<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>aAPC&#160;</td>
			<td>Dean A. Lee lab&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>a-MEM culture medium&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>Cat#12634</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-DYKDDDDK (FLAG Tag)</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#637308</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-anti-human TRA-1-81&#160;</td>
			<td>ThermoFisher</td>
			<td>17-8883-42&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD16</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#302015</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD43&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat# 560198&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD45&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat# 555485&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-GPC3</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#DB100B</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-NKG2D&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat# 558071&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>CellEvent Caspase-3/7 Green Detection Reagent&#160;</td>
			<td>Thermo fisher&#160;</td>
			<td>Cat#C10423</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Violet Cell Proliferation Kit&#160;</td>
			<td>Thermo fisher&#160;</td>
			<td>Cat#C34571</td>
			<td>Cell tracing fluorescent dye&#160;</td>
		</tr>
		<tr>
			<td>CryoStor solution&#160;</td>
			<td>Stem Cell Technologies</td>
			<td>https://www.stemcell.com/products/cryostor-cs10.html</td>
			<td>Cryopreservation medium</td>
		</tr>
		<tr>
			<td>CTS NK Xpander</td>
			<td>Gibco</td>
			<td>A5019001</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS NK-Xpander Medium</td>
			<td>Life Technologies</td>
			<td>Cat#A5019001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human NK Cell Enrichment Kit&#160;</td>
			<td>StemCell Technologies, Inc.&#160;</td>
			<td>Cat#19055</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma Aldrich</td>
			<td>E9508</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>Cat# 10437010</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-CD94&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#555888</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>GolgiPlug&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#555029</td>
			<td></td>
		</tr>
		<tr>
			<td>GolgiStop&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#554724</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix, GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>31765035</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>Cat#16050130</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum&#160;</td>
			<td>AB Sigma-Aldrich</td>
			<td>Cat#BP2525100</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Stem Cell NucleofectorTM Kit&#160;</td>
			<td>Lonza&#160;</td>
			<td>Cat# VPH-5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Human: HePG2 cells&#160;</td>
			<td>ATCC&#160;</td>
			<td>Cat#HB-8065</td>
			<td></td>
		</tr>
		<tr>
			<td>Human: HePG2-td-tomato-luc cells&#160;</td>
			<td>Dan S. Kaufman lab</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Human: iPS cells&#160;</td>
			<td>Dan S. Kaufman lab&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Human: SNU-449 cells&#160;</td>
			<td>ATCC</td>
			<td>Cat#CRL-2234</td>
			<td></td>
		</tr>
		<tr>
			<td>Human: SNU-449-td-tomato-luc cells&#160;</td>
			<td>Dan S. Kaufman lab</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>IncuCyte Caspase-3/7 Green Apoptosis Assay&#160;</td>
			<td>Essenbioscience&#160;</td>
			<td>Cat#4440</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr>
			<td>MP Biomedicals&#160;Human Serum, Type AB</td>
			<td>MP Biomedicals</td>
			<td>ICN2938249</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus</td>
			<td>StemCell Technologies, Inc.&#160;</td>
			<td>100-0276</td>
			<td></td>
		</tr>
		<tr>
			<td>NovoExpress software&#160;</td>
			<td>ACEA Biosciences&#160;</td>
			<td>https://www.agilent.com/en/product/research-flow-cytometry/flow-cytometry-software/novocyte-novoexpress-software-1320805</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-human SSEA-4 Antibody&#160;</td>
			<td>Biolegend</td>
			<td>330420</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD16&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#560995&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD226</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#559789</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD34&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat# 555822&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD45&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat# 555483&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD94&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#555888&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-cy7-CD56&#160;</td>
			<td>BioLegend</td>
			<td>Cat# 318318&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-FAS Ligand&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#564261&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-NKp30</td>
			<td>BD Biosciences</td>
			<td>Cat# 558407</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-NKp44&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#558563&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-NKp46</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#331908</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-NKp46&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#557991&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Peripheral blood buffy coat&#160;</td>
			<td>San Diego Blood Bank (https://www. sandiegobloodbank.org/)&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-TRAIL&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>Cat#565499&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>pKT2-mCAG-IRES-GFP-ZEO&#160;</td>
			<td>Branden Moriarity lab&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pMAX-GFP plasmid</td>
			<td>Lonza</td>
			<td>N/A</td>
			<td>GFP positive control&#160;</td>
		</tr>
		<tr>
			<td>Prism 9</td>
			<td>Graphpad&#160;</td>
			<td>Version 9</td>
			<td></td>
		</tr>
		<tr>
			<td>pSpCas9&#160;</td>
			<td>GenScript</td>
			<td>PX165</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer (10x)</td>
			<td>Biolegend</td>
			<td>Cat#420301</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human bFGF basic&#160;</td>
			<td>R&#38;D Systems&#160;</td>
			<td>Cat#4114-TC</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human BMP-4&#160;</td>
			<td>PeproTech</td>
			<td>Cat#120-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human FLT-3 Ligand&#160;</td>
			<td>PeproTech</td>
			<td>Cat# 300-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-15&#160;</td>
			<td>PeproTech</td>
			<td>Cat# 200-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-2&#160;</td>
			<td>PeproTech</td>
			<td>Cat# 200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-3&#160;</td>
			<td>PeproTech</td>
			<td>Cat#200-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-7&#160;</td>
			<td>PeproTech</td>
			<td>Cat# 200-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Nodal Protein</td>
			<td>R&#38;D Systems&#160;</td>
			<td>Cat#3218-ND-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human SCF&#160;</td>
			<td>PeproTech</td>
			<td>Cat# 300-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-&#946;1</td>
			<td>PeproTech</td>
			<td>Cat#100-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human VEGF&#160;</td>
			<td>PeproTech</td>
			<td>Cat# 100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma Aldrich</td>
			<td>214485</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff APEL 2 Medium</td>
			<td>StemCell Technologies, Inc.&#160;</td>
			<td>5270</td>
			<td>EB formation medium</td>
		</tr>
		<tr>
			<td>STEMdiff APEL2 Medium&#160;</td>
			<td>StemCell Technologies, Inc.&#160;</td>
			<td>Cat#05270</td>
			<td></td>
		</tr>
		<tr>
			<td>Super piggyBac Transposase expression vector</td>
			<td>SBI</td>
			<td>Cat#PB210PA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX AADvanced Dead Cell Stain Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>S10274, S10349</td>
			<td>Dead cell staining solution kit</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
	</tbody>
</table>

