, this behavior manifested in the outgrowth of stiffer metastatic cells in the rigid bone than in the soft lung, while in immunocompetent hosts, it led to preferential elimination of stiffer cancer cells and suppression of bone metastasis. Environmentally-induced cell stiffening and immune sensitization both required Osteopontin, a secreted glycoprotein that is upregulated during bone colonization. Analysis of patient metastases spanning mechanically distinct tissues revealed associations between environmental rigidity, immune infiltration, and cancer cell stiffness consistent with mechanically driven immunosurveillance. These results demonstrate how environmental mechanosensing modulates anti-tumor immunity and suggest a mechanoimmunological basis for metastatic site selection.

The ε4 variant of human apolipoprotein E (APOE4) is a key genetic risk factor for neurodegeneration in Alzheimer's disease and elevated all-cause mortality in humans. Understanding the factors and mechanisms that can mitigate the harmful effects of APOE4 has significant implications. In this study, we find that inactivating the VHL-1 (Von Hippel-Lindau) protein can suppress mortality, neural and behavioral pathologies caused by transgenic human APOE4 in Caenorhabditis elegans. The protective effects of VHL-1 deletion are recapitulated by stabilized HIF-1 (hypoxia-inducible factor), a transcription factor degraded by VHL-1. HIF-1 activates a genetic program that safeguards against mitochondrial dysfunction, oxidative stress, proteostasis imbalance, and endolysosomal rupture-critical cellular events linked to neural pathologies and mortality. Furthermore, genetic inhibition of Vhl reduces cerebral vascular injury and synaptic lesions in APOE4 mice, suggesting an evolutionarily conserved mechanism. Thus, we identify the VHL-HIF axis as a potent modulator of APOE4-induced neural pathologies and propose that targeting this pathway in nonproliferative tissues may curb cellular damage, protect against neurodegeneration, and reduce tissue injuries and mortality.

Phagocytosis is an intensely physical process that depends on the mechanical properties of both the phagocytic cell and its chosen target. Here, we employed differentially deformable hydrogel microparticles to examine the role of cargo rigidity in the regulation of phagocytosis by macrophages. Whereas stiff cargos elicited canonical phagocytic cup formation and rapid engulfment, soft cargos induced an architecturally distinct response, characterized by filamentous actin protrusions at the center of the contact site, slower cup advancement, and frequent phagocytic stalling. Using phosphoproteomics, we identified β2 integrins as critical mediators of this mechanically regulated phagocytic switch. Macrophages lacking β2 integrins or their downstream effectors, Talin1 and Vinculin, exhibited specific defects in phagocytic cup architecture and selective suppression of stiff cargo uptake. We conclude that integrin signaling serves as a mechanical checkpoint during phagocytosis to pair cargo rigidity to the appropriate mode of engulfment.

Immune cells have intensely physical lifestyles characterized by structural plasticity and force exertion. To investigate whether specific immune functions require stereotyped mechanical outputs, we used super-resolution traction force microscopy to compare the immune synapses formed by cytotoxic T cells with contacts formed by other T cell subsets and by macrophages. T cell synapses were globally compressive, which was fundamentally different from the pulling and pinching associated with macrophage phagocytosis. Spectral decomposition of force exertion patterns from each cell type linked cytotoxicity to compressive strength, local protrusiveness, and the induction of complex, asymmetric topography. These features were validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, live imaging of synaptic secretion, and in silico analysis of interfacial distortion. Synapse architecture and force exertion were sensitive to target stiffness and size, suggesting that the mechanical potentiation of killing is biophysically adaptive. We conclude that cellular cytotoxicity and, by implication, other effector responses are supported by specialized patterns of efferent force.

Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gβ₄ exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gβ₄-deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gβ₄. In Gβ₄ knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.

Spleen marginal zone (MZ) B cells are important for antibody responses against blood-borne antigens. The signals they use to detect exposure to blood are not well defined. Here, using intravital two-photon microscopy in mice, we observe transient contacts between MZ B cells and red blood cells that are in flow. We show that MZ B cells use adhesion G-protein-coupled receptor ADGRE5 (CD97) for retention in the spleen. CD97 function in MZ B cells depends on its ability to undergo autoproteolytic cleavage and signaling via Gα₁₃ and ARHGEF1. Red blood cell expression of the CD97 ligand CD55 is required for MZ B cell homeostasis. Applying a pulling force on CD97-transfected cells using an optical C-trap and CD55⁺ beads leads to accumulation of active RhoA and membrane retraction. Finally, we show that CD97 deficiency leads to a reduced T cell-independent IgM response. Thus, our studies provide evidence that MZ B cells use mechanosensing to position in a manner that enhances antibody responses against blood-borne antigens.

Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), remains a major medical problem. HBV has a high propensity for progressing to chronicity and can result in severe liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. CHB patients frequently present with viral coinfection, including human immunodeficiency virus type (HIV) and hepatitis delta virus. About 10% of chronic HIV carriers are also persistently infected with HBV, which can result in more exacerbated liver disease. Mechanistic studies of HBV-induced immune responses and pathogenesis, which could be significantly influenced by HIV infection, have been hampered by the scarcity of immunocompetent animal models. Here, we demonstrate that humanized mice dually engrafted with components of a human immune system and a human liver supported HBV infection, which was partially controlled by human immune cells, as evidenced by lower levels of serum viremia and HBV replication intermediates in the liver. HBV infection resulted in priming and expansion of human HLA-restricted CD8+ T cells, which acquired an activated phenotype. Notably, our dually humanized mice support persistent coinfections with HBV and HIV, which opens opportunities for analyzing immune dysregulation during HBV and HIV coinfection, and preclinical testing of novel immunotherapeutics.

Hepatitis B virus (HBV) only infects humans and chimpanzees, posing major challenges for modeling HBV infection and chronic viral hepatitis. The major barrier in establishing HBV infection in non-human primates lies at incompatibilities between HBV and simian orthologues of the HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Through mutagenesis analysis and screening among NTCP orthologues from Old World monkeys, New World monkeys and prosimians, we determined key residues responsible for viral binding and internalization, respectively and identified marmosets as a suitable candidate for HBV infection. Primary marmoset hepatocytes and induced pluripotent stem cell-derived hepatocyte-like cells support HBV and more efficient woolly monkey HBV (WMHBV) infection. Adapted chimeric HBV genome harboring residues 1-48 of WMHBV preS1 generated here led to a more efficient infection than wild-type HBV in primary and stem cell derived marmoset hepatocytes. Collectively, our data demonstrate that minimal targeted simianization of HBV can break the species barrier in small NHPs, paving the path for an HBV primate model.

Cytotoxic lymphocytes fight pathogens and cancer by forming immune synapses with infected or transformed target cells and then secreting cytotoxic perforin and granzyme into the synaptic space, with potent and specific killing achieved by this focused delivery. The mechanisms that establish the precise location of secretory events, however, remain poorly understood. Here we use single cell biophysical measurements, micropatterning, and functional assays to demonstrate that localized mechanotransduction helps define the position of secretory events within the synapse. Ligand-bound integrins, predominantly the α_Lβ₂ isoform LFA-1, function as spatial cues to attract lytic granules containing perforin and granzyme and induce their fusion with the plasma membrane for content release. LFA-1 is subjected to pulling forces within secretory domains, and disruption of these forces via depletion of the adaptor molecule talin abrogates cytotoxicity. We thus conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance their cytotoxic power and fidelity.

Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high-sequence homology of the HBV receptor, Na+/taurocholate co-transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP. Immunodeficient Fah