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

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 .......

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.......

Materials

Name	Company	Catalog Number	Comments
1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)	Avanti Polar Lipids	790404C-25mg	18:1 DGS-NTA(Ni) in chloroform
PIPETMAN L Multichannel P8x200L, 20-200 µL	Gilson	FA10011
1,2-dioleoyl-sn-glycero-3-phosphocholine	Avanti Polar Lipids	850375C-25mg	18:1 (Δ9-Cis) PC (DOPC) in chloroform
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 390	ATTO-TEC	AD 390-165	DOPE ATTO 390
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 488	ATTO-TEC	AD 488-165	DOPE ATTO 488
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 565	ATTO-TEC	AD 565-165	DOPE ATTO 565
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)	Avanti Polar Lipids	870273C-25mg	18:1 Biotinyl Cap PE in chloroform
200 µL yellow tips 10 x 96 Tips, Stack	Starlab	S1111-0206
5 mL polystyrene round-bottom tubes	Falcon®	352052
5.00 ± 0.05 µm non-functionalized silica beads	Bangs Laboratories Inc.	SS05003
96 Well Cell Cultture Plate U-bottom with Lid, Tissue culture treated, non-pyrogenic.	Costar®	3799	For FCM staining and co-culture of BSLB and cells.
96 Well Cell Cultture Plate V-bottom with Lid, Tissue culture treated, non-pyrogenic.	Costar®	3894	For 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 Centrifuge	Beckman Coulter		For normal in tube staining of biological samples for FCM
Aluminum Foil	Any brand		For protecting cells and BSLBs from light
anti-human CD154 (CD40L), clone 24-31	BioLegend	310815 and 310818	Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.
anti-human CD185 (CXCR5) Brilliant Violet 711, clone J252D4	BioLegend	356934	For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E
anti-human CD19 Brilliant Violet 421, clone HIB19	BioLegend	302234	For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E
anti-human CD2, clone RPA-2.10	BioLegend	300202	Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD2, clone TS1/8	BioLegend	309218	Brilliant Violet 421 conjugate.
anti-human CD252 (OX40L), clone 11C3.1	BioLegend		Alexa Fluor 647 conjugate
anti-human CD28, clone CD28.2	eBioscience	16-0289-85	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD317 (BST2, PDCA-1), clone 26F8	ThermoFisher Scientific, invitrogen	53-3179-42	Alexa Fluor 488 conjugate, we found this clone to be cleaner than clone RS38E.
anti-human CD38, clone HB-7	BioLegend	356624	Alexa Fluor 700 conjugate
anti-human CD38, clone HIT2	BioLegend	303514	Alexa Fluor 647 conjugate
anti-human CD39, clone A1	BioLegend		Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD4 Brilliant Violet 650, clone OKT4	BioLegend	317436	For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E
anti-human CD4, clone A161A1	BioLegend	357414 and 357421	PerCP/Cyanine5.5 and Alexa Fluor 647 conjugates, respectively
anti-human CD4, clone OKT4	BioLegend	317414 and 317422	PE/Cy7 and Alexa Fluor 647 conjugates, respectively
anti-human CD40, clone 5C3	BioLegend	334304	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD40, clone G28.5	BioLegend		Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD45, clone HI30	BioLegend	304056 and 368516	Alexa Fluor 647 and APC/Cy7 conjugates
anti-human CD47, clone CC2C6	BioLegend	323118	Alexa Fluor 647 conjugate
anti-human CD54 (ICAM-1), clone HCD54	BioLegend	322702	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD63 (LAMP-3), clone H5C6	BioLegend	353020 and 353015	PerCP/Cyanine5.5 and Alexa Fluor 647 conjugates, respectively
anti-human CD73, clone AD2	BioLegend	344002	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human CD80, clone 2D10	BioLegend	305216	Alexa Fluor 647 conjugate
anti-human CD81, clone 5A6	BioLegend	349512 and 349502	PE/Cy7 conjugate and labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester), respectively.
anti-human CD82, clone ASL-24	BioLegend	342108	Alexa Fluor 647 conjugate
anti-human CD86, clone IT2.2	BioLegend	305416	Alexa Fluor 647 conjugate
anti-human CD8a, clone HIT8a	BioLegend	300920	Alexa Fluor 700 conjugate
anti-human CD8a, clone SK1	BioLegend	344724	Alexa Fluor 700 conjugate
anti-human HLA-A/B/C/E, clone w6/32	BioLegend	311414 and 311402	Alexa Fluor 647 conjugate and Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human HLA-DR, clone L243	BioLegend	307656 and 307622	Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.
