Methods in cell biology最新文献

T cells expressing the γδ T-cell receptor (TCR) represent a numerically small proportion of total T cells. Unlike αβ T cells they are activated by non-peptide antigens independently of MHC-presentation. γδ T cells have been recognized as a favorable prognostic marker across different tumor entities. Recently, γδ T cells (in particular Vδ2 T cells), have gained attention because of their effective intrinsic anti-tumor reactivity. Moreover, their ability for MHC-independent activation and in vitro expansion to high numbers makes them attractive candidates for tumor immunotherapy by adoptive transfer. In this regard, the ex vitro identification of highly reactive γδ T cells upon stimulation enables us to specifically identify, isolate and expand γδ T cells which potentially represent those with high anti-tumor reactivity. CD137 and CD154 represent suitable markers for identifying specifically activated γδ T cells. In humans, the surface mobilization of CD137 and CD154 reveals antigen-specific activation of regulatory (Treg) and conventional CD4 T cells, respectively. We adapted this method for the analysis of Vδ2 T cells, in which the mobilization of both CD137 and CD154 can be used to investigate their activation, whereby CD137 and CD154 do not discriminate regulatory from conventional cells. Thus, this method provides a new way to rapidly analyze quick changes in Vδ2 T-cell activation and allows for using these markers for cell sorting and subsequent expansion of the specifically reacting Vδ2 T cells.

Sepsis is a systemic inflammatory response to infection, and its occurrence is associated with a poor prognosis in the context of multiorgan dysfunction syndrome (MODS). Although there are several animal models for the study of its etiology, the cecal ligation and puncture (CLP) model has been considered the "Gold standard" because it shows a high degree of similarity to the progression of human sepsis. Currently, it is one of the most frequently chosen options to search for therapeutic alternatives to diminish the progression and organ damage induced by sepsis. Here, we describe in depth the CLP technique in a rat model and its application in the study of acute renal failure (ARF), the most severe complication during sepsis.

Before being able to kill other cells, natural killer (NK) cells first have to establish contact with those targets. In case of a predominance of activating signals from the target cell over inhibitory ones, the killing process is initiated. It is possible, with a simple two-color flow cytometry method, to evaluate, for any given effector cell-target cell pair, the number of conjugates between both types of cells. The percentage obtained gives an idea of the amplitude of binding of the NK cells to the targets and might be expected to be indicative of the level of cytotoxicity. Nevertheless, there is no absolute correlation, as the percentages of conjugates are sometimes higher with relatively resistant targets than with the highly sensitive cell line K562. Practically, NK cells and target cells are stained with two differently fluorescent dyes and incubated together at the desired effector:target ratio (in our example, 1:1) for various periods of time (0, 10, 30min, etc.) at 37°C. After the incubation time, the cells are carefully introduced into the flow cytometer, where in principle three populations are distinguished: the single positive, unconjugated effector and target cells, respectively, and the double positive subset, which corresponds to the conjugates between both cell types. We describe here in detail the staining and cell culture protocols and procedures, and give several examples. Thus, the very cytotoxic NK leukemia cell line KHYG-1 versus the myeloid leukemia K562 (the "conventional" NK cell target) and the Burkitt lymphoma cell line Raji forms a high number of conjugates. In contrast, purified, non-activated, healthy donor-derived peripheral blood NK cells bind less to the targets, in accordance with their low (K562) or absent (Raji) cytotoxic activity.

Immunological synapses result from a T cell polarization process, requiring cytoskeleton remodeling. Actin and microtubules drive synapse architecture and the localization of intracellular organelles, including Golgi and endolysosomal compartments, ensuring the directional localization of synapse components. Microtubule remodeling includes the centrosome polarization and the formation of a radial microtubules network, extending from the centrosome to the synapse periphery. Concomitantly, a ring of filamentous actin forms at the synapse periphery. Microtubule and actin remodeling facilitate vesicle fusion at the synapse, enabling T cell effector functions. Analyzing structural subtleties of cytoskeleton remodeling at the immunological synapse is crucial to understand its role in T cell functions. It may also pinpoint pathological states related with cytoskeletal dysfunctions. Quantifying filamentous protein network properties is challenging due to their complex and heterogeneous architectures and the inherent difficulty of segmenting individual filaments. Here, we describe the development of an image processing approach aimed at quantifying microtubule organization at the immunological synapse without the need for filament segmentation. The method is based on the analysis of the spatial and directional organization of microtubules growing from the centrosome to the synapse periphery. It is applied to investigate the importance of Adenomatous polyposis coli (Apc), a polarity regulator and tumor suppressor, in immunological synapse structure and functions and its potential implication in anti-tumor immune responses. We provide an open-source napari plugin of the outlined methods for analyzing filamentous networks.

