J.W.H. thanks the NIH Cancer Nanotechnology Training Center at the Johns Hopkins Institute for NanoBioTechnology, the National Science Foundation Graduate Research Fellowship (DGE-1232825), and the ARCS foundation for fellowship support. This work was funded by support from the National Institutes of Health (P01-AI072677, R01-CA108835, R21-CA185819), TEDCO/Maryland Innovation Initiative, and the Coulter Foundation (JPS).

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+for+generating+CD8++T-cell+clones+for+immunotherapy+from+the+naive+repertoire.">In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naive repertoire.</a> Journal of immunological methods. 310 (1-2), 40-52 (2006).</li><li>Rudolf, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+costimulation+of+human+CD8+T+cells+by+anti-4-1BB+and+anti-CD28+on+synthetic+artificial+antigen+presenting+cells.">Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells.</a> Cancer immunology, immunotherapy : CII. 57 (2), 175-183 (2008).</li><li>Gulukota, K., Sidney, J., Sette, A., DeLisi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+complementary+methods+for+predicting+peptides+binding+major+histocompatibility+complex+molecules1.">Two complementary methods for predicting peptides binding major histocompatibility complex molecules1.</a> Journal of molecular biology. 267 (5), 1258-1267 (1997).</li><li>Castle, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+mutanome+for+tumor+vaccination.">Exploiting the mutanome for tumor vaccination.</a> Cancer research. 72 (5), 1081-1091 (2012).</li><li>Duan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+bioinformatic+profiling+of+mutational+neoepitopes+reveals+new+rules+to+predict+anticancer+immunogenicity.">Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity.</a> Journal of Experimental Medicine. 211 (11), 2231-2248 (2014).</li><li>Srivastava, P. K., Duan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+antigenic+fingerprint+of+each+individual+cancer+for+immunotherapy+of+human+cancer:+genomics+shows+a+new+way+and+its+challenges.">Harnessing the antigenic fingerprint of each individual cancer for immunotherapy of human cancer: genomics shows a new way and its challenges.</a> Cancer Immunology, Immunotherapy. 62 (5), 967-974 (2013).</li><li>Yadav, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+immunogenic+tumour+mutations+by+combining+mass+spectrometry+and+exome+sequencing.">Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing.</a> Nature. 515 (7528), 572 (2014).</li><li>Gros, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+neoantigen-specific+lymphocytes+in+the+peripheral+blood+of+melanoma+patients.">Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients.</a> Nature medicine. 22 (4), 433 (2016).</li></ol>

The authors declare the following competing financial interest(s): under a licensing agreement between NexImmune and The Johns Hopkins University, Jonathan Schneck is entitled to a share of royalty received by the university on sales of products described in this article. He was also a founder of NexImmune and owns equity in the company. He serves as a member of NexImmune's Board of Directors and scientific advisory board. The terms of these arrangements have been reviewed and approved by The Johns Hopkins University in accordance with its conflict of interest policies.

We have created a novel antigen-specific T cell isolation technology based on nanoparticle artificial antigen presenting cells (aAPCs). Nanoparticle aAPCs have peptide-loaded MHC on the surface that allows antigen-specific T cell binding and activation alongside co-stimulatory activation. aAPCs are also paramagnetic, and thus can be used to enrich rare antigen-specific T cells using a magnetic field. We have optimized and studied key nanoparticle properties of size, ligand density, and ligand choice and their influence o...

With the rise of many immunotherapies, there is a need to be able to characterize and control immune responses. In particular, the adaptive immune response is of interest because of the specificity and durability of the cells. Recently, chimeric-antigen-receptor T cell therapies have been approved for cancer therapy; however, the antigen-receptors are based off the common cell surface antigen CD19, instead of the antigens specific to the cancer1. Beyond the specificity, immunotherapies can also suffer from the lack of control, and limited understanding the dynamic immune response within cancer or autoimmunity.
One of the challenges of studying antigen-specific responses is their extremely low frequency, e.g., antigen-specific T cells are 1 of every 104 to 106 T cells2,3. Thus, to investigate which T cells are present or responding, the cells need to either be enriched and expanded, or their signal need to be amplified. It is expensive and difficult to maintain the feeder cells using current techniques that focus on the expansion of antigen-specific cells. Current techniques that focus on amplifying the signal of antigen-specific T cells, like the enzyme-linked immunospot (ELISPOT) assay, limit the re-use of those T cells4. Finally, because of low sensitivity, often these two techniques need to be combined for antigen-specific enumeration.
To address these issues, we have developed the magnetic nanoparticle-based artificial antigen presenting cell (aAPC)5,6,7,8. The aAPC can be functionalized with an antigen-specific signal-peptide loaded major histocompatibility complex (pMHC)-and co-stimulatory molecules-e.g., an anti-CD28 antibody-to both enrich antigen-specific T cells and then subsequently stimulate their expansion (Figure 1). The particles can thus be a cost-effective off-the-shelf product that can be both customized to meet antigen-specific stimulations yet standardized across experiments and patients. Performing the enrichment and expansion process results in hundreds to thousands-fold expansion of antigen-specific CD8+ T cells and can result in frequencies up to 60 percent after just one week, enabling the characterization or therapeutic use of the large number of cells. Herein, we describe how to make nanoparticle aAPCs, some critical design considerations in choosing the nanoparticle properties, and demonstrate some typical results from utilizing these particles in isolating and expanding rare antigen-specific CD8+ T cells.

