Biology subjects

Onfelt, B.

Publications and source records attributed to Onfelt, B..

4 recordsLinked to original sources

bioRxiv2025

Isolation of individual natural killer cells from deep, high-aspect ratio microwell arrays - an evaluative study

Immune cells exhibit functional heterogeneity beyond what is resolved by classical definitions of subpopulations based on cell surface expression of receptors. To develop efficient and personalized cell-based immunotherapies, we need to resolve this heterogeneity and understand the underlying parameters that dictate cellular responses to specific target cells. For this, new methods are required that can identify and harvest immune cells with specific functions, e.g., high cytotoxic potential, to form clonally expanded cells or to assess molecular or genetic signatures. In this study, we evaluate a system for non-destructive, live cell picking and release in deep, high-aspect ratio microwells and test it for isolation of individual natural killer (NK) cells. We assess its performance at retrieving and releasing beads from microwells and demonstrate its potential for single NK cell isolation with intact viability. We also implement a semi-automated workflow for functional single-cell screening of NK cell behavior in microwell arrays followed by single-cell identification and isolation, demonstrating the potential for functional screening and isolation of serial killing immune cells. Our evaluation concludes that this cell isolation system, in combination with microwell arrays, offers opportunities for improved understanding of NK cell biology with applications towards cell therapy. However, its limited throughput hinders large-scale applicability.

immunology↗

bioRxiv2024

A thermoplastic chip for correlative assays combining screening and high-resolution imaging of immune cell responses

Single-cell immune assays are developed for the identification and characterization of individual immune cell responses. Some methods provide snapshots of the phenotype of the cell, such as flow cytometry and single-cell RNA sequencing, whereas others, almost exclusively microscopy-based, can be used for longitudinal studies of individual cells. However, obtaining correlative data on cell dynamics and phenotype of individual immune cells is challenging but can provide more nuanced information of heterogeneous immune cell responses. In this work, we have addressed this challenge by developing an easy-to-use, disposable, thermoplastic microwell chip, designed to support screening and high-resolution imaging of single-cell behavior in two-and three-dimensional cell cultures. We show that the chip has excellent optical properties and we provide simple protocols for efficient long-term cell culture of suspension and adherent cells, the latter grown either as monolayers or as hundreds of single, uniformly-sized spheroids. We demonstrate the applicability of the system for single-cell analysis by correlating the dynamic cytotoxic response of single immune cells grown under different metabolic conditions to their intracellular cytolytic load at the end of the assay. Additionally, we illustrate highly multiplex cytotoxicity screening of tumor spheroids in the chip, comparing the effect of environment cues characteristic of the tumor microenvironment on natural killer (NK) cell-induced killing. Following the functional screening, we perform high-resolution 3D immunofluorescent imaging of infiltrating NK cells within the spheroid volumes.

immunology↗

bioRxiv2024

High-throughput analysis of membrane fluidity unveils a hidden dimension in immune cell states

Cell membranes undergo biophysical remodelling as an adaptation to the surroundings and to perform specific biological functions. However, the extent and relevance of such changes in human immune cells remain unknown, largely due to the lack of single-cell and multidimensional methodologies. Here, we apply a cytometry-based method to fill this gap by combining biophysical profiling with simultaneous analysis of immune cell markers. This platform reveals notable cell type-dependent plasma membrane order heterogeneity in immune cells. By sorting immune cells according to their membrane order and performing transcriptome and spatial surface proteome analyses together with functional tests, we show that plasma membrane order can be used to identify subsets of immune cells with distinct phenotypes and functional behaviours. Our findings demonstrate a broad heterogeneity of plasma membrane order in immune cells that will provide a more precise definition of immune cell states based on their biophysical properties in health and disease.

biophysics↗

bioRxiv2023

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 (B2M) expression is commonly employed to evade T cell-mediated rejection, although absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands CD54 and CD58 on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion of CD54 and CD58 in B2M-deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cells in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.

immunology↗