Genetic ablation of adhesion ligands mitigates rejection of allogeneic cellular immunotherapies.

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo.

Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells.

Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin ( ) expression is commonly employed to evade T cell-mediated rejection, although absence of triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands and on targets cells robustly dampens NK cell reactivity across all sub-populations.

A chimeric antigen receptor uniquely recognizing MICA/B stress proteins provides an effective approach to target solid tumors.

Background: The advent of chimeric antigen receptor (CAR) T cell therapies has transformed the treatment of hematological malignancies; however, broader therapeutic success of CAR T cells has been limited in solid tumors because of their frequently heterogeneous composition. Stress proteins in the MICA and MICB (MICA/B) family are broadly expressed by tumor cells following DNA damage but are rapidly shed to evade immune detection.

Methods: We have developed a novel CAR targeting the conserved α3 domain of MICA/B (3MICA/B CAR) and incorporated it into a multiplexed-engineered induced pluripotent stem cell (iPSC)-derived natural killer (NK) cell (3MICA/B CAR iNK) that expressed a shedding-resistant form of the CD16 Fc receptor to enable tumor recognition through two major targeting receptors.

The application of mechanobiotechnology for immuno-engineering and cancer immunotherapy.

Immune-engineering is a rapidly emerging field in the past few years, as immunotherapy evolved from a paradigm-shifting therapeutic approach for cancer treatment to promising immuno-oncology models in clinical trials and commercial products. Linking the field of biomedical engineering with immunology, immuno-engineering applies engineering principles and utilizes synthetic biology tools to study and control the immune system for diseases treatments and interventions. Over the past decades, there has been a deeper understanding that mechanical forces play crucial roles in regulating immune cells at different stages from antigen recognition to actual killing, which suggests potential opportunities to design and tailor mechanobiology tools to novel immunotherapy.

Mechanosensor Piezo1 mediates bimodal patterns of intracellular calcium and FAK signaling.

Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling.

Control of the activity of CAR-T cells within tumours via focused ultrasound.

Focused ultrasound can deliver energy safely and non-invasively into tissues at depths of centimetres. Here we show that the genetics and cellular functions of chimeric antigen receptor T cells (CAR-T cells) within tumours can be reversibly controlled by the heat generated by short pulses of focused ultrasound via a CAR cassette under the control of a promoter for the heat-shock protein. In mice with subcutaneous tumours, locally injected T cells with the inducible CAR and activated via focused ultrasound guided by magnetic resonance imaging mitigated on-target off-tumour activity and enhanced the suppression of tumour growth, compared with the performance of non-inducible CAR-T cells.

FRET-Based Ca Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound.

Fluorescence resonance energy transfer (FRET)-based biosensors have advanced live cell imaging by dynamically visualizing molecular events with high temporal resolution. FRET-based biosensors with spectrally distinct fluorophore pairs provide clear contrast between cells during dual FRET live cell imaging. Here, we have developed a new FRET-based Ca biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed).

Engineering light-controllable CAR T cells for cancer immunotherapy.

T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation.

Relationship between the site of thrombosis and the prevalence of pulmonary embolism in acute lower extremity deep venous thrombosis.

Objective: Lower extremity deep venous thrombosis (LEDVT) is common and can lead to pulmonary embolism (PE). Currently, the mechanism of how LEDVT causes PE is unclear. The aim of this study was to explore the relationship between the thrombus sites and PE in LEDVT patients.

An AND-Gated Drug and Photoactivatable Cre- System for Spatiotemporal Control in Cell-Based Therapeutics.

While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase.

Genetically Encoded FRET Biosensor for Visualizing EphA4 Activity in Different Compartments of the Plasma Membrane.

The EphA4 receptor tyrosine kinase is well-known for its pivotal role in development, cancer progression, and neurological disorders. However, how EphA4 kinase activity is regulated in time and space still remains unclear. To visualize EphA4 activity in different membrane microdomains, we developed a sensitive EphA4 biosensor based on Förster resonance energy transfer (FRET), and targeted it in or outside raft-like microdomains in the plasma membrane.

Acoustic mechanogenetics.

From basic studies in understanding the role of signaling pathways to therapeutic applications in engineering new cellular functions, efficient and safe techniques to monitor and modulate molecular targets from cells to organs have been extensively developed. The developmental advancement of engineering devices such as microscope and ultrasonic transducers allows us to investigate biological processes at different scales. Synthetic biology has further emerged recently as a powerful platform for the development of new diagnostic and therapeutic molecular tools.

Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors.

The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET).

Mechanogenetics for the remote and noninvasive control of cancer immunotherapy.

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells.

Laser-induced shockwave paired with FRET: a method to study cell signaling.

Cells within the body are subject to various forces; however, the details concerning the way in which cells respond to mechanical stimuli are not well understood. We demonstrate that laser-induced shockwaves (LIS) combined with biosensors based on fluorescence resonance energy transfer (FRET) is a promising new approach to study biological processes in single live cells. As "proof-of-concept," using a FRET biosensor, we show that in response to LIS, cells release intracellular calcium.

Quantitative FRET imaging to visualize the invasiveness of live breast cancer cells.

Matrix metalloproteinases (MMPs) remodel tumor microenvironment and promote cancer metastasis. Among the MMP family proteases, the proteolytic activity of the pro-tumorigenic and pro-metastatic membrane-type 1 (MT1)-MMP constitutes a promising and targetable biomarker of aggressive cancer tumors. In this study, we systematically developed and characterized several highly sensitive and specific biosensors based on fluorescence resonant energy transfer (FRET), for visualizing MT1-MMP activity in live cells.