Name: in vitro elisa test to evaluate rabies vaccine potency
Uploaded: 2020-05-11T12:30:00
Description: Watch this Scientific Journal Video about In Vitro ELISA Test to Evaluate Rabies Vaccine Potency at JoVE.com

Dr. Sylvie Morgeaux and Dr. Jean-Michel Chapsal must be acknowledged for their key participation to establish the ELISA assay on vaccine batches and to organize international Workshops and collaborative studies. We thank Sabrina Kali for critical reading of the manuscript. Dr. Pierre Perrin was responsible for the isolation and characterization of mAb-D1. This work has been mainly supported by Institut Pasteur funding.

1. Security precautions
NOTE: This method is applicable to both live RABV and inactivated vaccine.
<ol>
	<li>Use good Laboratory Practice and Safety procedures.</li>
	<li>Wear adequate Personal Protection Equipment (PPE) including disposable coat, gloves, mask, glasses, etc.</li>
	<li>When the live virus is titrated, use a class II biological safety cabinet.
	<ol>
		<li>Consider any material in contact with the samples (reagents, washing solutions, etc.) as infectious material.</li>
		<li>Tr...

The epitope recognized by the mAb-D1 is located in the antigenic site III of the RABV glycoprotein which is not only immuno-dominant for the induction of VNAbs but also involved in neurovirulence, pathogenicity40,41 and receptor recognition42. There is another important antigenic site along the glycoprotein, site II43, against which several MAbs have been isolated such as mAb-WI-111235. These...

Since more than 50 years, the NIH test1 is used as a gold standard method to evaluate the rabies vaccine potency before the batch release. This test consists of an intraperitoneal immunization of groups of mice with the vaccine to be tested followed by an intra-cerebral (IC) challenge 14 days later with the Challenge Virus Standard (CVS) strain of rabies virus (RABV). The potency is evaluated from the proportion of mice surviving the IC challenge. Although WHO2 and European Pharmacopeia3 still require the NIH test for assessing the vaccine potency, this test suffers several hurdles: results are highly variable4; infectious RABV is used during the challenge and this requires both technical skill and strict biosafety measures; large numbers of animals are used, and the severity of the challenge raises serious ethical concerns5. A less severe variation of this test has been developed: two weeks after the intra-peritoneal immunization, mice are not challenged by IC but bled and tested for the presence of specific RABV neutralizing antibodies (VNAbs) in their serum using an in vitro neutralization test. However, this test still requires sacrificing a large number of laboratory mice although it is already in use for the veterinary vaccines6,7 and has been considered for human vaccines8.
As of now, both International9 and European10 recommendations encourage manufacturers and National Control Laboratories (Official Medicine Control Laboratories - OMCLs) to implement the Replacement, Reduction, and Refinement of laboratory animal testing, referred as the 3Rs strategy. European Directive 2010/63/EU (in force since 2013/01/01) related to the protection and welfare of animals has also reinforced the constraints for vaccine manufacturers and laboratories involved in the Quality Control of rabies vaccines as well as in rabies research11. As a result, the development, validation, and use of alternative in vitro approaches have now become a priority. These are not only ethically sound but can also reduce the batch testing costs and shorten the time for results to hours instead of weeks3.
At the surface of the RABV particle, the glycoprotein adopts a trimeric form12,13,14,15,16. In rabies vaccine, this native trimeric form constitutes the major immunogen inducing VNAbs17 while the monomeric, soluble or denatured glycoproteins are poorly immunogenic18,19. Thus, the preservation of trimers of the glycoprotein along the vaccine production process is a good indicator for the preservation of an optimal immunogenic potential. Several immunochemical methods, such as the antibody-binding-test20,21, the single radial immunodiffusion (SRD) test22 and the ELISA test23,24,25,26,27 are recommended by the WHO Technical Report Series2 and the European monograph3 to quantify the antigen content in rabies vaccines. These are used by manufacturers to monitor the consistency of vaccine production and by the OMCL to assess the consistent formulation of batches of human vaccines28, even if the NIH test is still considered for the potency.
However, all these immunochemical methods are not equivalent. The SRD test requires a pre-treatment which may alter the membrane-anchored trimers and result in a soluble or denatured form of the glycoprotein22,29. Hence, SRD is not much efficient in discriminating between immunogenic and non-immunogenic glycoproteins resulting in an imperfect appraisal of the immunogenicity of a vaccine lot. By contrast, the ELISA test is more sensitive22, preserves the native structure of the glycoprotein, and is more appropriate to determine the content of the natively folded trimers of glycoprotein. The ELISA test can use either rabbit polyclonal or mouse monoclonal anti-glycoprotein antibodies purified or concentrated with ammonium sulfate. Studies have demonstrated good concordance between the NIH test and the antigen content evaluated by ELISA in vaccines and concluded that ELISA methods are suitable for the in vitro&#160;potency test. This advocates that ELISA tests might at least supplement or even replace the NIH test4,26,27,30,31,32,33. Today, the European Pharmacopoeia recommends the use of validated serological or immunochemical assays as alternatives to the NIH test3. The complete avoidance of animal use for vaccine potency has become a realistic perspective.
The method presented below is based on an indirect ELISA sandwich immunocapture using a mouse monoclonal antibody clone (mAb-D1) which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein15,34. This method was developed initially at the Institut Pasteur26,30 then optimized and validated by the Agence Nationale de S&#233;curit&#233; du M&#233;dicament et des produits de sant&#233; (ANSM) laboratory, i.e., the French OMCL4,33. The mAb-D1 is used both for sensitizing the plate and subsequently for detecting the captured antigen. This allows for the specific quantification of the glycoprotein trimers, i.e., the immunogenic RABV antigen. The mAb-D1 used for the detection is labeled with peroxidase, which is revealed in the presence of the substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content. It is of note that the same type of assay can be applied for different mAbs recognizing different antigenic sites of the RABV glycoprotein35. The method to obtain and purify or concentrate with ammonium sulfate anti-glycoprotein polyclonal rabbit immunoglobulins G (IgG) or monoclonal mouse globulins have been extensively described previously36 along with the method to conjugate antibodies with peroxidase37.