advances in human induced pluripotent stem cell-derived chimeric antigen receptor-expressing natural killer cells

Fortschritte bei humanen induzierten pluripotenten Stammzellen, die von chimären Antigenrezeptoren abgeleitet werden und natürliche Killerzellen exprimieren

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

advances-human-induced-pluripotent-stem-cell-derived-chimeric-antigen

Research

JoVE Journal

Immunology and Infection

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

387 Aufrufe.  University of California, San Diego. Hier stellen wir eine Methode zur Differenzierung und Erweiterung von humanen iPSC-abgeleiteten chimären Antigenrezeptoren (CAR)-exprimierenden natürlichen Killerzellen mit verbesserter Abtötung gegen verschiedene Malignome vor. Dieses Protokoll demonstriert die Differenzierung und Expansion von für natürliche Killer (NK) optimierten iPSC-abgeleiteten CAR-NK-Zellen und die Messung der Antitumoraktivität gegen verschiedene Tumorzelll...

Serie: Fortschritte bei humanen induzierten pluripotenten Stammzellen, die von chimären Antigenrezeptoren abgeleitet werden und natürliche Killerzellen exprimieren

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Gerichtete Differenzierung von induzierten pluripotenten Stammzellen in Richtung T-Lymphozyten

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Forschung

Immunologie und Infektion

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Künstliche Antigen-präsentierenden Zelle (AAPC) vermittelte Aktivierung und Expansion der Natural Killer-T-Zellen

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer Künstliche Antigen-präsentierenden Zellen (KaAPC) für effiziente<em&gt; In-vitro-</em&gt; Depletion von humanen Antigen-spezifischen T-Zellen

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-Resolution Imaging der natürlichen Killerzell Immunologische Synapse auf einem Glas-gestützten Planar Lipiddoppelschicht

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Entwicklung von Stammzellen abgeleiteten Antigen-spezifische regulatorische T-Zellen gegen Autoimmunität

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Durchflusszytometrische Analyse von natürlichen Killerzellen lytische Aktivität in menschlichem Vollblut

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Ein neuartiges Feeder-freie System für die Massenproduktion von murinen Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Ein Echtzeit-Potenz-Assay für chimerantigene Antigen-Rezeptor-T-Zellen, die auf feste und hämatologische Krebszellen abzielen

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

Verwendung von humaninduzierten, zellabgeleiteten Intestarz-Organoiden zur Untersuchung und Modifizierung des epithelialen Zellschutzes gegen Salmonellen und andere Krankheitserreger

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

Induzierte Differenzierung von M-Zell-ähnlichen Zellen in menschlichen Stammzell-abgeleiteten Ileal Enteroid Monolayern

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...