<table><tbody><tr><td>DimerX I: Recombinant Soluble Dimeric Human HLA-A2:Ig Fusion Protein</td><td>BD Biosciences</td><td>551263</td><td></td></tr><tr><td>DimerX I: Recombinant Soluble Dimeric Mouse H-2D[b]:Ig</td><td>BD Biosciences</td><td>551323</td><td></td></tr><tr><td>DimerX I: Recombinant Soluble Dimeric Mouse H-2K[b]:Ig Fusion Protein</td><td>BD Biosciences</td><td>550750</td><td></td></tr><tr><td>Vivaspin 20 MWCO 50 000</td><td>GE Life Sciences</td><td>28932362</td><td></td></tr><tr><td>Vivaspin 2 MWCO 50 000</td><td>GE Life Sciences</td><td>28932257</td><td></td></tr><tr><td>Purified Human Beta 2 Microglobulin</td><td>Bio-Rad</td><td>PHP135</td><td></td></tr><tr><td>nanomag-D-spio, NH2, 100 nm nanoparticles</td><td>Micromod</td><td>79-01-102</td><td></td></tr><tr><td>Super Mag NHS Activated Beads, 0.2 &amp;micro;m</td><td>Ocean Nanotech</td><td>SN0200&amp;nbsp;</td><td></td></tr><tr><td>Anti-Biotin MicroBeads UltraPure</td><td>Miltenyi</td><td>130-105-637</td><td></td></tr><tr><td>EZ-Link NHS-Biotin</td><td>ThermoFisher</td><td>20217</td><td></td></tr><tr><td>Sulfo-SMCC Crosslinker&amp;nbsp;</td><td>ProteoChem</td><td>c1109-100mg</td><td></td></tr><tr><td>2-Iminothiolane hydrochloride</td><td>Sigma-Aldrich</td><td>I6256 Sigma&amp;nbsp;</td><td></td></tr><tr><td>96 Well Half-Area Microplate, black polystyrene</td><td>Corning</td><td>3875</td><td></td></tr><tr><td>FITC Rat Anti-Mouse Ig, &amp;lambda;1, &amp;lambda;2, &amp;amp; &amp;lambda;3 Light Chain&amp;nbsp; Clone &amp;nbsp;R26-46&amp;nbsp;&amp;nbsp;</td><td>BD Biosciences</td><td>553434</td><td></td></tr><tr><td>FITC Mouse Anti-Armenian and Syrian Hamster IgG&amp;nbsp; Clone &amp;nbsp;G192-1</td><td>BD Biosciences</td><td>554026</td><td></td></tr><tr><td>B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J (transgenic PMEL) mice</td><td>Jackson Laboratory</td><td>005023</td><td></td></tr><tr><td>C57BL/6J (B6 wildtype) mice</td><td>Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>CD8a+ T Cell Isolation Kit, Mouse</td><td>Miltenyi</td><td>130-104-075</td><td></td></tr><tr><td>MS Columns</td><td>Miltenyi</td><td>130-042-201</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi</td><td>130-042-401</td><td></td></tr><tr><td>Streptavidin-Phycoerythrin, SAv-PE</td><td>Biolegend</td><td>405203</td><td></td></tr><tr><td>N52 disk magnets of 0.75 inches&amp;nbsp;</td><td>K&amp;amp;J Magnetics</td><td>DX8C-N52</td><td></td></tr><tr><td>APC anti-mouse CD8a Antibody, clone 53-6.7</td><td>Biolegend</td><td>100711</td><td></td></tr><tr><td>LIVE/DEAD Fixable Green Dead Cell Stain Kit, for 488 nm excitation&amp;nbsp;</td><td>ThermoFisher</td><td>L-34969</td><td></td></tr></tbody></table>

enrich and expand rare antigen-specific t cells with magnetic nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

To complete a successful enrichment and expansion of antigen-specific T cells, the peptide-loaded MHC-Ig and co-stimulatory molecules should be successfully attached to the aAPC particle. Based on the 3 methods of particle attachment, we provide some representative data for a successful conjugation procedure outcome (Figure 5a). Indeed, if the ligand density is too low, then there will not be effective stimulation of antigen-specific CD8+ T cells where this o...

Watch this Scientific Journal Video about Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles at JoVE.com

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

enrich-expand-rare-antigen-specific-t-cells-with-magnetic

Research

JoVE Journal

Engineering

11.4K Views.  Johns Hopkins University. Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy. 

Video: Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

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.

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 ...

Immunology and Infection

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Bioengineering

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

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 ...

Isolation and Characterization of Antigen-Specific T Cells in Mouse Lungs

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Author Spotlight: Optimized Protocol for Detecting Antigen-Specific T Cells in Mouse Lungs Using Tetramers

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...

Magnetic Activated Cell Sorting (MACS): Isolation of Thymic T Lymphocytes

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Defence against pathogens depends on surveillance by the immune system. This system is complex and comprises many cell types, each one with specific functions. This complex composition enables immune responses to a large diversity of pathogens and injuries. Adaptive immunity allows specific responses against specific pathogens. The majority of cells responsible for this type of immunity are the lymphocytes (B cells and T cells). Usually, B cells respond to extracellular infections (such as bacterial infections), and T cells respond to intracellular infections (such as viral infections). The different types of cells in lymphocyte populations can be characterized by the combination of cell surface proteins they express and/or by a panel of secreted cytokines.
Magnetic sorting allows enrichment of targeted cell populations using magnetic properties and expression of one or several cell surface proteins (1, 2). This technique consists of three steps. First, the cells are incubated with magnetic beads that are coupled with one or several monoclonal-specific antibodies. Cells that express surface proteins that bind to these antibodies attach to the magnetic beads. Then, the targeted cell populations are captured with a magnet. To finish, the targeted cells are eluted from the magnet. At the end, two sorting products are obtained, one containing unlabeled cells and the second containing the target cells coupled with the magnetic beads. Columns can be used to improve the efficiency of magnetic sorting. In the column, a non-magnetic element lengthens the path of cell through the column. Hence, cell flow is slowed down, facilitating cell capture by the magnet.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10495/10495fig1.jpg" /> 
Figure 1: Schematic representation of magnetic separation. Thymic leukocytes are stained with anti-CD3 biotinylated antibodies. After washing, streptavidin (SAV) coupled beads specifically fix the biotin on anti-CD3 antibodies. (1) Cells are transferred in a column. (2) The magnet does not retain unlabeled cells, while CD3-positive cells remain in the column. Finally, the column is separated from the magnet and (3) CD3-positive cells are eluted in medium. <a href="https://www.jove.com/files/ftp_upload/10495/10495fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are two types of magnetic sorting (3). In positive sorting, cells of interest are captured with the magnetic beads. In negative sorting, unwanted cells are removed by capturing with the magnetic beads carrying the appropriate antibodies. This MACS technique permits good enrichment of targeted cells and improves the percentage of recovered cells from 1-20% to 60-98% in an organ. After sorting, it is necessary to verify cell purity and sorting by different methods (e.g. flow cytometry). The MACS technique is ideal to enrich a target population for other experiments such as cell culture or cell cycle analysis.
In this lab exercise, we demonstrate how to isolate thymic leukocytes and thereafter enrich thymic CD3-positive cells from the mix using magnetic cell sorting technique.

MACS: Isolation of Thymic T Lymphocytes

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur ...

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T-Cell Enrichment: A Technique to Isolate T-Cells from Mixed Cell Population by Magnetic Separation

Source: Nguyen, H. D., et al. <a href="https://www.jove.com/t/60083">Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease.</a> J. Vis. Exp. (2020).
This video describes T-cells enrichment from the spleen of a mouse by negative selection using a magnetic separator. The technique involves targeting the other cells in the sample for depletion using antibodies or ligands directed against specific cell surface antigens.

Source: Nguyen, H. D., et al. Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease. J. Vis. Exp. ...