eoe sub articles

EoE Sub Articles

1. Preparation of target cells that express red fluorescent protein in their nuclei
<ol>
	<li>Obtain a target mouse cancer cell line from an appropriate source. 
	NOTE: In this protocol, a highly metastatic derivative of E0771 mouse mammary tumor cells (E0771-LG) is used. Parental E0771 cells originate from C57BL/6 mice.</li>
	<li>Thaw and maintain a vial of E0771-LG cells with Dulbecco&#39;s Modified Eagles Medium (DMEM) including 10% (v/v) fetal bovine serum (FBS) in a cell cult...

an in vitro assay for studying the cytotoxicity of pre-activated cd8+ t cells against cancer cells

Source: Kitamura, T., et al. <a href="https://app.jove.com/v/58841">Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells.</a> J. Vis. Exp. (2019)
This video showcases an apoptosis assay in cancer cells induced by pre-activated effector T cells. By co-culturing these cells with cancer cells marked with fluorescent nuclei, a caspase-cleavable fluorogenic substrate is added. Apoptotic cancer cells will display a different color in their nuclei, leading to dual fluorescence. This dual fluorescence is indicative of cancer cells undergoing apoptosis.

<img alt="Figure 1" src="/files/ftp_upload/21904/21904_Figure1.jpg" /> 
Figure 1. Gating strategy to isolate suppressor cells from the metastatic lung. (A) Representative dot plots to isolate monocytic myeloid-derived suppressor cells (M-MDSCs) and metastasis-associated macrophages (MAMs). The threshold of Ly6C level to distinguish MAMs (Ly6Clow) and M-MDSCs (Ly6Chigh) is based on that of resident alveolar macrophages (RMAC). (<...

An In Vitro Assay for Studying the Cytotoxicity of Pre-Activated CD8+ T Cells against Cancer Cells

1. Preparation of target cells that express red fluorescent protein in their nuclei

	Obtain a target mouse cancer cell line from an appropriate source.
	...

Take two CD8+ T cell populations: effector&#160; CD8+ T cells pre-activated through antibody-mediated engagement of T cell and co-stimulatory receptors and suppressed CD8+ T cells inhibited by myeloid-derived suppressor cells.
Add these T cells to basement-membrane-coated wells with cancer cells expressing a nuclear-restricted fluorescent protein.
Pipette a fluorogenic&#160; caspase-3 substrate &#8212; a DNA-binding fluorogenic dye conjugated to a caspase-3 recognition site-containing peptide.
Incubate. Effector T cell receptors bind to the class I MHC-peptide complex on the cancer cells, triggering the release of cytotoxic granules containing perforins and granzymes.
Perforins create cancer cell membrane pores, allowing granzyme entry and caspase activation, initiating apoptosis.
Activated caspases additionally cleave the fluorogenic caspase substrate, releasing the dye that binds to DNA and fluoresces.
Under a fluorescence microscope, cancer cells co-cultured with effector T cells exhibit dual nuclear fluorescence, indicating cancer cell apoptosis.
In contrast, cancer cells co-cultured with suppressed T cells display only nuclear protein fluorescence, suggesting non-apoptotic cells.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

JoVE Journal

Cancer Research

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

An In Vitro Assay for Studying the Cytotoxicity of Pre-Activated CD8⁺ T Cells against Cancer Cells

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