Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

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

Summary

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Abstract

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

Introduction

Scientific knowledge of the structural features of T-cell immune synapses and their link to the functional activity of T cells has been generated primarily from the study of cell lines and clones derived from PB. To what degree these findings relate to primary T cells obtained from blood or human lymphoid tissues remains unclear, as the synaptic interfaces of T cells residing in lymphoid and other tissues have not been analyzed thus far. Importantly, emerging data suggest that tissue-resident and lymphoid-organ-derived T cells may have significant differences in their phenotype and functional activity compared to those in PB6,

Protocol

This study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants, and blood and lymph node samples were acquired with the approval of the Institutional Review Board at the University of Pennsylvania (IRB#809316, IRB# 815056). All human subjects were adults. Cord blood samples were kindly provided by Labor and Delivery of the Department of Obstetrics & Gynecology at Thomas Jefferson University. All samples were de-identified.

1. Isolation of CD4+ T Cells for an Image Analysis

  1. Thaw a 1 mL aliquot containing 107 frozen peripheral b....

Representative Results

First, we compared the structure of the synaptic interface formed by activated cord-blood-derived CD8+ T cells exposed to lipid bilayers built either in traditional large-scale flow cell systems (see the Table of Materials for details)1,2,3,4 or in multichannel flow slides. The bilayers contained fluorescent-labeled anti-CD3 and .......

Discussion

The novel technique described here utilizes similar reagents required to build planar bilayers in the conventional flow cell5 and can be successfully applied to perform the imaging of primary human T cell–bilayer interfaces3,4,15. The technique offers a significant reduction in the fluorescent molecules usage and requires 10–20x fewer T cells as compared to a flow cell system.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the R01AI118694 NIH grant to Michael R. Betts, which includes sub-award 566950 to Yuri Sykulev. We thank the Sidney Kimmel Cancer Center Bioimaging Shared Resource for their excellent support.

....

Materials

NameCompanyCatalog NumberComments
CD4 T cell isolation kit, humanMiltenyl Biotec130-096-533
CD8 T cells Isolation Kit, humanMiltenyl Biotec130-096-495
DOPCAvanti Polar Lipids850375C
DOGS NTAAvanti Polar Lipids790528C
Biotinyl Cap PEAvanti Polar Lipids870273C
Human Serum AlbuminOctapharma USANDC 68982-643-01
sticky-Slide VI 0.4ibidi80608
Coverslips for sticky-Slidesibidi10812
Bioptech FCS2 ChamberBioptech060319-2-03
anti-CD3 antibodyThermo Fisher Scientific16-0037-81OKT3 clone, hybridoma cells are available from ATCC
anti- CD28 antibodyGenetexGTX146649.3 clone
CaseinSigmaC5890
Biotin-PEO4-NHSThermo Fisher Scientific21329
DMSOSigmaD2650-5
Alexa Fluor 488 protein labeling kit with column for labeled protein purificationThermo Fisher ScientificA10235
Alexa Fluor 568 protein labeling kit with column for labeled protein purificationThermo Fisher ScientificA10238
Amersham Cy5 NHS EsterGE Life SciencePA15101
pMT/V5-His A, B, C Drosophila Expression VectorsThermo Fisher ScientificV412020
pcopneo, G418 Drosophila expression vector for positive selectionATCC37409
Serum free Drosophial media Insect-XPRESSLonza12-730Q
Hybridoma YN1/1.7.4ATCCCRL1878The hybridoma secrets antibody against ICAM-1.
Cyanogen bromide-activated-Sepharose 4BSigma-AldrichC9142Utilized for preparation of Sepharose with covelently bound anti-ICAM antibody.
MasterFlex tangential flow concentratorCole-Parmer77601-60 7592-40Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant
Centramate Lab Tangential Flow SystemsPall LaboratoryFS002K10 OS010T12 FS005K10Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant
Ni-NTA AgaroseQIAGEN30210
Dialysis tubingSpectra/Por131384
Papain from papaya latexSigmaP3125
mouse anti-human antibody against CD107aBD Bioscences555798Clone H4A3
Ansell Natural Blue GlovesFisher Scientific19-014-539
Nalgene Polypropylene Scissor-Type ForcepsThermo Fisher Scientific6320-0010
StreptavidinProZymeSA10
Confocal microscopeNikonNikon TiE inverted microscope equipped with PFS for long-term image stability control, 60x oil objectives, 4 lasers with excitation lines at 405, 458, 488, 514, 561, and 640 nm, 2 GaAsP detectors and 2 high sensitivity PMTs, DIC transmitted light, Programmable X,Y,Z stage for multiple positions and stitching of large areas, time lapse functions, Tokai-Hit temperature and CO2-controlled chamber for live imaging, and anti-vibration isolation table
TIRF microscopeAndorAndor Revolution XD system equipped with Nikon TIRF-E illuminator, Lasers with 405,488,561 and 640 lines, DIC transmitted light, Yokogawa CSU-X1 spinning disk head for confocal imaging, 100/1.49 NA objective, Andor iXon X3 EM-CCD camera, objective heater, and a piezoelectric motorized stage with Perfect Focus System (PFS)
MetaMorph Premier Image Analysis SoftwareMolecular devices

References

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

T CellsPeripheral BloodLymphoid TissueSynaptic InterfaceICAM 1Nickel II chlorideCaseinLiposomesBilayerFlow ChamberMicroscopy

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved