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
<ol>
	<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...

<ol>
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The authors have no conflict of interest to declare.

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...

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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

3.9K 조회수.  The University of Oxford. 면역 시 냅 스의 출력의 연구 는 현미경 방법으로 주로 제한 되었습니다. BSLB는 유동 세포측정, 현미경 검사법, 프로테오믹스 및 RNA 염기서열 분석 기술을 포함하여 T 세포 면역 시냅스의 출력을 연구하는 데 사용할 수 있는 공구의 레퍼토리를 확장했습니다. 이와 같이, BSLB는 다양한 면역학 질문에서 사용할 수 있으며 T 세포 출력의 조성 및 생화학적 조절을 식별할 수 있다.