anti-human ICAM-1, clone HCD54	BioLegend	322702	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human ICOS, clone C398.4A	BioLegend	313516	Armenian Hamster IgG
anti-human ICOSL, clone MIH12	BioLegend	329611	Alexa Fluor 647 conjugate
anti-human ICOSL, clone MIH12	eBioscience	16-5889-82	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human LFA-1, clone TS1/22	BioLegend	Produced in house	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human OX40, clone Ber-ACT35 (ACT35)	BioLegend	350018	Alexa Fluor 647 conjugate
anti-human PD-1 , clone EH12.2H7	BioLegend	135230 and 329902	Alexa Fluor 647 conjugate and Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human PD-L1, clone 29E.2A3	BioLegend	329702	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human PD-L2, clone 24F.10C12	BioLegend	329611	Alexa Fluor 647 conjugate
anti-human PD-L2, clone MIH18	BioLegend	345502	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
anti-human TCRab, clone IP26	BioLegend	306712 and 306714	Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.
antti-human CD156c (ADAM10), clone SHM14	BioLegend	352702
antti-human CD317 (BST2, Tetherin), clone RS38E	BioLegend	348404	Alexa Fluor 647 conjugate
Armenian Hamster IgG Alexa Fluor 647 Isotype control, clone HTK888	BioLegend	400902	Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
BD Cytometer Setup and Tracking beads	Becton Dickinson & Company (BD)	641319	Performance track of instruments before quantitative FCM
BD FACSDiva	Becton Dickinson & Company (BD)	23-14523-00	Acquisition software
Bovine Seum Albumin	Merck, Sigma-Aldrich	A3294
CaCl2, Calcium chloride	Merck, Sigma-Aldrich	C5670	anhydrous, 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 powder	Merck, Sigma-Aldrich	C5890
Dynabeads Human T-Activator CD3/CD28	ThermoFisher Scientific, Gibco	11132D
DynaMag-2	ThermoFisher Scientific, Invitrogen™	12321D	For the removal of Dynabeads Human T-Activator CD3/CD28 in volumes less than 2 mL
DynaMag™-15	ThermoFisher Scientific, Invitrogen™	12301D	For the removal of Dynabeads™ Human T-Activator CD3/CD28 in volumes less than 15 mL
Fetal Bovine Serum Qualified, One Shot	ThermoFisher Scientific, Gibco	A3160801	Needs heat inactivation for 30 min at 56 oC
Ficoll-Paque PLUS	Cytiva, GE Healthcare	GE17-1440-02	Sterile solution of polysaccharide and sodium diatrizoate for lymphocyte isolation
Fixable Viability Dye eFluor 780	eBiosciences	65-0865-14	For the exclusion of dead cells during analyses
FlowJo	Becton Dickinson & Company (BD)	Version 10.7.1	Analysis software
Grant Bio MPS-1 Multi Plate Shaker	Keison Products	MPS-1	For 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, Gibco	15630-056
HEPES, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid.	Merck, Sigma-Aldrich	H4034	For 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 Steel	ThermoFisher Scientific	51026282	For culturing and expanding purified CD4+ and CD8+ T cells.
Hula Mixer® Sample Mixer	ThermoFisher Scientific, Life Technologies	15920D	Vertical, 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 solution	Merck, Sigma-Aldrich	12667-M
Human TruStain FcX Fc Receptor Blocking Solution	BioLegend	422302	Fc Receptor Blocking Solution for blocking of Fc Receptors from biologically relevant samples
Innovatis CASY cell counter and analyzer TT	Biovendis Products GmbH		For the counting of cells and the determination of cell size and volume based on the exclusion of electric current.
