Methods in cell biology最新文献

Histones are essential nuclear proteins that package eukaryotic DNA into chromosomes, play a vital role in gene regulation, DNA replication, DNA repair and chromosome condensation. Understanding histone modifications is crucial for grasping biological and disease-related processes. Specific alterations in histone modifications serve as sensitive and selective biomarkers for conditions like cancer, impacting both tumor and immune cells and affecting their interactions. Indeed, the interest in histone modifications is growing in the field of tumor immunology and immunotherapy. Different techniques have been developed to characterize histone proteins and their modifications. Here, we present a simple acid extraction protocol to identify and quantify histones. The workflow described here can be used to detect and measure histone proteins or specific residues of histone, even capturing changes resulting from treatment with epigenetic drugs (Epi-drugs) or other drugs in in different human cancer cell line models.

T cell receptor (TCR) stimulation of T lymphocytes by antigen bound to the major histocompatibility complex (MHC) of an antigen-presenting cell (APC), together with the interaction of accessory molecules, induces the formation of the immunological synapse (IS), the convergence of secretion vesicles toward the centrosome, and the polarization of the centrosome to the IS. Upon IS formation, an initial increase in cortical filamentous actin (F-actin) at the IS takes place, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. These reversible, cortical actin cytoskeleton reorganization processes that characterize a mature IS occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells and cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. Besides, IS formation constitutes the basis of a signaling platform that integrates signals and coordinates molecular interactions that are necessary for an appropriate antigen-specific immune response. In this chapter we deal with the three-dimensional (3D) analysis of the synaptic interface architecture, as well as the analysis of the localization of different markers at the IS.

Limited therapeutic options for triple-negative breast cancer (TNBC) patients prompted the exploration of advanced immunotherapeutic approaches in this cancer entity. γδ T cells started gaining attention for their remarkable ability to suppress skin cancer, which rapidly extended to other cancer entities. This special T cells represent a suitable immune population to be used in adoptive T cell transfer approaches. Combining characteristics of both αβ T cells and natural killer (NK) cells, these unique T cells exhibit swift cancer cell elimination independent of MHC class I antigen presentation. The distinct advantage of γδ T cell immunotherapy lies in its HLA-unrestricted nature, enabling the utilization of cells from healthy donors. Up to date, many studies demonstrate that also expanded γδ T cells from breast cancer patients exhibit enhanced cytotoxicity and cytokine release in vitro, paving the way for γδ T cell-based therapies. The approach outlined below offers an alternative method for conducting in vitro cytotoxicity assays, utilizing γδ T cells as the effector cell population and breast cancer stem cells as the target.

Cytotoxic lymphocytes, such as cytotoxic T cells and natural killer (NK) cells, are instrumental in the recognition and eradication of pathogenic cells, notably those undergoing malignant transformation. Cytotoxic lymphocytes establish direct contact with cancer cells via the formation of a specialized cell-cell junction known as the lytic immunological synapse. This structure serves as a critical platform for lymphocytes to integrate surface signals from potential cancer cells and to direct their cytolytic apparatus toward the confirmed targets. Conversely, cancer cells evolve synaptic defense strategies to evade lymphocyte cytotoxicity. This chapter delineates protocols using imaging flow cytometry to examine and quantify important subcellular processes occurring within cytotoxic lymphocytes and cancer cells engaged into an immunological synapse. These processes encompass the spatial redistribution of cytoskeletal components, vesicles, organelles and cell surface molecules. We specifically describe methods to generate and select conjugates between MDA-MB-231 breast cancer cells or K-562 leukemic cells and either the NK-92MI cell line or primary human NK cells. In addition, we detail procedures to evaluate the synaptic polarization of the actin cytoskeleton, CD63-positive vesicular compartments, MHC class I molecules, as well as the microtubule-organizing center in effector cells.

Over the last decades, intensive research studies have been focused on describing how the immunological synapse is formed, the intracellular mechanisms that control lytic granules formation, and even further, the steps toward granule polarization before the killing event is achieved. These convoluted processes pose significant experimental challenges since the components' sizes are smaller than the diffraction limit of the conventional fluorescent microscopy techniques and their highly dynamic nature. Here, we describe a procedure to perform a quantitative analysis of the protein markers of these lytic granules by using τau-STED imaging and 3D-quantitative colocalization of lytic granule markers. The innovative technology offered by τau-STED microscopy and unbiased imaging analysis is a great tool that could be applied to further our understanding of lytic granule composition and localization and study other dynamic processes at the immunological synapses.

Understanding human T-cell antigen recognition in health and disease is becoming increasingly instrumental for monitoring T-cell responses to pathogen challenge and for the rational design of T-cell-based therapies targeting cancer, autoimmunity and organ transplant rejection. Here we showcase a quantitative imaging platform which is based on the use of planar glass-supported lipid bilayers (SLBs). The latter are functionalized with antigen (peptide-loaded HLA) as adhesion and costimulatory molecules (ICAM-1, B7-1) to serve as surrogate antigen presenting cell for antigen recognition by T-cells, which are equipped with T-cell antigen receptors (TCRs) sequenced from antigen-specific patient T-cells. We outline in detail, how the experimental use of SLBs supports recoding and analysis of synaptic antigen engagement and calcium signaling at the single cell level in response to user-defined antigen densities for quantitative comparison.

Natural killer (NK) cells are cytotoxic innate lymphoid cells that play critical roles in the mitigation of viral infections and cancer through the secretion of cytolytic granules and immunomodulatory cytokines. Abnormalities in NK function can lead to viral infections, autoimmunity, and cancer. The current protocol provides an NK isolation technique to study the signaling pathways downstream to the Killer cell immunoglobulin-like receptors (KIR) that serve as key human NK cell function regulators. This procedure enables investigating mechanisms specific to individual KIRs to improve our understanding of NK cell function in health and disease.

T cell adhesion kinetics are a powerful indicator of target cell recognition during the cell-cell exploration process and formation of the immunological synapse facilitating cell communication and activation through specific intercellular molecular interactions. Various techniques have been used to document these binding kinetics, which foreshadow the dynamics of immunological synapse formation. Here, optical tweezers were used for studying at the level of single cells, the adhesion kinetics of human leukemia T lymphocyte cell line (CEM) to mouse mast cell line (P815) used as a tumor cell model. The P815 FcγRII receptors were saturated with the mouse anti-human CD3ɛ immunoglobulin G (OKT3) for initiating the T cell-P815 interaction through the engagement of the T cell CD3 nucleating the TCR complex formation structuring the synapse. Methods were developed to assess the time required to turn a contact between a T cell and a tumor cell into a stable interaction, and thus initiate the synapse formation. Single T cells were manipulated with the optical tweezers while the tumor cells were adhered to the glass surface under culture conditions. Three adhesions scenario were investigated by exerting either repetitive contacts engaging the same area of the two cells, repetitive contacts engaging the same area of the T cell but different areas on the tumor cell surface, or rolling the T cell over the tumor cell surface. With these methods, we observed that the median time of contact of CEM on P815 decreased in the presence of anti-CD3 OKT3 from 46s to 1.3s and the median rolling distance decreased from 50μm to 1.8μm prior the T cell immobilization. T cell adhesion speed assays can be used for measuring their lack of early response, identifying molecules involved in cell adhesion, or screening potential modulators. The techniques and quantitative methods, described here for studying T cell/target cell interaction based on manipulations using optical tweezers, can be generalized to all types of immunological or virological synapses as between T cell/dendritic cell, cytotoxic T cell/target, T cell/macrophage, T cell/B cell, NK cell/target, immune cell/infected cell and others.

This chapter presents a series of quantitative analyses that can be used to study the formation of the immune synapse (IS) in B cells. The methods described are automated, consistent, and compatible with open-source platforms. The IS is a crucial structure involved in B cell activation and function, and the spatiotemporal organization of this structure is analyzed to provide a better understanding of its mechanisms. The analyses presented here can be applied to other immune cells and are accessible to researchers of diverse fields. In addition, the raw data derived from the results can be further explored to perform quantitative measurements of protein recruitment and tracking of intracellular vesicles. These techniques have the potential to enhance not only our understanding of the IS in B cells but also in other cell models.