eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of PBMCs and enrichment/purification of NK cells
NOTE: Other methods for the isolation of peripheral blood mononuclear cells (PBMCs) and enrichment/purification can also be performed.
<ol>
	<li>Draw 200 mL of blood under regulated conditions into a tube containing heparin.</li>
	<li>Add 15 mL of the density gradient medium into a 50 mL tube with a porous barrier incorporated. Spin the tube at 1000 x g for 30 s.</li>
	<li>Add 12.5 mL of blood into the tube, followed by another 12.5 mL of PBS, and gently invert 3x. Spin the tube at 800 x g for 15 min at room temperature (RT) with no breaks.</li>
	<li>Prepare a 50 mL conical tube with 40 mL of PBS for each tube with a porous barrier while the cells are spinning.</li>
	<li>Carefully remove the tubes and inspect after centrifugation. Check for a thin, visibly white layer of PBMCs above the porous barrier, between the 20 mL and 25 mL demarcations on the 50 mL tube.</li>
	<li>Carefully remove as much liquid as possible from the top using a pipette without disturbing the thin, white PBMC layer.</li>
	<li>With a clean 10 mL serological pipette, gently remove the PBMC layer in addition to the clear, yellowish liquid layer down to the filter of the tube.</li>
	<li>Expel the PBMCs and liquid into the 50 mL conical containing PBS prepared in step 1.4. Spin tubes at RT and 300 x g for 5 min.</li>
	<li>Aspirate the PBS wash and add an additional 40 mL of PBS. Spin tubes at RT and 300 x g for 5 min.</li>
	<li>After washing, count the cells and resuspend them in 40 &#181;L of 2% BSA/PBS per 1 x 107 cells.</li>
	<li>Proceed to the isolation of NK cells using the method of choice. Choices include manual or automated magnetic bead-based sorting. Fluorescence-activated cell sorting can also be performed.</li>
	<li>Remove a small aliquot of cells to determine the purity of NK cells by flow cytometry. Gating strategy includes the following: gate on live cells based on forward vs. side scatter profile, then assess the purity based on NK markers (e.g., CD3, CD56) in the specific live population. Typical purity is &#62;95%.</li>
	<li>After isolation is completed, spin cells at RT and 300 x g for 5 min to pellet and aspirate.</li>
	<li>Resuspend the cell pellet to 1 x 107 cells/mL in RPMI 1640 with nutrients, 10% heat-inactivated FBS, 1 mM sodium pyruvate, 55 mM 2-ME, and 10 mM HEPES (pH = 7.2).</li>
</ol>
2. Antibody-mediated activation of NK cells via Fc&#947;RIIIa
<ol>
	<li>Set a refrigerated microcentrifuge to 4 &#176;C.</li>
	<li>Dispense 1 x 106 (100 &#181;L of) resuspended NK cells into 1.5 mL tube(s) and place on ice. 
	NOTE: Cells can be dispensed into a 96 well U-bottom plate if there are numerous samples.</li>
	<li>Prepare 1 &#181;L of 100 &#181;g/mL rituximab (or antibody of interest) per sample and add 1 &#181;L to the cells for a final concentration of 1 &#956;g/mL antibody. 
	NOTE: Other molecules can be added to the cells at this point or earlier for the assessment of combination effects. Here, ritixumab was used for stimulation. In prior studies, small molecule inhibitors have been added to stimulations.</li>
	<li>Incubate the sample on ice for 30 min. During incubation, prepare 50 &#956;L of 50 &#181;g/mL anti-human &#954; light chain antibody per sample in media and warm to 37 &#176;C on a heat block or in a water bath.</li>
	<li>After incubation of cells on ice, add 1 mL of ice-cold media and spin at 135 x g for 5 min at 4 &#176;C. Wash again with 1 mL of ice-cold media.</li>
	<li>Aspirate the supernatant after the last wash and add 50 &#181;L of the anti-human &#954; light chain mixture prepared in step 2.4.</li>
	<li>Immediately incubate samples for the end-user-determined timepoints at 37 &#176;C on a heat block or in a water bath.</li>
	<li>Stop stimulation by adding 1 mL of ice-cold media and immediately spin samples in a refrigerated centrifuge at 135 x g for 5 min. Wash once more with 1 mL of ice-cold media.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>50-980-487</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse HRP antibody</td>
			<td>Cell Signaling Technologies</td>
			<td>7076</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS instrument</td>
			<td>Miltenyi</td>
			<td></td>
			<td>NK cell isolation method; another isolation instrument may be used</td>
		</tr>
		<tr>
			<td>CD107a APC antibody</td>
			<td>Biolegend</td>
			<td>328620</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V, fatty acid free</td>
			<td>Millipore Sigma</td>
			<td>10775835001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokine 30-Plex Human Panel</td>
			<td>Thermo Fisher Scientific</td>
			<td>LHC6003M</td>
			<td>chemokine/cytokine method; another chemokine/cytokine analysis method may be used</td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Hyclone</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human &#954; light chain 
			antibody</td>
			<td>Millipore Sigma</td>
			<td>AP502</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>pAKT (S473) antibody</td>
			<td>Cell Signaling Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pERK1/2 antibody</td>
			<td>Cell Signaling Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pPRAS40 antibody</td>
			<td>Cell Signaling Technologies</td>
			<td>13175</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-actin HRP antibody</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>NK cell isolation kit</td>
			<td>Miltenyi</td>
			<td>130-092-657</td>
			<td>NK cell isolation kit; another isolation kit may be used</td>
		</tr>
	</tbody>
</table>

an in vitro artificial activation assay for studying fc receptor activation by antibodies

Source: Petriello, A. et al. <a href="https://app.jove.com/v/61144">Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies.</a> J. Vis. Exp. (2020)
This video demonstrates Fc&#947;RIIIa-driven events initiated by therapeutic antibodies in human natural killer cells. The artificial stimulation platform facilitates the exploration of downstream effector functions, encompassing cytoskeletal rearrangement, degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of PBMCs and ...

Take a suspension of natural killer, or NK, cells.
Add anti-CD20 monoclonal antibodies and incubate.
In the absence of target cancer cells expressing the CD20 antigen, the Fc region of the antibodies bind to the Fc&#947;RIIIa receptors on the NK cell surface.
Post-incubation, add chilled media. Centrifuge and remove unbound antibodies.
Next, add anti-human &#954; light chain antibodies. Incubate.
These secondary antibodies bind to the &#954; light chain of the monoclonal antibodies.
The antibody crosslinking mimics target antigen binding, triggering an intracellular signaling cascade.
This causes artificial stimulation of NK cells in the absence of cancer cells.
Activated NK cells undergo cytoskeletal rearrangement and release cytotoxic granules.
Further, the cells produce chemokines and cytokines.
Following the desired duration, add chilled media to stop cell stimulation.
Centrifuge the tube. Collect the supernatant and cell pellet for downstream analysis of antibody-mediated effector NK cell functions.

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Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

62 Views. Source: Petriello, A. et al. Assessment of Human Natural Killer Cell Events Driven by FcγRIIIa Engagement in the Presence of Therapeutic Antibodies. J. Vis. Exp. (2020) This video demonstrates FcγRIIIa-driven events initiated by therapeutic antibodies in human natural killer cells. The artificial stimulation platform facilitates the exploration of downstream effector functions, encompassing cytoskeletal rearrangement, degranulation, chemokine/cytokine production, and signaling pathway...

Video: An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies - Concept

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

JoVE Journal

Cancer Research

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Immunology and Infection

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies

Transcript

An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells