Source: Céspedes, P. F., et al. Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses. J. Vis. Exp. (2022)
This video illustrates the reconstitution of synthetic antigen-presenting cells through Bead-Supported Lipid Bilayers and their application in assessing the synaptic output generated by activated T cells.
1. Protein density calibrations on BSLBs (Bead-supported lipid bilayers)
2. Performing synaptic transfer experiments between T cells and BSLBs
3. Measuring the synaptic transfer of particles to BSLBs
Figure 1: Absolute quantification of proteins on the surface of APCs. (A) Example of quantitative flow cytometry measurements of ICAM-1 on the surface of tonsillar B cells (Foll. Bc) and helper T cells (TFH). (i-vii) Gating strategy for analyzing single CXCR5+ Bc and TFH isolated from human palatine tonsils. Shown is the sequential gating strategy for identifying single, live events contained within the continuous window of acquisition. (iii-iv) black arrows indicate doublets. (viii) overlaid histograms showing the cell surface expression of ICAM-1 (teal histograms) compared to FMO controls (grey histograms) and FMO controls labeled with relevant isotypes (black histograms, which overlap with the grey histograms) of the populations shown in (vii). Arrows indicate the direction for the nested gating strategy used to identify CXCR5+ B cells (Bc; CD19+) and TFH (CD4+). (B) Extraction of absolute molecules on the surface of tonsillar cells from MFI requires regression analyses of MESF benchmark beads acquired using the same instrument setting as the cells shown in A. (i-v) Shown is the sequential gating strategy for identifying single, live events contained within the continuous window of acquisition. (vi) Gating and measurement of MFIs from different standard MESF populations. (vii) shown are overlaid histograms of the MESF populations identified in (vi). The values displayed on the top right represent the MFIs for each of the 5 MESF populations (blank, 1, 2, 3, and 4). (viii) Linear regression of MESF over cMFI for the MESF populations shown in (vii). Shown is the slope (b) for extracting MESF bound to cells from data in A. (C) In the extraction of the number of molecules, follow simple mathematical operations starting with the application of the slope calculated in (viii) from measured MESF cMFI (cMFIM) and reference MESF values (MESFR). To extract the MESF bound to cells (MESFcells), divide the corrected MFI of cells (cMFIcells) by the calculated slope. Then, to calculate the number of molecules bound to cells (Molec.cells), divide MESFcells by the F/P of the detection (quantification) antibody. Finally, to calculate the molecular density on the surface of cells (Dcells), divide Molec.cells by the estimated cell surface area (CSAE). Abbreviations: X = independent variable; Y = dependent variable (measured fluorescence), cMFIM = measured corrected MFI; MESFR = reference MESF values; MESFcells = estimated MESF per cell; cMFIcells = corrected MFI cells; Molec.cells = estimated molecules per cell. Dcells = estimated density on cells; CSAE = estimated Cell Surface Area.
Figure 2: Reconstitution of BSLB with recombinant ICAM-1 and the measurement of particulate transfer to BSLBs. (A, i-vi) Flow cytometry analysis of BSLBs reconstituted with increased densities of recombinant monomeric ICAM-1 12-His (rICAM-1). (i-v) As in Figure 1, focus the gating strategy on single BSLBs within the continuous window of acquisition. Note the gap immediately before the time continuum gate, which was excluded to prevent errors of measurement. (vi) Good protein quality often results in the homogeneous coating of BSLB at high concentrations, with the observation of narrow fluorescence distributions (low Coefficient of Variation, see histograms in vi). (B) Regression analyses of ICAM-1 reference concentration (CR) over measured density (DM). Use the slope to calculate target concentrations of protein (CT) to achieve the density of cells (Dcells) measured in the experiments in Figure 1. Abbreviations: 12-His = 12-histidines tag; DM = measured molecular densities; CR = reference concentrations of the rICAM-1; CT = target concentration (to be interpolated); Dcells = densities measured in cells (see also Fig. 1C).
Figure 3: Measurement of T cell synaptic particles transferred to BSLBs. (A; i-v) Flow diagram showing the critical steps for the co-culturing of T cells with BSLBs reconstituting model membranes and the subsequent measurement of particle transfer with flow cytometry. (iv) Blue and dark yellow diagrams show the relative distribution and location of cells and BSLBs in b-iparametric flow cytometry plots. (v) Fluorescence distribution histograms displaying the relative gain of fluorescence of agonistic BSLBs (dark yellow) compared to null BSLBs (grey). (B) Exemplary synaptic transfer experiment. (i-vi) Shown is the gating strategy to identify single BSLBs and cells within the continuous acquisition window. Violet arrows indicate the direction of analysis, which continues in C. (C) (i) Focus the analyses on the MFI of single cells (blue) and single BSLBs (yellow). (ii) Equations to calculate the normalized synaptic transfer (NST%, top) and Tmax% (bottom) from the cMFI calculated for BSLB and cells. (iii-vi) Overlaid histograms showing the change of fluorescence intensity distributions for cells (blue shades) and BSLBs (yellow shades) across different densities of the T-cell activating anti-CD3ε-Fab, including non-activating (grey) and activating with either 250 (soft color value) or 1,000 (high color value) molec./µm2. Numbers in different color values represent the NST% measured for the BSLB histograms shown in yellow. The overlaid histograms show the overarching hierarchy in the synaptic transfer of T cell vesicles positive for different markers. For this composition of BSLBs (200 molec./µm2 of ICAM-1 and increasing densities of anti-CD3ε-Fab), tSVs are transferred to BSLB with TCR+(iii) > CD81+(iv) > CD4+(v) > CD28+(vi). As demonstrated in previous articles, TCR and CD81 are components of SEs and are transferred with comparatively higher efficiencies to CD4, despite the latter being expressed at comparatively higher surface levels. SE shedding results in the loss of cell surface CD81 and TCR and the gain of these signals on BSLBs (open purple arrows for 250 molec./µm2, and closed purple arrows for 1,000 molec./µm2 in yellow histograms). (D) Improper cooling down of conjugates leads to cells ripping off the SLB from silica beads as seen from comparing input beads (left biparametric plot) and conjugates subjected to rapid cooling down to 4 °C from 37 °C (right biparametric plot). Compare also with Figure 3B panel (vi). Abbreviations: PRF1 = perforin 1; NST% = normalized synaptic transfer; Tmax% = percent of maximum observed transfer (in control or reference condition); tSVs: trans-synaptic vesicles; SEs: synaptic ectosomes.
The authors have nothing to disclose.
96 Well Cell Cultture Plate U-bottom with Lid, Tissue culture treated, nonpyrogenic | Costar® | 3799 | For FCM staining and co-culture of BSLB and cells. |
96 Well Cell Cultture Plate V-bottom with Lid, Tissue culture treated, nonpyrogenic. | 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 | |
Multiwell 6 well Tissue culture treated with vacuum gas plasma | Falcon | 353046 | For culturing and expanding purified CD4+ and CD8+ T cells. |
Casein from bovine milk, suitable for substrate for protein kinase (after dephosphorylation), purified powder | |||
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 Sho | ThermoFisher Scientific, Gibco | A3160801 | Needs heat inactivation for 30 min at 56 oC |
anti-human CD154 (CD40L), clone 24-31 | BioLegend | 310815 and 310818 | Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively |
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 |