measuring the activation of fc-mediated effector functions by ha antibodies

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

To assess the ability of specific antibodies of interest to target the hemagglutinin-expressing cells, replace the supernatant of the virally infected cell cultures with 25 microliters of antibody-dependent cell-mediated cytotoxicity, or ADCC, assay medium.
Next, in a separate 96-well plate, perform 4-fold dilutions of the antibodies of interest in triplicate and fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic, and taking care to include the background and no antibody controls.
When all of the antibody has been diluted, transfer 25 microliters of the diluted antibody to the appropriate corresponding wells in the experimental cell plate and place the plate in the cell culture incubator. After 15 minutes, add 7.5 times 10 to the 4th modified effector Jurkat cells, expressing the appropriate Fc receptor per well in 25 microliters of ADCC medium for a final volume of 75 microliters of ADCC medium per well.
Return the plate to the cell culture incubator for 6 hours, thawing luciferase substrate buffer in a 37-degree Celsius water bath during the last hour of the incubation. When the buffer is ready, add 75 microliters of luciferase substrate buffer to all of the wells except the background control well, and read the plate on a luminometer. The fold induction of antibody-dependent cell-mediated cytotoxicity in the transfected or infected cell cultures can then be calculated.

measuring-activation-fc-mediated-effector-functions-ha

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1. Expression of Influenza Virus Hemagglutinin via Transfection
<ol>
	<li>Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a white tissue culture treated 96-well plate and let it sit for 4 h in a 37 &#176;C incubator (with 5% CO2).</li>
	<li>Transfect cells with 100 ng of DNA coding for viral hemagglutinin and 0.2 &#956;L of transfection reagent per well in a total volume of 50 &#956;L. 
	NOTE: Both plasmid and transfectant reagent are diluted in 1x Opti-Minimum Essential Media (MEM) Reduced Serum Medium.</li>
	<li>After incubating plates in a 37 &#176;C incubator with 5% CO2 for 18 h, remove transfection media.</li>
</ol>
2. Assessing the FcRg/NFAT-mediated Activation of Luciferase Activity by Monoclonal Antibodies or Sera
<ol>
	<li>First, replace the growth media of transfected cells with 25 &#181;L of assay media (1x RPMI 1640 supplemented with 4% (vol/vol) low IgG fetal bovine sera).</li>
	<li>In a separate 96-well plate, perform four-fold dilutions of the antibodies of interest starting at 30 &#956;g/mL using the assay media. Perform dilutions in triplicates, and an example plate layout is illustrated in Figure 1A. Ensure that a control that excludes antibodies (no antibody control) and a background control (assay buffer alone) is included. 
	NOTE: For both the &#8216;no antibody&#39; and background controls, add 25 &#181;L of assay buffer instead of the antibody.</li>
	<li>Transfer 25 &#956;L of the serially diluted antibodies from Step 2.2 to corresponding wells on the plate of transfected cells.</li>
	<li>Incubate plate in a 37 &#176;C incubator (with 5% CO2) for 15 min.</li>
	<li>Add 25 &#956;L of the appropriate modified effector Jurkat cell expressing murine FcgRIV or human FcgRIIIa (75,000 cells/well). The total volume for each well should be 75 &#181;L, and the starting final concentration of the antibody is at 10 &#181;g/mL (Figure 1B). 
	NOTE: For the background control, add 25 &#181;L of assay buffer instead of effector cells. To examine antibody-dependent cell-mediated phagocytosis, a modified Jurkat cell expressing the human FcgRIIa can be used.</li>
	<li>Incubate the plate in a 37 &#176;C incubator with 5% CO2 for 6 h.</li>
	<li>Thaw and warm luciferase substrate buffer in 37 &#176;C water for about 1 h before use. 
	NOTE: To make the luciferase substrate buffer, reconstitute the lyophilized luciferase assay substrate in the provided assay buffer. The reconstituted substrate buffer can be stored at -30 &#176;C to -10 &#176;C for up to six weeks.</li>
	<li>Add 75 &#956;L/well of luciferase substrate buffer to all wells except for the background control.</li>
	<li>Read on a luminometer.</li>
	<li>Calculate fold induction with the following equation: Fold induction = (Induced value-Background value) / (No mAb value-Background value).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>Human embryonic kidney cells</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>1X Opti-MEM Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>51985-034</td>
			<td></td>
		</tr>
		<tr>
			<td>White tissue culture treated 96-well plate</td>
			<td>Corning, Inc.</td>
			<td>3917</td>
			<td>Assay plates</td>
		</tr>
		<tr>
			<td>10X MEM</td>
			<td>Gibco</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr>
			<td>Jurkat cell expressing murine FcgRIV</td>
			<td>Promega</td>
			<td>M1201</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr>
			<td>Jurkat cell expressing human FcgRIIIa</td>
			<td>Promega</td>
			<td>G7010</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr>
			<td>Jurkat cell expressing human FcgRIIa</td>
			<td>Promega</td>
			<td>G9901</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>BioTek</td>
			<td>Synergy H1 Multi-Mode reader</td>
			<td>Luminescence plate reader</td>
		</tr>
		<tr>
			<td>Bio-Glo Luciferase Assay System</td>
			<td>Promega</td>
			<td>G7940</td>
			<td>Contains luciferase assay buffer and luciferase assay substrate</td>
		</tr>
	</tbody>
</table>

Source: Bailey, M. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

<img alt="Figure 1" src="/files/ftp_upload/22083/22083_Figure1.jpg" /> 
Figure 1: A schematic of a sample plate layout and composition of the experimental groups. (A) The starting concentration for each antibody sample is at 10 &#181;g/mL and serially diluted across the plate (column 1 to column 12) 4-fold. Each sample is done in triplicates. Row G is reserved for the no mAb control, and Row H is the background. (B) The final volume for each experimental group before the addition of luciferase substrate buffer is 75 &#181;L. Of note, the sample and &#8216;no mAb groups&#8217; have 25 &#181;L of effector cells added, while the background control groups have 75 &#181;L of assay media.

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

Take serial dilutions of an antibody targeting the Influenza virus hemagglutinin, or HA, a glycoprotein consisting of a globular head and a stalk.
Add the dilutions onto transfected mammalian cells expressing surface-bound HA. The antibodies bind to the HA stalk regions, forming immune complexes.
Introduce engineered T cells expressing Fc receptors and encoding a luciferase reporter under the control of the transcription factor NFAT.
During incubation, the HA-bound antibody binds to the Fc receptor, while the HA head binds to sialic acid, triggering T cell activation and increasing cytosolic calcium concentration.
This activation initiates a signaling cascade that results in the dephosphorylation of NFAT, triggering its nuclear translocation.
NFAT binds to its response elements in the genome, driving luciferase expression.
Add a cell-permeable bioluminescent substrate. Luciferase oxidizes the substrate, producing a signal.
Decreased luminescence with decreasing antibody concentration confirms HA stalk-specific antibody-mediated T cell activation.

40 次观看. 测量 HA 抗体对 Fc 介导的效应子功能的激活

视频: 测量 HA 抗体对 Fc 介导的效应子功能的激活 - 实验

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

通过抗原肽和Fc-III三氧化二甲物的双功能结合阻断靶向抗体

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...

免疫与感染

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

微胶质细胞抗体介导的陶氏清除研究

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

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Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies