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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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).

Introduction

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 ne....

Protocol

1. Measurement of cell surface protein densities with quantitative flow cytometry

  1. Prepare 0.22 µm-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 µm pore filter unit to remove serum impurities and store at 4 °C.
  2. Recover the cells and sediment them by centrifugation at 300 .......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)Avanti Polar Lipids790404C-25mg18:1 DGS-NTA(Ni) in chloroform

PIPETMAN L Multichannel P8x200L, 20-200 µL
GilsonFA10011
1,2-dioleoyl-sn-glycero-3-phosphocholineAvanti Polar Lipids850375C-25mg 18:1 (Δ9-Cis) PC (DOPC) in chloroform
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 390ATTO-TECAD 390-165DOPE ATTO 390
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 488ATTO-TECAD 488-165DOPE ATTO 488
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 565ATTO-TECAD 565-165DOPE ATTO 565
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)Avanti Polar Lipids870273C-25mg18:1 Biotinyl Cap PE in chloroform
200 µL yellow tips 10 x 96 Tips, StackStarlabS1111-0206
5 mL polystyrene round-bottom tubesFalcon®352052
5.00 ± 0.05 µm non-functionalized silica beadsBangs Laboratories Inc.SS05003
96 Well Cell Cultture Plate U-bottom with Lid, Tissue culture treated, non-pyrogenic.Costar®3799For FCM staining and co-culture of BSLB and cells.
96 Well Cell Cultture Plate V-bottom with Lid, Tissue culture treated, non-pyrogenic.Costar®3894For FCM staining of cells or beads in suspension.
Alexa Fluor 488 NHS Ester (Succinimidyl Ester)Thermo Fisher Scientific, Invitrogen™A20000
Alexa Fluor 647 NHS Ester (Succinimidyl Ester)Thermo Fisher Scientific, Invitrogen™A37573 and A20006
Allegra X-12R CentrifugeBeckman CoulterFor normal in tube staining of biological samples for FCM
Aluminum FoilAny brandFor protecting cells and BSLBs from light
anti-human CD154 (CD40L), clone 24-31BioLegend310815 and 310818Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.
anti-human CD185 (CXCR5) Brilliant Violet 711, clone J252D4BioLegend356934For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E
anti-human CD19 Brilliant Violet 421, clone HIB19BioLegend302234For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E
anti-human CD2, clone RPA-2.10BioLegend300202Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD2, clone TS1/8BioLegend309218Brilliant Violet 421 conjugate.
anti-human CD252 (OX40L), clone 11C3.1BioLegendAlexa Fluor 647 conjugate
anti-human CD28, clone CD28.2eBioscience16-0289-85Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD317 (BST2, PDCA-1), clone 26F8ThermoFisher Scientific, invitrogen53-3179-42Alexa Fluor 488 conjugate, we found this clone to be cleaner than clone RS38E.
anti-human CD38, clone HB-7BioLegend356624Alexa Fluor 700 conjugate
anti-human CD38, clone HIT2BioLegend303514Alexa Fluor 647 conjugate
anti-human CD39, clone A1BioLegendLabeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD4 Brilliant Violet 650, clone OKT4BioLegend317436For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E
anti-human CD4, clone A161A1BioLegend357414 and 357421PerCP/Cyanine5.5 and Alexa Fluor 647 conjugates, respectively
anti-human CD4, clone OKT4BioLegend317414 and 317422PE/Cy7 and Alexa Fluor 647 conjugates, respectively
anti-human CD40, clone 5C3BioLegend334304Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD40, clone G28.5BioLegendLabelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD45, clone HI30BioLegend304056 and 368516Alexa Fluor 647 and APC/Cy7 conjugates
anti-human CD47, clone CC2C6BioLegend323118Alexa Fluor 647 conjugate
anti-human CD54 (ICAM-1), clone HCD54BioLegend322702Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD63 (LAMP-3), clone H5C6BioLegend353020 and 353015PerCP/Cyanine5.5 and Alexa Fluor 647 conjugates, respectively
anti-human CD73, clone AD2BioLegend344002Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD80, clone 2D10BioLegend305216Alexa Fluor 647 conjugate
anti-human CD81, clone 5A6BioLegend349512 and 349502PE/Cy7 conjugate and labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester), respectively.
anti-human CD82, clone ASL-24BioLegend342108Alexa Fluor 647 conjugate
anti-human CD86, clone IT2.2BioLegend305416Alexa Fluor 647 conjugate
anti-human CD8a, clone HIT8aBioLegend300920Alexa Fluor 700 conjugate
anti-human CD8a, clone SK1BioLegend344724Alexa Fluor 700 conjugate
anti-human HLA-A/B/C/E, clone w6/32BioLegend311414 and 311402Alexa Fluor 647 conjugate and Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human HLA-DR, clone L243BioLegend307656 and 307622Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.
anti-human ICAM-1, clone HCD54BioLegend322702Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human ICOS, clone C398.4ABioLegend313516Armenian Hamster IgG
anti-human ICOSL, clone MIH12BioLegend329611Alexa Fluor 647 conjugate
anti-human ICOSL, clone MIH12eBioscience16-5889-82Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human LFA-1, clone TS1/22BioLegendProduced in houseLabelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human OX40, clone Ber-ACT35 (ACT35)BioLegend350018Alexa Fluor 647 conjugate
anti-human PD-1 , clone EH12.2H7BioLegend135230 and 329902Alexa Fluor 647 conjugate and Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human PD-L1, clone 29E.2A3BioLegend329702Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human PD-L2, clone 24F.10C12BioLegend329611Alexa Fluor 647 conjugate
anti-human PD-L2, clone MIH18BioLegend345502Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human TCRab, clone IP26BioLegend306712 and 306714Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.
antti-human CD156c (ADAM10), clone SHM14BioLegend352702
antti-human CD317 (BST2, Tetherin), clone RS38EBioLegend348404Alexa Fluor 647 conjugate
Armenian Hamster IgG Alexa Fluor 647 Isotype control, clone HTK888BioLegend400902Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
BD Cytometer Setup and Tracking beadsBecton Dickinson & Company (BD)641319Performance track of instruments before quantitative FCM
BD FACSDivaBecton Dickinson & Company (BD)23-14523-00Acquisition software
Bovine Seum AlbuminMerck, Sigma-AldrichA3294
CaCl2, Calcium chlorideMerck, Sigma-AldrichC5670anhydrous, BioReagent, suitable for insect cell culture, suitable for plant cell culture, ≥96.0%
Casein from bovine milk, suitable for substrate for protein kinase (after dephosphorylation), purified powderMerck, Sigma-AldrichC5890
Dynabeads Human T-Activator CD3/CD28ThermoFisher Scientific, Gibco11132D
DynaMag-2ThermoFisher Scientific, Invitrogen™12321DFor the removal of Dynabeads Human T-Activator CD3/CD28 in volumes less than 2 mL
DynaMag™-15ThermoFisher Scientific, Invitrogen™12301DFor the removal of Dynabeads™ Human T-Activator CD3/CD28 in volumes less than 15 mL
Fetal Bovine Serum Qualified, One ShotThermoFisher Scientific, GibcoA3160801Needs heat inactivation for 30 min at 56 oC
Ficoll-Paque PLUSCytiva, GE HealthcareGE17-1440-02Sterile solution of polysaccharide and sodium diatrizoate for lymphocyte isolation
Fixable Viability Dye eFluor 780eBiosciences65-0865-14For the exclusion of dead cells during analyses
FlowJoBecton Dickinson & Company (BD)Version 10.7.1Analysis software
Grant Bio MPS-1 Multi Plate ShakerKeison ProductsMPS-1For the mixing of either cells during stainings or BSLBs during staning or protein loading (as an alternative to orbital agitation)
HEPES Buffer Solution (1 M)ThermoFisher Scientific, Gibco15630-056
HEPES, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid.Merck, Sigma-AldrichH4034For preparation of HBS/HAS or HBS/BSA buffer. BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
HERACell 150i CO2 incubator, 150 L, Electropolished Stainless SteelThermoFisher Scientific51026282For culturing and expanding purified CD4+ and CD8+ T cells.
Hula Mixer® Sample MixerThermoFisher Scientific, Life Technologies15920DVertical, variable-angle laboratory mixer used for the mixing of BSLBs and lipid master mix, blocking solutions, protein master mix and small scale antibody stainings.
Human Serum Albumin, 30% aqueous solutionMerck, Sigma-Aldrich12667-M
Human TruStain FcX Fc Receptor Blocking SolutionBioLegend422302Fc Receptor Blocking Solution for blocking of Fc Receptors from biologically relevant samples
Innovatis CASY cell counter and analyzer TTBiovendis Products GmbHFor the counting of cells and the determination of cell size and volume based on the exclusion of electric current.
KCl, Potassium chlorideMerck, Sigma-AldrichP5405Powder, BioReagent, suitable for cell culture
L-Glutamine 200 mM (100x)ThermoFisher Scientific, Gibco25030-024
MgCl2, Magessium chlorideMerck, Sigma-AldrichM2393BioReagent, suitable for cell culture, suitable for insect cell culture
Microtube Insert for 24 x 1.5/2.0 mL tubesKeison ProductsP-2-24Microtube insert for Grant Bio MPS-1 Multi Plate Shaker
Mini IncubatorLabnet International I5110A-230VFor the incubation (co-culturing) of BSLB and cells in the absence of CO2
Minimum Essential Medium Non-Essential Amino AcidsThermoFisher Scientific, Gibco11140-035
Mouse IgG polyclonal antibody controlMerck, Sigma-AldrichPP54Used as positive control for the measurement of antibodies bound to mouse IgG capture bead standards
Mouse IgG1, k Isotype, clone MOPC-21BioLegend400129, 400112, 400130, 400144, 400128 and 400170Alexa Fluor 488, PE, Alexa Fluor 647, Alexa Fluor 700, APC/Cyanine7 and Brilliant Violet 785  conjugates, respectively.
Mouse IgG1, k Isotype, clone X40Becton Dickinson & Company (BD), Horizon562438Brilliant Violet 421 conjugate.
Mouse IgG1, κ Isotype control, clone P3.6.2.8.1eBioscience14-4714-82Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
Mouse IgG2a, k Isotype, clone MOPC-173BioLegend400240Alexa Fluor 647 conjugate
Mouse IgG2b, k Isotype, clone MPC-11BioLegend400330 and 400355Alexa Fluor 647 and Brilliant Violet 785 conjugates, respectively
Multiwell 6 well Tissue culture treated with vacuum gas plasmaFalcon353046For culturing and expanding purified CD4+ and CD8+ T cells.
Na2HPO4, Disodium PhosphateMerck, Sigma-AldrichS7907
NaCl, Sodium chlorideMerck, Sigma-AldrichS5886BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
NiSO4, Nickel(II) sulfateMerck, Sigma-Aldrich656895For saturating NTA sites; added during the blocking process
Penicillin Streptomycin [+]10,000 units Penicillin; [+] 10,000 µg/mL StreptomycinThermoFisher Scientific, Gibco15140-122
Phosphate Buffered Saline pH 7.4, sterileThermoFisher Scientific, Gibco10010No Ca2+ or Mg2+ added
Polyethersulfone (PES) Filter unitThermo Scientific Nalgene UY-06730-43Hydrophilic PES membrane with low protein binding facilitates the filtering of solutions with high protein content
PURESHIELD argon ISO 14175-I1-ArBOC Ltd.11-YFor the protection of lipid stocks stored at +4 ºC.
Purified StreptavidinBioLegend280302
Quantum Alexa Fluor 488 MESF beadsBangs Laboratories Inc.488Benchmark beads for the interpolation of Alexa Fluor 488 molecules bound to cells and/or BSLB
Quantum Alexa Fluor 647 MESF beadsBangs Laboratories Inc.647Benchmark beads for the interpolation of Alexa Fluor 647 molecules bound to cells and/or BSLB
Rat anti-mouse IgG Kappa Light Chain, clone OX-20ThermoFisher Scientific, invitrogenSA1-25258Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
Recombinant human IL-2Peprotech200-02-1MG
RMPI Medium 1640 (1x); [-] L-GlutamineThermoFisher Scientific, Gibco31870-025
Rosette Human B Cell Enrichment CocktailSTEMCELL Technologies15064Isolation of B cells for measuring densities of proteins in purified cell populations
Rosette Human CD4+ T Cell Enrichment CocktailSTEMCELL Technologies15022C.1
Rosette Human CD4+CD127low T Cell Enrichment CocktailSTEMCELL Technologies15361Pre-enrichment of CD4+ CD127Low T cells for the downstream isolation of Tregs by FACS.
Rosette Human CD8+ T Cell Enrichment CocktailSTEMCELL Technologies15063
RPMI Medium 1640 (1x); [-] Phenol RedThermoFisher Scientific, Gibco11835-063For the incubation (co-culturing) of BSLB and cells in the absence of CO2. Phenol red-free media reduces the autofluorescence of cells in flow cytometry and microscopy based measurements.
Sodium Pyruvate (100 mM)ThermoFisher Scientific, Gibco11360-070
Sprout mini centrifugeFisherScientific, Heathrow Scientific LLC120301Benchtop microcentrifuge used to wash silica beads and BSLB in 1.5 mL Eppendorf tubes.
Sterile cappeed 5 mL polystyrene round-bottom tubesFalcon352058
UltraComp eBeads Compensation BeadsThermoFisher Scientific, invitrogen01-2222-42
Zeba Spin Desalting Columns 7K MWCOThermo Fisher Scientific, Invitrogen™89882

References

  1. Cespedes, P. F., Beckers, D., Dustin, M. L., Sezgin, E. Model membrane systems to reconstitute immune cell signaling. FEBS Journal. 288 (4), 1070-1090 (2021).
  2. Choudhuri, K., et al. Polarized release of T....

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