Name: preparation of bead-supported lipid bilayers to study the particulate output of t cell immune synapses
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Description: Watch this Scientific Journal Video about Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses at JoVE.com

We are grateful to our laboratory members and the Kennedy Institute of Rheumatology community for constructive scientific discussions, especially our flow cytometry facility manager Jonathan Webber. This work was funded by Wellcome Trust Principal Research Fellowship 100262Z/12/Z, the ERC Advanced Grant (SYNECT AdG 670930), and the Kennedy Trust for Rheumatology Research (KTRR) (all three to MLD). PFCD was supported by EMBO Long-Term Fellowship (ALTF 1420-2015, in conjunction with the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Sklodowska-Curie Actions) and an Oxford-Bristol Myers Squibb Fellowship.

1. Measurement of cell surface protein densities with quantitative flow cytometry
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	<li>Prepare 0.22 &#181;m-filtered human flow cytometry buffer (hFCB) by adding EDTA (to a final 2 mM concentration) and human AB serum (to a final 10%) to sterile phosphate-buffered saline (PBS), pH 7.4 (see Table 1). Filter the solution using a 0.22 &#181;m pore filter unit to remove serum impurities and store at 4 &#176;C.</li>
	<li>Recover the cells and sediment them by centrifugation at 300 &#21...

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Abcam Available from: <a target="_blank" href="https://www.abcam.com/protocols/counting-cells-using-a-haemocytometer">https://www.abcam.com/protocols/counting-cells-using-a-haemocytometer</a> (2021)</li><li>Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+compensation+for+flow+cytometry:+visualization+artifacts,+limitations,+and+caveats.">Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats.</a> Cytometry. 45 (3), 194-205 (2001).</li><li>Dam, T., Junghans, V., Humphrey, J., Chouliara, M., Jonsson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+T+cells+is+induced+by+binding+to+nickel-chelating+lipids+in+supported+lipid+bilayers.">Calcium signaling in T cells is induced by binding to nickel-chelating lipids in supported lipid bilayers.</a> Frontiers in Physiology. 11, 613367 (2020).</li><li>Nguyen, R., Perfetto, S., Mahnke, Y. 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T., Trotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+controls,+instrument+setup,+and+the+determination+of+positivity.">Flow cytometry controls, instrument setup, and the determination of positivity.</a> Cytometry A. 69 (9), 1037-1042 (2006).</li><li>Balint, S., 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=Supramolecular+attack+particles+are+autonomous+killing+entities+released+from+cytotoxic+T+cells.">Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells.</a> Science. 368 (6493), 897-901 (2020).</li><li>Cespedes, P. 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BSLBs are versatile tools for studying the particulate output of T cells stimulated with model APC membranes. The flexibility of the method allows the reconstitution of complex and reductionist membrane compositions to study the effects of ligands and their signals on the secretion of tSVs and supramolecular attack particles and their components. We have tested this technology on various T cells, including preactivated TH, CTL, Tregs, and CART15. This protocol also works for the measurement of syn...

The immunological synapse (IS) is a pivotal molecular structure formed at the interface of cells engaged in physical contact that facilitates the regulated exchange of juxtracrine information. Different ISs have been described in the literature, and a growing body of evidence suggests these molecular hubs are a conserved feature of cellular networks. Various immune cells, including B cells, natural killer cells, dendritic cells, macrophages, and T cells, exchange information via the assembly of short-lived contacts1. Multiomic studies are advancing the understanding of novel subsets of leukocytes and stromal cells driving pathogenic cellular networks and expressing surface proteins with unknown functions. As synthetic APCs, BSLBs allow the direct investigation of the functional role of individual proteins in the integration of activating signals, namely antigens and costimulation/corepression, by T cells and the resulting release of effector particles referred to as signal four.
This paper describes the protocols and critical technical points to consider while using BSLBs to mimic the surface composition of model APCs. The protocols for the quantitative measurement of immune receptors and other surface proteins on APCs are presented along with the protocol for the reconstitution of synthetic APCs containing these measured quantities. Then, the steps required for coculturing T cells and BSLB are presented along with the protocol for the quantitative measurement of trans-synaptic particle transfer using flow cytometry. Most remarkably, BSLBs facilitate studying a plasma membrane-derived population of tSVs termed synaptic ectosomes (SEs). T-cell antigen receptor-enriched (TCR+) SEs are shed in response to TCR triggering2 and efficiently captured by BSLBs3, representing an excellent readout to assess the agonistic properties of antigens and the modelled membrane composition. CD63+ exosomes and supramolecular attack particles (SMAPs) are also released by stimulated T cells and captured by BSLBs. They can be used as additional readouts of activation and the resulting exocytic and lytic granule secretion by T cells. The mobilization of exocytic vesicles to the interacting pole of the T cell also facilitates the directional release of cytokines, such as IL-2, IFN-&#947;, and IL-10 in response to activation4,5,6,7,8. Although T-cell released cytokines can also be detected on BSLBs, a more dedicated study is currently under development to validate the quantitative analysis of cytokine release at the immunological synapse.
To interrogate how specific membrane compositions influence T cells&#39; synaptic output requires defining the physiological density of the target membrane component. Flow cytometry-based quantifications of cell surface proteins are an essential step in this protocol and require: 1) the use of antibodies with known numbers of fluorochromes per antibody (F/P), and 2) benchmark beads providing a standard reference for interpolating fluorochrome molecules from measured mean fluorescence intensities (MFIs).
These benchmark standards consist of five bead populations, each containing an increasing number of equivalent soluble fluorochromes (MESFs), which span the dynamic range of arbitrary fluorescence detection. These standard populations yield discrete fluorescence peaks, facilitating the conversion of arbitrary fluorescence units into MESFs by simple linear regression. The resulting MESFs are then used alongside antibody F/P values to calculate the average number of bound molecules per cell (or BSLB in later steps). The application of estimated cell surface areas to the average number of detected molecules then enables the calculation of physiological densities as molecules/&#181;m2. This quantification protocol can also be adapted to the measurement of protein densities on T cells and the biochemical reconstitution of membrane compositions mediating the formation of homotypic T cell synapses (i.e., T-T synapses9). If needed, the valency of antibody binding can be further estimated by using recombinant targets labeled with known numbers of fluorochromes per molecule. Then, the antibody-binding valency can be calculated for the same BSLB population by simultaneously comparing the number of bound fluorescent proteins and quantification antibodies (using two different quantification fluorochromes and MESF standards).
The reconstitution of APC membranes requires the assembly of supported lipid bilayers (SLBs) on silica beads1. Liposome stocks containing different phospholipid species can be harnessed to form a versatile lipid-bilayer matrix, enabling the anchoring of recombinant proteins with different binding chemistry (the preparation of liposomes is detailed in 10). Once the physiological density (or densities) of the relevant ligand &#34;on cells&#34; is defined, the same flow cytometry protocol is adapted to estimate the concentration of recombinant protein needed to coat BSLBs with the target physiological density. Two different anchoring systems can be used either in combination or separately.
First, SLB containing a final 12.5 mol% of Ni2+-containing phospholipids is sufficient to provide approximately 10,000 His-tag binding sites per square micron10 and works well to decorate BSLBs with most commercially available proteins whose physiological densities do not exceed this maximum loading capacity. The second loading system harnesses biotin-containing phospholipids (as mol%) to load biotinylated anti-CD3e Fab (or HLA/MHC monomers) via streptavidin bridges. The combination of these two BSLB decoration methods then enables the flexible tailoring of BSLBs as synthetic APCs. For highly complex APC surface compositions, the mol% of phospholipids and proteins can be increased to load as many proteins as the question at hand requires. Once the working concentrations of proteins and mol% of biotinylated phospholipids are defined, BSLBs can be assembled to interrogate the synaptic output of T cells with multiparametric flow cytometry.

preparation of bead-supported lipid bilayers to study the particulate output of t cell immune synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

FCM for absolute protein quantification on the cell surface 
The reconstitution of BSLBs presenting physiological densities of ligands requires the estimation of total protein densities on the modeled cell subset. To reconstitute BSLBs, include any relevant ligand expected to play a role in the signaling axis of interest alongside proteins supporting the adhesion and functional interaction between BSLB and cells, such as ICAM-1 and costimulatory molecules, e.g., CD40, CD58, and B7 receptors (CD80 an...