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	<tbody>
		<tr height="34">
			<td height="34" style="height:35px;width:465px;">Class II Biological Safety Cabinet</td>
			<td style="width:340px;">ThermoFisher Scientific</td>
			<td style="width:262px;">10445753</td>
			<td style="width:456px;">if titrating live virus</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:465px;">Clear Flat-Bottom Immuno Nonsterile 96-Well Plates, 400 &#181;L, MAXISORP</td>
			<td>ThermoFisher Scientific</td>
			<td>439454</td>
			<td>good for binding to the loaded antibody</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:85px;width:465px;">Equip Labo Polypropylene Laboratory Fume Hood</td>
			<td>ThermoFisher Scientific</td>
			<td>12576606</td>
			<td>for the preparation of sulfuric acid</td>
		</tr>
		<tr height="112">
			<td height="112" style="height:113px;width:465px;">Immunology Plate Strong 
			Adsorption MAXISORP Flat Bottom 
			Well F96</td>
			<td style="width:340px;">Dutscher</td>
			<td>55303</td>
			<td>good for binding to the loaded antibody</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;">Microplate Sealing Tape(100 sheets)</td>
			<td>ThermoFisher Scientific</td>
			<td>15036</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:465px;">Microplate&#160; single mode reader Sunrise</td>
			<td style="width:340px;">TECAN</td>
			<td style="width:262px;"></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;">Microplate shaker-incubator</td>
			<td style="width:340px;">Dutscher</td>
			<td>441504</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:465px;">Microplate washer Wellwash</td>
			<td>ThermoFisher Scientific</td>
			<td>5165000</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:465px;">Multichannel pipette (30-300 &#181;L) 12 channels</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:262px;">4661180N</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="103">
			<td height="103" style="height:104px;width:465px;">Single Channel pipettes (Kit 2 : Finnpipettes F2 0.2-2 &#956;L micro, 2-20 &#956;L, 20-200 &#956;L &#38; 100-1000 &#956;L)</td>
			<td>ThermoFisher Scientific</td>
			<td>4700880</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro elisa test to evaluate rabies vaccine potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

In the following example the reference vaccine Lot 09, consisting of purified inactivated rabies virus particles (PV vaccine strain), is used. The glycoprotein trimers (10 &#181;g/mL) content in this has been established after the determination of the total amount of viral proteins (BCA test) and evaluation of the percentage of the glycoprotein by SDS-polyacrylamide gel electrophoresis. Alternatively, a calibrated reference vaccine, e.g., the WHO 6th International Standard (IS)...

Watch this Scientific Journal Video about In Vitro ELISA Test to Evaluate Rabies Vaccine Potency at JoVE.com

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

In the aim of reducing animal testing and because of the variability of the NH test, WHO encourages to develop in vitro approaches for evaluating rabies vaccine potency. This ELISA test shows a good concordance with a classical NH test in recognizing the immunogenic form of the glycoprotein, shows a high sensitivity, and can be performed within only one day. Evaluation of rabies vaccine potency in vitro by ELISA is an alternative to the in vivo NH test.It is promoted by the manufacturers and national control laboratories. It's critical to distribute pre-sized volumes in duplicate for each dilution and to well dry the plate on absorbent paper after washes to avoid To begin this procedure, put on adequate personal protection equipment including disposable coat, gloves, mask, and glasses. Treat the contaminated materials by immersing it in a solution of 2.6%sodium hypochlorite for 30 minutes for decontamination.Next, set out a 96-well immunoassay plate and add 200 microliters of the monoclonal antibody diluted in the carbonate buffer to each well. Cover the plate with adhesive film and incubate the microplate at 37 degrees Celsius for three hours in a humidified environment. Then carefully aspirate and transfer the well content into a recipient containing a solution of 2.6%sodium hypochlorite.Invert the microplate and let it dry on absorbent paper at room temperature for five minutes. First, add 300 microliters of the passivation buffer to each well. Cover the plate with adhesive film and incubate at 37 degrees Celsius for 30 minutes.After this, transfer the contents of the well to one containing a solution of 2.6%sodium hypochlorite. To wash the plate, add 300 microliters of the washing buffer to each well. Then aspirate carefully and transfer the well content into a recipient containing a solution of 2.6%sodium hypochlorite.Repeat this washing process five more times to extensively wash the sensitized plate. After this, invert the microplate and let it dry on a piece of absorbent paper at room temperature for one minute. Reconstitute the reference vaccine in one milliliter of distilled water such that the final concentration of rabies virus glycoprotein is 10 micrograms per milliliter.Prepare a 10-fold dilution of the reconstituted reference vaccine in the diluent to reach a rabies virus glycoprotein concentration of one microgram per milliliter. Next, perform six serial two-fold dilutions of this reference vaccine in the diluent as outlined in table one of the text protocol. Distribute 200 microliters of the diluent in duplicate wells to serve as a blind control and 200 microliters per well for each reference vaccine dilution in duplicate.Then prepare a 10-fold dilution of the tested vaccine in the diluent and prepare seven two-fold serial dilutions of the tested vaccine in diluent as outlined in table two of the text protocol. Distribute 200 microliters of each tested vaccine dilution in duplicate to each well of the microplate. Cover the microplate with adhesive film and incubate at 37 degrees Celsius for one hour.After this, remove the film. Aspirate carefully and transfer the contents of each well into a recipient containing a 2.7%sodium hypochlorite solution. To wash the plate, add 300 microliters of washing buffer to each well, aspirate carefully and transfer the contents of each well into a recipient containing a 2.6%sodium hypochlorite solution.Repeat the washing process five more times to remove the unbound antigen and conserve the G-protein trimers bound to the coated antibody. Invert the washed microplate and let it dry on a piece of absorbent paper at room temperature for one minute. Then distribute 200 microliters of the recommended dilution of peroxidase labeled D1 monoclonal antibody in diluent to each well.Cover the microplate with adhesive film and incubate at 37 degrees Celsius for one hour. Next, remove the film, aspirate carefully, and transfer the contents of each well into a recipient containing a 2.6%sodium hypochlorite solution. To wash the microplate, add 300 microliters of washing buffer to each well.Aspirate carefully and transfer the contents of each well into a recipient containing 2.6%sodium hypochlorite solution. Repeat this washing process five more times to remove the unbound peroxidase labeled antibody. Invert the washed microplate and let it dry on a piece of absorbent paper at room temperature for one minute.Next, distribute 200 microliters of substrate-chromogen solution into each well. Seal the microplate with film and incubate at room temperature in the dark for 30 minutes. Then add 50 microliters of stopping solution into each well to stop the reaction.Carefully wipe the bottom of the microplate and place it in the spectrophotometer. Determine the optical density at 492 nanometers for all of the wells used. Collect this data in a spreadsheet file for analysis.After this, create plots for both the reference vaccine curve and the optical density values as outlined in the text protocol. In this study, the in vitro ELISA test is used to evaluate rabies vaccine potency. Representative optical density values from a typical experiment are shown here.These values are used to draw the reference vaccine curve by plotting the mean optical density for the different dilutions of the reference vaccine on the vertical axis and the concentration of the glycoprotein on the horizontal axis. The glycoprotein content of the tested vaccine is then estimated using this curve. The evaluation is precise for dilutions that have a mean optical density value in the liner area of the reference vaccine curve.Taking into account the dilution of one-to-40, the vertical projection of OD 1534 on the x-axis corresponds to 500 nanograms per milliliter. So the tested vaccine is estimated at 20 micrograms per milliliter of glycoprotein. When the in vitro potency is established, it is necessary to compare the tested vaccine to the reference six WHO international standard calibrated in international units.The same method can be used with other antibodies to enlarge detection of more vaccine strains including those used in veterinary vaccines which are classically more variable. Antibodies can also be used in competition for titration of antirabies antibodies in equine or human serum. Precautions must be taken with vaccines that are not inactivated or with viruses.Be sure to use personal protective equipment, wear gloves and decontaminate everything properly. Also, be careful with tablets of as they are carcinogenic and with the acidic sodium hypochlorite solution.

in-vitro-elisa-test-to-evaluate-rabies-vaccine-potency

Research

JoVE Journal

Immunology and Infection

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The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

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In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

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Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

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A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

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The direct rapid immunohistochemical test (DRIT) offers a World Organization for Animal Health and World Health Organization (OIE/WHO) recognized alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis. This test allows for field-based applications that can be performed in approximately 1 h upon brain impressions using light microscopy.

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, ...

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Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

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Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to&#160;opsono-phagocytic killing assays. An advantage of the opsono-adherence&#160;assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.&#160;&#160;&#160;&#160;&#160;&#160;

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is an alternative method to the opsono-phagocytic killing assay to evaluate the opsonic functions of antibodies in vaccine development.

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...

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 ...