KCl, Potassium chloride	Merck, Sigma-Aldrich	P5405	Powder, BioReagent, suitable for cell culture
L-Glutamine 200 mM (100x)	ThermoFisher Scientific, Gibco	25030-024
MgCl2, Magessium chloride	Merck, Sigma-Aldrich	M2393	BioReagent, suitable for cell culture, suitable for insect cell culture
Microtube Insert for 24 x 1.5/2.0 mL tubes	Keison Products	P-2-24	Microtube insert for Grant Bio MPS-1 Multi Plate Shaker
Mini Incubator	Labnet International	I5110A-230V	For the incubation (co-culturing) of BSLB and cells in the absence of CO2
Minimum Essential Medium Non-Essential Amino Acids	ThermoFisher Scientific, Gibco	11140-035
Mouse IgG polyclonal antibody control	Merck, Sigma-Aldrich	PP54	Used as positive control for the measurement of antibodies bound to mouse IgG capture bead standards
Mouse IgG1, k Isotype, clone MOPC-21	BioLegend	400129, 400112, 400130, 400144, 400128 and 400170	Alexa Fluor 488, PE, Alexa Fluor 647, Alexa Fluor 700, APC/Cyanine7 and Brilliant Violet 785  conjugates, respectively.
Mouse IgG1, k Isotype, clone X40	Becton Dickinson & Company (BD), Horizon	562438	Brilliant Violet 421 conjugate.
Mouse IgG1, κ Isotype control, clone P3.6.2.8.1	eBioscience	14-4714-82	Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
Mouse IgG2a, k Isotype, clone MOPC-173	BioLegend	400240	Alexa Fluor 647 conjugate
Mouse IgG2b, k Isotype, clone MPC-11	BioLegend	400330 and 400355	Alexa Fluor 647 and Brilliant Violet 785 conjugates, respectively
Multiwell 6 well Tissue culture treated with vacuum gas plasma	Falcon	353046	For culturing and expanding purified CD4+ and CD8+ T cells.
Na2HPO4, Disodium Phosphate	Merck, Sigma-Aldrich	S7907
NaCl, Sodium chloride	Merck, Sigma-Aldrich	S5886	BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
NiSO4, Nickel(II) sulfate	Merck, Sigma-Aldrich	656895	For saturating NTA sites; added during the blocking process
Penicillin Streptomycin [+]10,000 units Penicillin; [+] 10,000 µg/mL Streptomycin	ThermoFisher Scientific, Gibco	15140-122
Phosphate Buffered Saline pH 7.4, sterile	ThermoFisher Scientific, Gibco	10010	No Ca2+ or Mg2+ added
Polyethersulfone (PES) Filter unit	Thermo Scientific Nalgene	UY-06730-43	Hydrophilic PES membrane with low protein binding facilitates the filtering of solutions with high protein content
PURESHIELD argon ISO 14175-I1-Ar	BOC Ltd.	11-Y	For the protection of lipid stocks stored at +4 ºC.
Purified Streptavidin	BioLegend	280302
Quantum Alexa Fluor 488 MESF beads	Bangs Laboratories Inc.	488	Benchmark beads for the interpolation of Alexa Fluor 488 molecules bound to cells and/or BSLB
Quantum Alexa Fluor 647 MESF beads	Bangs Laboratories Inc.	647	Benchmark beads for the interpolation of Alexa Fluor 647 molecules bound to cells and/or BSLB
Rat anti-mouse IgG Kappa Light Chain, clone OX-20	ThermoFisher Scientific, invitrogen	SA1-25258	Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)
Recombinant human IL-2	Peprotech	200-02-1MG
RMPI Medium 1640 (1x); [-] L-Glutamine	ThermoFisher Scientific, Gibco	31870-025
Rosette Human B Cell Enrichment Cocktail	STEMCELL Technologies	15064	Isolation of B cells for measuring densities of proteins in purified cell populations
Rosette Human CD4+ T Cell Enrichment Cocktail	STEMCELL Technologies	15022C.1
Rosette Human CD4+CD127low T Cell Enrichment Cocktail	STEMCELL Technologies	15361	Pre-enrichment of CD4+ CD127Low T cells for the downstream isolation of Tregs by FACS.
Rosette Human CD8+ T Cell Enrichment Cocktail	STEMCELL Technologies	15063
RPMI Medium 1640 (1x); [-] Phenol Red	ThermoFisher Scientific, Gibco	11835-063	For 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, Gibco	11360-070
Sprout mini centrifuge	FisherScientific, Heathrow Scientific LLC	120301	Benchtop microcentrifuge used to wash silica beads and BSLB in 1.5 mL Eppendorf tubes.
Sterile cappeed 5 mL polystyrene round-bottom tubes	Falcon	352058
UltraComp eBeads Compensation Beads	ThermoFisher Scientific, invitrogen	01-2222-42
Zeba Spin Desalting Columns 7K MWCO	Thermo Fisher Scientific, Invitrogen™	89882