Watch this Scientific Journal Video about Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses at JoVE.com

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

The study of the output of the immune synapse has been restricted mostly to microscopic methods. BSLBs have expanded our repertoire of tools that can be used to study the output of T cell immune synapses, including flow cytometry, microscopy, proteomics, and RNA sequencing technologies. As such, BSLBs can be used in a wide variety of immunology questions and in identifying the composition and biochemical regulation of the T cell output.This includes, for instance, the study of chimeric antigen receptors, drugs that inhibit specific signaling pathways, or the genetic ablation of genes of interest. One of the most critical aspects of this protocol is to optimize the protein calibrations and the multicolor flow cytometry panel required for tracking the trans-synaptic particle of interest. This requires the systematic iteration of protein concentrations, antibody concentrations, and instrument settings.Begin by diluting one microliter of bead solution with 1, 000 microliters of PBS. Count the beads using a hemocytometer chamber and calculate their concentration per milliliter. Then, calculate the volume of silica beads needed for 500, 000 final BSLBs per titration point.Transfer the required volume of silica beads to a sterile 1.5-milliliter microcentrifuge tube. Wash the silica beads three times with one milliliter of sterile PBS and centrifuge the beads. Prepare three volumes of liposome master mix containing a final 12.5-mole percent of nickel-containing phospholipids.Use the liposome master mix to resuspend the washed silica beads. Then, gently mix it by pipetting up and down half of the total volume. Using a sterile technique, add argon or nitrogen gas to the tube to displace air and protect the lipids from oxidation during mixing.Add argon to the 0.4-millimolar lipid stocks. Store the stock and manipulate using a sterile technique. Move the BSLBs to the vertical mixer and mix for 30 minutes at room temperature using orbital mixing at 10 RPM.Then, spin down the beads by centrifuging for 15 seconds at room temperature on a bench-top minicentrifuge. Block the formed BSLBs by adding one milliliter of 5%BSA containing 100-micromolar of nickel sulfate to saturate NTA sites on the BSLBs. Wash three times using HBS-HSA buffer to remove the excess blocking solution.Take a new U-bottom or V-bottom 96-well plate and prepare two-fold serial dilutions of the proteins. Resuspend the prepared BSLBs in a volume such that 100 microliters of HBS-HSA buffer contain 500, 000 BSLBs. Then, take another U-bottom or V-bottom 96-well plate and transfer 100 microliters of the BSLB suspension to the wells in such a way that each well receives 500, 000 BSBLs.Spin down the second plate at room temperature for two minutes at 300x G.Discard the supernatant and transfer 100-microliter volumes from the protein titration plate to the plate containing the sedimented BSLBs. Mix gently, avoiding excess bubble generation while pipetting. Use aluminum foil to protect it from light and incubate at room temperature for 30 minutes at 1, 000 RPM.Then, wash the plate three times by adding HBS-HSA buffer to the plate and spin it down at room temperature for two minutes at 300x G.Count the cells after washing. Then, dilute them to a final concentration of 2.5 million per milliliter using synaptic transfer assay medium. Once the BSLBs are loaded with the protein mix, wash the BSLBs twice with HBS-HSA buffer to remove the excess unbound proteins.Resuspend 500, 000 BSLBs per well in 200 microliters of HBS-HSA buffer. Transfer 100 microliters of BSLBs per well to a new U-bottom 96-well plate to make a duplicate, such that the final amount of BSLB per well is 250, 000. Spin down the BSLBs at 300x G for two minutes at room temperature.Discard the supernatant and then resuspend the BSLBs using 100 microliters of T cell suspension. Mix gently to prevent the formation of bubbles. Incubate the co-cultures for 90 minutes at 37 degrees Celsius.Protect the cells from light and cool down the co-cultures by first incubating them at room temperature for a minimum of 15 minutes. Centrifuge the co-cultures at room temperature for five minutes at 500x G.Discard the supernatant and resuspend the co-cultures in calcium and magnesium ion-free 2%BSA PBS at room temperature for blocking. Place the cells on ice for 45 minutes and protect them from light.Prepare the antibody master mix using