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.
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.
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 PB6,7. This has further solidified the need to better understand the features of the T-cell synaptic interface in primary human T cells.
To this end, we have developed a novel mini-scale approach exploiting lipid bilayers built into multichannel flow slides enabling us to perform the imaging of T-cell/bilayer interfaces with less than 105 primary T cells isolated from human PB and LN. This novel technique allows the study of the biophysical properties of primary human T-cell synaptic interfaces in order to better model and understand in vivo cell-cell interactions.
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
2. Components for the Preparation of the Planar Lipid Bilayers
3. Formation of Glass-supported Planar Lipid Bilayers
4. Imaging of the T Cells Interaction with the Planar Bilayer
5. Image Analysis
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 ICAM-1 at 50 molecules/µm2 and 300 molecules/µm2, respectively. CD8+ T cells derived from human cord blood were activated by a stimulation with plate-bound anti-CD3 and anti-CD28 antibodies13,14. There was no difference between CD8+ T cells that formed classical immunological synapses on the lipid bilayers built in either the flow cell or the multichannel flow slide (Figure 1). All other experiments were performed with lipid bilayers made in the multichannel flow slides.
Next, we examined the ability of PB- and LN-derived human CD4+ T cells to form synaptic interfaces with the planar lipid bilayers and release granules. We exploited TIRF microscopy to maximize the vertical resolution at the T cell-bilayer interface to visualized degranulating cells and to evaluate the kinetics of the granule release. We observed 4 groups of cells for both the LN- and PBMC-derived CD4+ T cells: some T cells established mature immune synapses, but either did or did not release granules, other T cells released the granules without a formation of mature synapses, and still other T cells neither formed synapses nor released the granules (Figure 2)7. The difference between LN- and PBMC-derived CD4+ T cells became apparent when the kinetics of the granule release was evaluated. PBMC-derived CD4+ T cells were able to begin releasing granules almost twice as fast as LN-derived CD4+ cells (Figure 3)7. Overall, the data demonstrate that despite a heterogeneity of LN- and PBMC-derived CD4+ T cells, the latter contained a fraction of T cells capable of rapid degranulation.
Figure 1: Comparison of the flow slide system and conventional flow chamber. (A) This panel shows the multichannel flow slide allowing an assembly of 6 glass-supported planar lipid bilayers. Each bilayer is connected to the delivery and the exit ports. Less than 1 x 105 cells are sufficient for the analysis. (B) This panel shows the conventional flow chamber system allowing an assembly of 1 glass-supported planar lipid bilayer. 2 x 106 cells are required for the analysis. The other panels show (C and D) representative TIRF microscopy and (E and F) confocal with interference reflection microscopy (IRM) images of the interface developed by activated cord-blood-derived CD8 T cells. The cells were exposed to a lipid bilayer containing ICAM-1 (300 molecules/µm2) and anti-CD3 antibodies (50 molecules/µm2) acquired (C and E) in flow slides or (D and F) in a conventional flow system under similar conditions. The TIRF microscopy and confocal with IRM images are taken using 100X and 60X magnification objectives, respectively. The percentage of T cells that form mature synapses on the bilayers built in either flow slide or conventional flow chamber was very similar but varies between experiments from 75% to 90%. In all images, Cy-5-labelled ICAM-1 molecules are shown in blue; Alexa-Fluor-488-labeled anti-CD3 antibodies are shown in green; Alexa-Fluor-568-labeled anti-CD107a antibodies bound to CD107a are shown in red; IRM images are shown in cyan; and the scale bars are 10 µm in length. Please click here to view a larger version of this figure.
Figure 2: Structure of a T cell–bilayer interface and the pattern of degranulation by lymph node and peripheral blood mononuclear CD4+ T cells. LNMC- or PBMC-derived cells were sorted to separate CD4+ T cells that were exposed to the bilayers containing 50 molecules/µm2 of anti-CD3 antibodies and 300 molecules/µm2 of ICAM-1 protein. The interface between the cells and the bilayers was imaged by TIRF microscopy. The scale bars are 5 µm. (A) These representative images of a T cell – bilayer interface demonstrate the structure of the T cell–bilayer interfaces and degranulation pattern. (B) These diagrams show a representation of LNMC- and PBMC-derived CD4+ T cells with a different structure of synaptic interfaces and patterns of granule release. Please click here to view a larger version of this figure.
Figure 3: Dynamic changes of the structure of a T cell–bilayer interface and the kinetics of a degranulation of lymph node and peripheral blood mononuclear CD4+ T cells. (A) This panel shows time-dependent changes of a T cell–bilayer interface and the appearance of released granules. The scale bars are 5 µm. (B) This panel shows a quantitation of the kinetics of a granule release by LN-derived CD4+ T cells (closed circles) and PBMC-derived CD4+ T cells (open circles). Each individual circle indicates the time of the first appearance of a detectable granule release by individual cells. The median and IQR (interquartile range) are shown for all scatter plots. Mann-Whitney tests were performed to compare differences between the indicated groups of T cells. * P <0.05, ** P <0.01. Please click here to view a larger version of this figure.
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 system5, creating the opportunity to analyze primary human T cells from blood and other tissues.
The number of injected cells used in this study allowed us to image about 50 cells per imaging field with a 60X objective. A larger concentration resulted in cell aggregation, reducing the number of cells suitable for analysis. We could further reduce the number of injected cells 10–50x, but the lower limit of the cell number depends on cell heterogeneity, on the number of imaging fields, and on the experimental design. Some investigators have previously used bilayers built in an open chamber with a glass bottom that required a similar number of cells for analysis16. However, the closed system described here, with a channel height of 0.1 – 0.4 mm, allows for rapid cell sedimentation to synchronize the cell attachment and analysis of the T-cell response without the risk of liquid evaporation. The sticky slide system further permits the formation of up to three bilayers per channel. Thus, the same cell sample could be used for the analysis of cell behavior on distinct bilayers with a different composition of stimulatory, adhesion, and costimulatory molecules.
Some precautions should be taken during the assembly of the sticky slides and coverslips for a successful bilayer formation. Particularly, the coverslips should be completely dry, as even a small amount of liquid left on the glass surface could result in leakage during the bilayer washing procedure. Importantly, it is essential to even the attached coverslip at the edges of the contact with the outer ring of polypropylene scissor-type forceps to avoid leakage (as described in step 3.4.1). It is also important to avoid any bubbling in the entry ports of the channels. If any bubbles enter a channel, they are almost impossible to remove and ruin the bilayer in the channel. Similarly, it is important to avoid any fast pipetting, since the turbulent flow of liquid may introduce bubbles into the channel and damage the bilayer.
A release of cytolytic granules is detected by the appearance of CD107a at the T cell-bilayer interface. Fab regions of Alexa-Fluor-568-labeled anti-CD107a antibodies are added to CD8 T cells prior to the loading on the bilayers. TIRF microscopy exploits an evanescent wave to illuminate the area about 100 nm above the bilayer in the volume defined as evanescent volume, allowing to increase the vertical resolution of the imaging. Labeled antibody Fabs, which diffuse very rapidly within the evanescent volume at 37 °C, do not generate a detectable fluorescent signal. During the membrane fusion of specialized lysosomes containing lytic molecules with the cell membrane, CD107a membrane protein appears on the T cells contact surface in distinct locations. The Fabs get bound to the CD107s protein at that time. Attached to the CD107a protein, fluorescent-labeled Fab(s) form immobile clusters that generate a bright fluorescence detectable by TIRF microscopy, indicative of T cell degranulation.
The authors have nothing to disclose.
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.
CD4 T cell isolation kit, human | Miltenyl Biotec | 130-096-533 | |
CD8 T cells Isolation Kit, human | Miltenyl Biotec | 130-096-495 | |
DOPC | Avanti Polar Lipids | 850375C | |
DOGS NTA | Avanti Polar Lipids | 790528C | |
Biotinyl Cap PE | Avanti Polar Lipids | 870273C | |
Human Serum Albumin | Octapharma USA | NDC 68982-643-01 | |
sticky-Slide VI 0.4 | ibidi | 80608 | |
Coverslips for sticky-Slides | ibidi | 10812 | |
Bioptech FCS2 Chamber | Bioptech | 060319-2-03 | |
anti-CD3 antibody | Thermo Fisher Scientific | 16-0037-81 | OKT3 clone, hybridoma cells are available from ATCC |
anti- CD28 antibody | Genetex | GTX14664 | 9.3 clone |
Casein | Sigma | C5890 | |
Biotin-PEO4-NHS | Thermo Fisher Scientific | 21329 | |
DMSO | Sigma | D2650-5 | |
Alexa Fluor 488 protein labeling kit with column for labeled protein purification | Thermo Fisher Scientific | A10235 | |
Alexa Fluor 568 protein labeling kit with column for labeled protein purification | Thermo Fisher Scientific | A10238 | |
Amersham Cy5 NHS Ester | GE Life Science | PA15101 | |
pMT/V5-His A, B, C Drosophila Expression Vectors | Thermo Fisher Scientific | V412020 | |
pcopneo, G418 Drosophila expression vector for positive selection | ATCC | 37409 | |
Serum free Drosophial media Insect-XPRESS | Lonza | 12-730Q | |
Hybridoma YN1/1.7.4 | ATCC | CRL1878 | The hybridoma secrets antibody against ICAM-1. |
Cyanogen bromide-activated-Sepharose 4B | Sigma-Aldrich | C9142 | Utilized for preparation of Sepharose with covelently bound anti-ICAM antibody. |
MasterFlex tangential flow concentrator | Cole-Parmer | 77601-60 7592-40 | Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant |
Centramate Lab Tangential Flow Systems | Pall Laboratory | FS002K10 OS010T12 FS005K10 | Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant |
Ni-NTA Agarose | QIAGEN | 30210 | |
Dialysis tubing | Spectra/Por | 131384 | |
Papain from papaya latex | Sigma | P3125 | |
mouse anti-human antibody against CD107a | BD Bioscences | 555798 | Clone H4A3 |
Ansell Natural Blue Gloves | Fisher Scientific | 19-014-539 | |
Nalgene Polypropylene Scissor-Type Forceps | Thermo Fisher Scientific | 6320-0010 | |
Streptavidin | ProZyme | SA10 | |
Confocal microscope | Nikon | Nikon 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 microscope | Andor | Andor 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 Software | Molecular devices |