Cancer immunotherapy has revolutionized cancer treatment by harnessing the immune system's potential to combat cancer. Among the various strategies in this field, the use of killed tumor cells (KC) induced by immunogenic cell death (ICD) inducers has gained attraction. This approach involves the treatment of cancer cells in vitro, followed by the subcutaneous injection of these killed cells into tumor-bearing mice. ICD induction triggers the exposure and release of damage-associated molecular patterns (DAMPs) and neoantigens, activating both innate and adaptive immune responses against cancer. Vaccination assays with immunocompetent mice and syngeneic cancer cells are considered the gold standard for identifying ICD inductors, as they effectively demonstrate the immunized host's capacity to achieve tumor rejection, typically showing more than 50% of protection. Despite significant progress in understanding ICD mechanisms, translating these findings into clinical practice faces challenges. Controversially, some reports indicate ICD induction with <50% protection in prophylactic vaccination. This variability in ICD interpretation can lead to "false positives" or overestimations of the immunogenicity of cell death induced by antitumor treatments, potentially complicating its clinical translation. Thus, rigorous adherence to the gold standard is necessary, and complementary experiments to assess the immunogenicity of cell death are advantageous. Here, we present a protocol to confirm the immunogenicity and therapeutic effectiveness of cell death induced by an ICD-inducer and evaluate its ability to reduce tumor burden in an established syngeneic mouse model.

In recent years, significant advancements have been achieved in the development of multiplex imaging methodologies for immunophenotyping, enabling a comprehensive characterization of the complexity of tumor microenvironments. Imaging mass cytometry combines the detection of over 40 cellular targets with spatial information, enabling the identification of not only which cells are present in a tissue but also their localization relative to each other. Here, we present an easy-to-implement imaging mass cytometry workflow that ranges from antibody selection and testing to running a full panel. Additionally, we discuss the standard steps of IMC analysis and the currently available tools. Overall, the protocols proposed here are directly applicable to characterize immune contextures in a variety of tissues.

Myeloid-derived suppressor cells (MDSCs) ameliorate inflammation by inhibiting T cell responses. In pathological conditions, such as autoimmunity, chronic infections or cancer they accumulate in the periphery. In cancer, MDSCs can also be part of the tumor microenvironment and are associated with a worse prognosis and limited response to immunotherapy. Nowadays attempts are made to specifically target MDSCs in cancer therapy. Still, the role of MDSCs in standard cancer treatment modalities, such as radiotherapy remains mostly elusive. Here, we describe a flow cytometry-based method to determine and monitor monocytic and granulocytic-derived MDSCs directly from whole blood in an easy, fast and reliable assay. As specific surface markers for MDSCs are lacking, the assay follows a gating strategy that excludes successively the main immune cells types and analyzes the remaining events for a set of molecules that are expressed on MDSCs. This assay is especially appropriate for longitudinal analyses and clinical trials and is suitable for being integrated into more complex immunophenotyping panels to generate a comprehensive immune status.

During hypoxia, tissues are subjected to an inadequate oxygen supply, disrupting the balance needed to maintain normal function. This deficiency can occur due to reduced oxygen delivery caused by impaired blood flow or a decline in the blood's ability to carry oxygen. In tumors, hypoxia and vascularization play crucial roles, shaping their microenvironments and influencing cancer progression, response to treatment and metastatic potential. This chapter provides guidance on the use of non-invasive imaging methods including Positron Emission Tomography and Magnetic Resonance Imaging to study tumor oxygenation in pre-clinical settings. These imaging techniques offer valuable insights into tumor vascularity and oxygen levels, aiding in understanding tumor behavior and treatment effects. For example, PET imaging uses tracers such as [18F]-fluoromisonidazole (FMISO) to visualize hypoxic areas within tumors, while MRI complements this with anatomical and functional images. Although directly assessing tumor hypoxia with MRI remains challenging, techniques like Blood Oxygen Level Dependent (BOLD) and Dynamic Contrast-Enhanced MRI (DCE-MRI) provide valuable information. BOLD can track changes in oxygen levels during oxygen challenges, while DCE-MRI offers real-time access to perfusion and vessel permeability data. Integrating data from these imaging modalities can help assess oxygen supply, refine treatment strategies, enhance therapeutic effectiveness, and ultimately improve patient outcomes.

The tumor microenvironment (TME) consists of complex interactions between cellular and extracellular components, among which the immune system is known to play an integral role in disease progression and response to therapy. Cytokines and chemokines are cell signaling proteins used by immune cells to communicate with each other as well as with other cell types in the body. These proteins control systemic and local immune responses and levels of cytokines/chemokines in the TME have been associated with tumor outcomes. However, cytokines and chemokines have varied expression across cell types, tumors, and host conditions. Therefore, approaches to effectively study the production of these proteins at the single-cell level in the TME are needed to fully elucidate the mechanisms governing the anti-cancer immune response. Here, we detail a protocol to assess the production of cytokines/chemokines across leukocyte populations in mouse tumors using RNA flow cytometry. Importantly, this method can be adapted with minimal changes to study various mouse and human tumors, other RNA analytes, and non-tumor tissues.