ice-cold 0.22-micrometer-filtered 2%BSA and PBS as a staining buffer. This master mix will provide extra blocking. Spin down the co-cultures at 500x G for five minutes and four degree Celsius.Discard the supernatants, and then use a multi-channel pipette to resuspend the cells in the staining master mix containing optimized antibody concentrations. Include isotype-labeled cells and BSLBs, fluorescent and non-fluorescent BSLBs, and cells and BSLBs stained alone. Mix gently by pipetting up and down half of the volume.Incubate for 30 minutes on ice and protect them from light. Wash the cells and BSLBs twice using ice-cold 2%BSA PBS. Spin them down at 500x G for five minutes at four degrees Celsius.After centrifugation, check the co-culture sedimented on the bottom of the wells. Then, resuspend the washed co-cultures in 100 microliters of PBS and acquire immediately. Activate the log scale for side-scattered light and the time-of-flight parameter for side-and forward-scattered lights.Acquire MESF standards, ensuring both the dim-set and brightest populations fall in the linear range of the instrument. Then, acquire compensation samples, calculate compensation, and apply the compensation matrix to the experiment. Acquire and save a minimum of 20, 000 total MESF standards for each quantification channel.Avoid using FACS sheath fluid or similar to resuspend MESF beads, as these contain salts and alcohols that will destroy the fluorochromes, leading to wider fluorescence peaks and high error. For acquisition using high-throughput samplers, set instrument acquisition to Standard, set sample acquisition to 80 microliters. Sample Flow Rate between two and three microliters per second, sample Mixing Volume at 50 microliters, sample Mixing of 150 microliters per second, and mixing per well between three and five.Acquire a minimum of 10, 000 single BSLBs per sample. Finally, export the FCS files. An example of quantitative flow cytometry measurements of ICAM-1 on the surface of tonsillar B cells and helper T-cells is shown here.The representative images describe the gating strategy for analyzing single CXCR5 B cells and follicular-helper T cells isolated from human palatine tonsils. This sequential gating strategy identifies single live events within the continuous acquisition window. The doublets, the cell surface expression of ICAM-1 compared to FMO controls and FMO controls labeled with relevant isotypes of the populations, are shown here.Gating and measurement of MFIs from different standard MESF populations in the overlaid histograms of the MESF populations are shown here. The values represent the MFIs for each of the five MESF populations. Linear regression of MESF over cMFI for the MESF populations are presented here.The slope extracts MESF bound to cells from the FCM data of ICAM-1 on the surface of tonsillar B cells in TFH. The conversion to absolute molecular densities follows these simple mathematical operations. The representative images show the flow cytometry analysis of BSLBs reconstituted with increased densities of recombinant monomeric ICAM-1 12 histidines.Regression analyses of ICAM-1 reference concentration over measured density are shown here. The slope is used to calculate target concentrations of protein to achieve the density of cells. The flow diagrams show the critical steps for co-culturing T cells with BSLBs, reconstituting model membranes in the subsequent measurement of particle transfer with flow cytometry.The gating strategy to identify single BSLBs and cells within the continuous acquisition window is shown here. One of the most important aspects of this protocol is that, to maintain reproducibility of the results, each time you change protein or lipid stocks, you need to perform new calibration analysis. To discover new effective molecules secreted by different T cell subsets, BSLBs can be further isolated by fluorescence-activated cell sorting as subjected to transcriptomic and proteomic analysis.Our colleagues within the University of Oxford have used this technology to study and demonstrate new receptor-ligand interactions, and also to study the effects of genetic ablations on the output of the T cell immune synapse.

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Immunology and Infection

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.
Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.
Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.
Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.
This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...